UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE · NATAL 2016 . 2 CLARISSA DE ALMEIDA MOURA...
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1 UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA CLARISSA DE ALMEIDA MOURA APRENDIZAGEM ESPAÇO-TEMPORAL E EFEITOS DAS CONDIÇÕES LUMINOSAS NO APRENDIZADO DO PEIXE PAULISTINHA NATAL 2016
Transcript of UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE · NATAL 2016 . 2 CLARISSA DE ALMEIDA MOURA...
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE BIOCIÊNCIAS
PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA
CLARISSA DE ALMEIDA MOURA
APRENDIZAGEM ESPAÇO-TEMPORAL E EFEITOS DAS CONDIÇÕES
LUMINOSAS NO APRENDIZADO DO PEIXE PAULISTINHA
NATAL
2016
2
CLARISSA DE ALMEIDA MOURA
APRENDIZAGEM ESPAÇO-TEMPORAL E EFEITOS DAS CONDIÇÕES
LUMINOSAS NO APRENDIZADO DO PEIXE PAULISTINHA
DEFESA DA DISSERTAÇÃO
APRESENTADA AO PROGRAMA DE
PÓS-GRADUAÇÃO EM
PSICOBIOLOGIA DA UNIVERSIDADE
FEDERAL DO RIO GRANDE DO
NORTE, COMO REQUISITO PARA A
OBTENÇÃO DO TÍTULO DE MESTRE
EM PSICOBIOLOGIA (ÁREA:
COMPORTAMENTO ANIMAL).
Orientadora: Profa. Dra. Ana
Carolina Luchiari
NATAL
2016
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Catalogação da Publicação na Fonte. UFRN / Biblioteca Setorial do Centro de Biociências
Moura, Clarissa de Almeida.
Aprendizagem espaço-temporal e efeitos das condições luminosas no aprendizado do peixe
paulistinha / Clarissa de Almeida Moura. – Natal, RN, 2016.
86 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. Aprendizagem espaço-temporal. – Dissertação. 2. Memória. – Dissertação. 3. Ritmo circadiano. –
Dissertação. I. Luchiari, Ana Carolina. II. Universidade Federal do Rio Grande do Norte. III. Título.
RN/UF/BSE-CB CDU 159.953
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APRENDIZAGEM ESPAÇO-TEMPORAL E EFEITOS DAS CONDIÇÕES
LUMINOSAS NO APRENDIZADO DO PEIXE PAULISTINHA
CLARISSA DE ALMEIDA MOURA
Natal, 03 de março de 2016.
Banca Avaliadora
______________________________
Profª Dra. Ana Carolina Luchiari
Departamento de Fisiologia- UFRN
Orientadora
____________________________________
Prof. Dr. Mário André Leocadio Miguel
Departamento de Fisiologia- UFRN
Membro Interno
____________________________________
Prof. Dr. Rodrigo Egydio Barreto
Departamente de Fisiologia – UNESP Botucatu
Membro externo
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AGRADECIMENTOS
Ao mestre Jesus por guiar meus passos e iluminar minha mente e minhas mãos, me
fazendo pensar e agir de maneira sensata em cada detalhe dessa pesquisa.
À minha orientadora Ana Carolina Luchiari, que acreditou em meu potencial, abriu
as portas para meu caminho na ciência, me preparou não só como pesquisadora, mas
como futura docente, e não mediu esforços para me ajudar sempre que precisei.
Às minhass amigas e companheiras de laboratório, Bábara, Mayara, Priscilla e
Diana por toda força, palavras de carinho e incetivo nas horas certas.
Aos meus ajudantes na pesquisa, Vanessa, Jéssica Pollyana, Mara, Celisa, Fred e
Joanny por cada tempo dedicado e esforço em me ajudar com a coleta de dados. Sem
vocês, esse trabalho jamais estaria completo!
À Bruna Del Vechio, por ajudar nas correções e com os conhecimentos da
Cronobiologia, e ao professor Mário Miguel, pela sua disponibilidade e cordialidade em
me ajudar com as análises.
À Tales, meu companheiro no amor e nos estudos, por sua companhia nos finais de
semana de coleta, dias de estudos, e sua ajuda com os gráficos.
À minha família, que por torcer tanto pelo meu sucesso, não medem esforços para
me ver alcançar os objetivos. Mãe, pela paciência e preocupação. Pai, pelo esforço em
ajudar e incentivo para que eu jamais desistisse. E, antes de tudo, pela educação que
vocês me deram, pois para mim, não há nada de mais precioso na vida!
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RESUMO GERAL
Para processar a informação ambiental e perceber o tempo, os indivíduos utilizam-se de
pistas ambientais, como luz e temperatura, que servem como guias para o relógio
interno. O mecanismo temporizador endógeno é chamado relógio circadiano endógeno,
o qual comanda uma grande variedade de ritmos diários bioquímicos, fisiológicos e
comportamentais presentes nos organismos. Com isso, os animais podem antecipar
eventos espaço-temporalmente distribuídos e usar essa informação para organizar as
atividades diárias, o que é uma vantagem adaptativa para os indivíduos, já que muitos
fatores ambientais apresentam variação circadiana. Aprendizagem espaço-temporal (do
inglês: "time-place learning’’-TPL) é a habilidade de associar lugares com importantes
eventos biológicos em diferentes horas do dia. Em nosso estudo utilizamos como
modelo o peixe paulistinha (Danio rerio), conhecido por ser altamente social, para testar
aprendizagem espaço-temporal baseada em reforço social. Além disso, objetivamos
averiguar os efeitos das condições de claro constante e escuro constante na
aprendizagem espaço-temporal, e se nessas condições, a atividade do peixe paulistinha é
alterada. Para isso, testamos três diferentes condições (n=10): grupo claro-escuro (CE),
grupo claro constante (CC) e grupo escuro constante (EE) durante 30 dias da seguinte
maneira: diariamente, um grupo de 5 peixes paulistinha foi introduzido em um
recipiente localizado no compartimento da manhã (um dos lados do aquário), às 8:00h e
retirado às 9:00h, e em outro recipiente do compartimento da tarde (lado oposto do
aquário), às 17:00h e removido às 18:00h, servindo como estímulo para que o peixe
experimental ocupasse o compartimento onde o grupo fosse colocado. O
comportamento foi filmado nos dois horários, 15 minutos antes e durante os 60 minutos
de exposição ao estímulo, no 15º e no 30ª dia, porém neste último, os peixes foram
filmados sem a presença do estímulo a fim de averiguarmos a aprendizagem espaço-
temporal. Por fim, para saber a influência das três condições luminosas na atividade dos
peixes, filmamos os últimos 6 dias de teste, para registrar o padrão de atividade. Nossos
resultados mostraram que em ciclo claro-escuro (CE) o peixe paulistinha apresenta TPL,
bem como é capaz de antecipar a hora e local do estímulo (grupo de coespecíficos),
enfatizando a importância do estímulo social para a aprendizagem. Em condições de
claro constante e escuro constante, o peixe paulistinha não apresentou aprendizagem
espaço-temporal. Ademais, após 30 dias em condições luminosas constantes (claro
constante e escuro constante), o peixe paulistinha mantém ritmo circadiano, porém em
claro constante sua atividade é aumentada e seu ritmo atividade-repouso é alterado,
através de um padrão de atividade distribuída homogeneamente ao longo das 24h, ao
invés de concentrada na subjetiva fase clara, como nos grupos de ciclo claro-escuro e
escuro constante, os quais conservam o padrão de atividade diurno da espécie.
Palavras chave: aprendizagem espaço-temporal, memória, estímulo social, ritmo
circadiano.
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SUMÁRIO
1. INTRODUÇÃO GERAL...................................................................................... 8
Sistema temporizador endógeno e sincronizadores.................................................. 8
Ritmos em peixes...................................................................................................... 11
Ritmo no peixe paulistinha........................................................................................ 12
Pistas sociais como sinalizadores.............................................................................. 14
Aprendizagem espaço-temporal................................................................................ 15
2. OBJETIVOS ........................................................................................................ 20
Objetivo Geral........................................................................................................ 20
Objetivos Específicos ............................................................................................... 20
3. ARTIGO 1.......................................................................................................... 21
ABSTRACT .......................................................................................................... 21
INTRODUCTION..................................................................................................... 22
MATERIAL AND METHODS................................................................................ 23
Animals and procedures............................................................................................ 23
Experimental design……………………………………………………………….. 24
Statistical analysis..................................................................................................... 26
RESULTS................................................................................................................. 26
Responsiveness on day 15......................................................................................... 26
Determination of learning on day 30........................................................................ 27
DISCUSSION........................................................................................................... 29
REFERENCES........................................................................................................... 33
4. ARTIGO 2.............................................................................................................. 45
ABSTRACT............................................................................................................... 45
INTRODUCTION...................................................................................................... 46
MATERIAL AND METHODS.................................................................................. 48
Animals and procedures............................................................................................. 48
Experimental design................................................................................................... 48
Activity registry.......................................................................................................... 50
Statistical analysis....................................................................................................... 51
RESULTS................................................................................................................... 52
Constant light (LL)..................................................................................................... 52
Constant dark (DD)..................................................................................................... 53
Activity registry.......................................................................................................... 54
DISCUSSION............................................................................................................. 55
REFERENCES........................................................................................................... 59
5. CONCLUSÃO GERAL.......................................................................................... 75
6. REFERÊNCIAS GERAIS...................................................................................... 76
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INTRODUÇÃO GERAL
Sistema temporizador endógeno e sincronizadores
A rotação do planeta influencia marcadamente não só a rotina, como a fisiologia
e processos moleculares complexos dos organismos, nos quais seus ritmos acompanham
os ciclos dia/noite. Foi o pesquisador Franz Halberg que em 1959 definiu o termo ritmo
circadiano (Latin: circa=cerca; dies=dia) para referir-se aos ritmos biológicos diários
que possuem período de cerca de 24 horas. Durante décadas, acreditou-se que a
ritmicidade não era uma característica interna dos seres vivos, mas dependia do ciclo
claro/escuro para existir. Porém, diversos estudos que mantinham os organismos em
condições constantes de iluminação concluíram que a ritmicidade é endógena e
geneticamente determinada (Pfeffer 1915 apud Bunning & Chandrashekaran 1975;
Kleinhoonte 1929 apud Schawassmann 1971; Bünning 1935). A capacidade endógena
de gerar ritmo permite as espécies uma antecipação a recursos necessários para
sobrevivência (Pittendrigh 1960). Com o passar do tempo, relação entre sistema rítmico
e funcionamento de relógio levou a necessidade de estudos mais aprofundados sobre o
que hoje é conhecido como relógio biológico endógeno, no qual permite o
processamento e monitoramento das atividades que variam diariamente (Pittendrigh
1993).
Para processar a informação ambiental e perceber o tempo, os indivíduos
utilizam-se de pistas ambientais externas, como luz e temperatura, que servem como
guia para esse relógio interno (Dunlap 1999). A luz é um dos sincronizadores (ou
zeitgebers) mais importantes para guiar osciladores internos (Hastings 1991). Segundo
Zhdanova e Reebs (2006), as três maiores propriedades de um sistema circadiano
dependente de luz incluem fotorrecepção, oscilação intrínseca, e a habilidade de
perceber informações de fase circadiana do ambiente local (através de osciladores
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periféricos, por exemplo). Em condições ambientais constantes, como luz constante ou
escuro constante, o indivíduo pode apresentar um padrão de livre curso nos seus ritmos,
variando em um período um pouco maior ou um pouco menor, em torno das 24 horas
diárias (Marques & Menna-barreto 2003a). O ritmo de livre curso, gerado pelos
osciladores endógenos dos seres vivos, tem sua fase e frequência ajustados pelos
agentes sincronizadores ambientais. A esse processo, dar-se o nome de arrastamento
(Pittendrigh 1981). Esses agentes sincronizadores (zeitgebers) podem, em alguns casos,
alterar o ritmo de um indivíduo transitoriamente, sem provocar mudança constante.
Nessa condição, denominada mascaramento (Aschoff 1960), as vias sensoriais captam a
informação ambiental externa e evocam comportamento rítmico independente do
relógio biológico.
O relógio endógeno (ou marcapasso) engloba um sistema de temporização
autorregulatório e oscilante, com estruturas anatomicamente definidas e osciladores
codificados no DNA (Fildman 1982; Hall &Rosbash 1987; apud Marques & Menna-
barreto, 2003b). Em mamíferos, o marcapasso central está localizado numa estrutura do
hipotálamo (parte do diencéfalo) denominada núcleo supraquiasmático (NSQ). A
informação luminosa do ambiente é captada através da retina, que se conecta com o
NSQ através do trato retino-hipotalâmico. Assim, a atividade rítmica do NSQ pode ser
arrastada pela luz ambiental (Moore, 1996). À frente, o NSQ se conecta com a coluna
intermédio-lateral da medula espinhal e a partir desta, a informação luminosa chega à
glândula pineal através de nervos simpáticos. A informação chegada à glândula pineal
altera a secreção de melatonina, bem como seus efeitos e duração no organismo, que
dependem da duração do período escuro da alternância dia/noite. A melatonina
circulante tem seu perfil plasmático variável de acordo com a duração fase escura, que
podem ser mais longa ou curta dependendo da estação do ano. Sendo assim, a
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melatonina pode funcionar como um sinalizador hormonal do ciclo ambiental claro-
escuro para o meio interno do organismo (Marques & Menna-Barreto 2003c). Portanto,
esse hormônio atua como importante sinalizador para a orientação temporal circadiana.
Para perceber a luz, os vertebrados possuem fotorreceptores distribuídos de
maneiras diferentes pelo corpo (Menaker et al. 1997). Enquanto os mamíferos possuem
fotorreceptores localizados exclusivamente na retina (Nelson & Zucker 1981),
vertebrados não mamíferos, como peixes, possuem fotorreceptores na retina, glândula
pineal e partes internas do cérebro (Foster et al. 1994). Li et al. (2012) mostraram que a
remoção de células fotorreceptoras da pineal no peixe paulistinha (Danio rerio) afetam
não apenas a sensitividade da retina, mas também os ritmos de outras atividades, como
comportamento locomotor, sugerindo que essas células tenham função de marcadores
centrais da regulação comportamental e fisiológica do animal. Em moluscos, insetos,
répteis, pássaros e mamíferos existe associação entre os osciladores internos e os
fotorreceptores, enfatizando a predominância da luz no arrastamento de ciclos
circadianos (Zordan et al. 2000), e essa relação pode indicar a co-evolução entre o
sistema de percepção de luz e relógio circadiano endógeno (Amaral et al. 2014; Shen et
al. 2011).
Apesar da luz ser considerada um dos mais importantes agentes sincronizadores,
a temperatura também funciona como sincronizador, modulando os ritmos circadianos
na maioria dos organismos, de cianobactérias a vertebrados (Rensing & Ruoff 2002).
Neste caso, a fotofase coincide com a termofase (fase de luz fase de alta temperatura)
e a escotofase com a cicrofase (fase de escuro fase de baixa temperatura). As
transições de temperatura fria para quente estão relacionadas com o amanhecer, e
transições de temperatura quente para fria com o crepúsculo (Johnson et al. 2004). Em
estudo realizado com o peixe paulistinha (Danio rerio), López-Olmeda et al. (2006)
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observaram arrastamento do ritmo circadiano tanto para fotofase (quando a temperatura
era constante), quanto para termociclos (quando a luz era constante), com maior
atividade locomotora durante o dia, independente das fases do ciclo de temperatura.
Estes autores também relataram que o peixe apresentou livre curso, com período (t) de
23,3 horas, quando submetido a ritmo de luz fraca constante e temperatura constante.
Outro forte agente sincronizador é a atividade alimentar (Boulos & Terman
1980). Alimentação restrita à hora do dia influencia o sistema de temporização
circadiano através de um sistema oscilador independente do núcleo supraquiasmático
em mamíferos (Stephan 2002). Sob essas condições, muitas espécies desenvolvem
atividade alimentar antecipatória. A ingestão de alimento provoca respostas fisiológicas
que podem atuar como estímulos de arrastamento, influenciando o ritmo tanto do
relógio circadiano central, como dos periféricos (Mistlberger 2011). Quando o ciclo
claro-escuro e o ritmo de alimentação estão em conflito, apenas alguns órgãos
permanecem sincronizados ao ciclo luminoso circadiano, provavelmente para preservar
outras funções, como a mensuração do dia e noite, a ritmicidade anual e a dependência
do arrastamento fótico do sistema nervoso central (Damiola et al. 2000; Stokkan et al.
2001).
Ritmos em peixes
Como muitos organismos, os peixes possuem ritmo circadiano de atividade,
osciladores periféricos espalhados em uma variedade de tecidos, e osciladores centrais
cuja estrutura geral ainda não está bem esclarecida (Reebs 2002; Zhdanova & Reebs
2006). Assim como nos mamíferos, o ritmo circadiano de atividade em peixes
sincronizam com oscilações diárias de pistas ambientais (Manteifel et al. 1978; Muller
1978). Também similar aos mamíferos (Moore-Ede et al. 1976), pelo menos dois
agentes sincronizadores podem estar envolvidos no controle do ritmo circadiano em
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peixes: ciclo claro-escuro e atividade alimentar, indicando um sistema com múltiplos
osciladores na relação temporal dos peixes com o seu ambiente (Boujard & Leatherland
1992). Em algumas espécies de peixes, como Lota lota (Muller 1973; Kavaliers 1980a),
Fundulus heteróclitos (Kavaliers 1980b) e Couesius plumbeus (Kavaliers 1978), o ritmo
circadiano de atividade é mantido em livre curso sob condições luminosas constantes. A
melatonina, principal hormônio relacionado ao ciclo circadiano, e os seus principais
órgãos de produção (glândula pineal e retina), também possuem importante papel no
ritmo circadiano e arrastamento em peixes (Zhdanova & Reebs 2006). No peixe
paulistinha, por exemplo, a glândula pineal se desenvolve poucas horas após a
fertilização e logo se torna responsiva à luz, secretando melatonina em resposta à fase
escura (Danilova et al. 2004; Kazimi & Cahill 1999). Diferentes formas de enzimas
envolvidas na produção de melatonina são expressas na glândula pineal e na retina
desses animais (Benyassi et al. 2000).
Como citado anteriormente, outro fator de forte influência no ritmo em peixes é
a atividade alimentar (Boujard & Leatherland, 1992). Os peixes podem ser classificados
quanto à atividade alimentar em noturnos, diurnos e crepusculares, de acordo com os
respectivos turnos de forrageio. Quando o alimento é disponibilizado em horário restrito
do dia, peixes, como outros animais, podem antecipar o local e horário do recurso
alimentar (Chen & Tabata 2002; Reebs & Lague 2000). Quando mantidos em condições
ambientais constantes (luminosidade, temperatura), peixes podem sincronizar a
atividade locomotora ao padrão temporal de disponibilidade de comida (Geet et al.
1994; Naruse & Oishi 1994).
Ritmo no peixe paulistinha
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Por serem animais diurnos, o peixe paulistinha apresenta alta atividade durante o dia
e atividade reduzida durante a noite (Hurd et al. 1998). No estado de sono, a atividade
natatória é reduzida, bem como funções cognitivas e visuais (Paciorek & Mcrobert
2012). Em condições de escuro constante, o peixe paulistinha exibe atividade
locomotora rítmica e aumenta a atividade durante o dia subjetivo (Hurd et al. 1998).
Porém, nessas condições, a atividade locomotora geral se apresenta reduzida em relação
aos animais expostos ao ciclo claro-escuro (CE), podendo ocorrer perda de ritmo (Tovin
et al. 2012). Em luz constante, a maioria dos indivíduos se torna permanentemente
ativos em relação aos do CE, o que também pode levar à perda de ritmo (Elbaz et al.
2013). O estudo de Moore & Whitmore (2014), mostrou que em condições de escuro
constante (EE), o peixe paulistinha apresenta significante oscilação circadiana. Além
disso, os resultados desse estudo revelaram áreas cerebrais e periféricas (como coração)
do peixe paulistinha contendo fotorreceptores e marcadores circadianos diretamente
responsivos à luz, dentre eles expressão de genes, como cry1a e per2.
O peixe paulistinha possui alguns genes relacionados com a regulação do ritmo
circadiano homólogos a alguns genes de mamíferos (Delaunay et al. 2000; Kobayashi et
al. 2000), como uma variedade de tipos cry, clock, bmal e per (Kobayashi et al. 2000;
Whitmore et al. 1998; Cermakian et al. 2000; Pando et al. 2001). Dentre esses genes,
cry1a, per2 e per3 são conhecidos por serem intensamente responsivos à luz (Ziv &
Gothilf 2006; Tamai et al. 2007; Moore & Whitmore 2014). O sistema de relógio
circadiano desses animais possui osciladores responsivos à luz presentes na glândula
pineal do SNC, na qual possui projeções para demais regiões do cérebro (Yáñez et al.
2009), e osciladores periféricos espalhados por diversos tecidos do corpo (Whitmore et
al. 1998). Esse sistema é visto como altamente descentralizado (Cermakian et al. 2000;
Whitmore et al. 1998), fazendo-se necessárias análises detalhadas das estruturas neurais
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deste relógio (Moore & Whitmore 2014). Até mesmo em culturas celulares há presença
de osciladores responsivos à luz (Pando et al. 2001; Whitmore et al. 2000). Há registros
de que o relógio biológico do peixe paulistinha é herdado através de genes maternos e
se encontra ativo nas primeiras etapas da embriogênese (Delaunay et al. 2000).
A glândula pineal do peixe paulistinha controla a síntese de melatonina
independente de outras estruturas relacionadas ao relógio interno (Cahill 1996). Assim
como primatas diurnos, a melatonina diminui a atividade locomotora dos peixes,
induzindo estado semelhante ao sono (Zhdanova et al. 2002). Quanto a fisiologia do
sono, o trabalho de Sigurgeirsson et al. (2013) mostrou que o peixe paulistinha pode
exibir uma homeostase e pressão de sono mais fracas em comparação com mamíferos, e
a privação de sono não é muito eficaz em alterar sua resposta ao estresse. O trabalho de
Yokogawa et al. (2007) revelou o peixe paulistinha dorme durante a noite quando
exposto tanto ao ciclo claro-escuro (CE), quanto em escuro constante (EE). Entretanto,
até uma semana na condição de luz constante (150 lux), a luz parece suprimir o sono do
peixe, fazendo desaparecer o ritmo sono-vigília, retornando progressivamente em torno
de 1 a 2 semanas.
Pistas sociais como sinalizadores
Pistas sociais são conhecidas por influenciar o sistema circadiano em diversas
espécies (Mrosovsky 1988). O termo ‘pista social’ é aplicado em situações em que
qualquer sinal periódico emitido de um animal para outro tem a capacidade de arrastar
ritmos, seja um estímulo visual, acústico, olfatório ou tátil (Rajaratnam & Redman
1999). A pista social pode ter papel importante na orientação temporal dos indivíduos
dentro do grupo (Calhoun 1975; Farr & Andrews 1978). Por exemplo, saguis comuns
(Callithrix jacchus) em ambiente de ciclo claro-escuro tem o perfil de atividades
circadianas modulado por pistas sociais resultantes da interação entre animais dentro de
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um grupo (Melo et al. 2013). Em humanos, atividades diárias de rotina, como trabalho e
recreação, também podem arrastar o ritmo circadiano (Ehlers et al. 1988, 1993; Elmore
et al. 1994; Monk et al. 1990).
Em peixes, a vida em grupo oferece diversas vantagens, como menor risco de
predação (Landeau & Terborgh, 1986; Godin et al. 1988), maior troca de informação
social (Mathis et al. 1995) e otimização de forrageio (Ryer & Olla 1992). Muitos peixes
adotam um comportamento de agregação social denominado formação de cardume (ou
schooling em inglês) (Pitcher & Parrish 1993), que demanda alta taxa de troca de
informações entre os membros do grupo. O paulistinha apresenta forte preferência pelo
grupo social (Gerlai et al. 2000; Paciorek & Mcrobert 2012; Luchiari et al. 2015),
formam cardume no ambiente natural desde o estado larval, e demonstram alta
capacidade de distinção entre coespecíficos e heteroespecíficos, o que o torna um
excelente modelo para estudar o comportamento social (Engeszer et al. 2007).
O estudo de Paciorek & Mcrobert (2012) mostrou que variações no fotoperíodo
alteram o comportamento de agregação diária do peixe paulistinha: no ciclo claro-
escuro (12:12 CE; luzes ligando as 8:00 h), os peixes se agregam menos por volta da
meia noite, aumentando este comportamento com o passar do dia, e atingindo pico
máximo de agregação às 20 horas (horário que as luzes desligam). Já em luz constante
ou escuro constante, não mostraram diferenças na agregação ao longo do dia (Paciorek
& Mcrobert 2012). Esse estudo sugere que pistas ambientais luminosas do ciclo claro-
escuro (ascender e apagar das luzes) são importantes na regulação do comportamento
social.
Aprendizagem espaço-temporal
Devido ao movimento de rotação da Terra em torno de seu próprio eixo ser de
24 horas, a grande variedade de seres vivos é capaz de prever flutuações de eventos
16
ambientais recorrentes (ex.: variação de luz, disponibilidade de alimento, etc.) dentro
desse período (Kuhlman et al. 2015). Antecipar eventos espaço-temporalmente
distribuídos e usar essa informação para organizar as atividades diárias é uma vantagem
adaptativa para os indivíduos, já que muitos fatores ambientais apresentam variação
circadiana (Mulder et al. 2013).
Aaprendizagem espaço temporal (do inglês time-place learning - TPL) é a
habilidade de associar lugares com importantes eventos biológicos em diferentes horas
do dia (Widman et al. 2000; Wilkie 1995). Em nosso estudo, utilizamos o paradigma da
aprendizagem espaço-temporal, no qual a localização de um estímulo foi apresentada
sempre em determinado horário do dia e determinado local do tanque experimental.
Neste paradigma, os animais são treinados por um período de tempo em sessões diárias
que ocorrem sempre em horários fixos, no intuito de aprender a visitar ou evitar locais
específicos apenas nos horários em que foram treinados, e, em seguida os animais são
testados para a aprendizagem do local e horário do estímulo (Mulder et al. 2013).
Existem quatro diferentes estratégias de aprendizagem espaço-temporal:
circadiana, pista contextual, ordinal e tempo intervalar (Carr & Wilkie 1997). Na
estratégia circadiana, o indivíduo aprende que a hora do evento tem periodicidade fixa,
e pode associá-la com as diferentes fases de um oscilador circadiano endógeno. Dessa
forma, ele aprende à prever a hora que esses eventos geralmente ocorrem. Na estratégia
de pista contextual, não há associação temporal, e o animal apenas aprende a visitar ou
evitar o local “a” na presença de uma pista contextual (ex. ascender das luzes). Nesse
caso, é preciso excluir qualquer pista de discriminação (diferenças entre sessões) para
que o animal não utilize essa estratégia.
Na estratégia ordinal, o animal antecipa eventos que ocorrem em certa ordem
dentro de um período de tempo, associando a sequência desses eventos (ex. primeiro
17
evento ocorreu no local “a”, segundo no local “b”, e assim por diante), ou seja, ele
estabelece uma rota diária de alternância. Essa estratégia possui duas variantes:
temporal e não-temporal. Na temporal, a sequência é reestabelecida diariamente, e na
não-temporal, a sequência é memorizada dia após dia, de forma alternada
sequencialmente. Pular a primeira sessão do dia é uma maneira para identificar o uso da
estratégia ordinal, pois ao invés de visitar o segundo local na segunda sessão, o animal
visitará o primeiro local.
Na estratégia de tempo intervalar, o indivíduo associa um intervalo de tempo
relativo à apresentação de uma ou mais pistas ambientais (ex. o animal aprende que em
certo intervalo de tempo após o ascender das luzes, deve visitar o local “a”, e em
intervalo de tempo anterior ao apagar das luzes, ele deve visitar o local “b”). Essa
estratégia não está relacionada com a atividade de genes relógio, ou seja, é independente
do sistema circadiano (Lewis et al. 2003; Papachristos et al. 2011). Para averiguar o uso
da estratégia de tempo intervalar, pistas externas devem ser eliminadas, por exemplo,
testando os animais em luz ou escuro constante. Se, nessas condições, a aprendizagem
não ocorrer, pode ser indicativo de que o indivíduo utilizou a estratégia de tempo
intervalar, ou alguma outra estratégia que não seja a circadiana. Caso a aprendizagem
persista, independente de pistas externas, é muito provável o uso de uma estratégia
circadiana.
Há evidências que a aprendizagem espaço-temporal diária dependa do sistema
circadiano e da ligação deste com a memória associativa (Mulder et al. 2013). Segundo
Biebach (1989), quando um animal é treinado para visitar diferentes locais em
diferentes horas do dia, mais do que o arrastamento de um oscilador circadiano interno
deve estar envolvido, pois a associação entre hora e local deve também ser aprendida.
Na TPL diária, a associação do tempo com o local e a natureza do evento guia a tomada
18
de decisão comportamental. O animal é estimulado a lembrar do tempo, pois o tempo é
que discrimina as escolhas corretas e incorretas. A escolha correta depende da
memorização e retenção da memória do período específico que o evento ocorreu,
baseada em encontros prévios. Na estratégia circadiana, a escolha correta sobre a
localização do estímulo implica na consulta automática a um relógio biológico interno
(Mulder et al., 2013).
Os primeiros experimentos de TPL foram feitos em abelhas, e os pesquisadores
observaram que elas conseguem encontrar alimentos específicos em determinadas horas
do dia (Wahl 1932), em vários horários (Koltermann 1974), e se o alimento está
disponível em dois diferentes lugares em dois períodos do dia, aprendendo a associar
esses dois locais aos dois horários referentes à cada um deles (Finke 1958). Outros
estudos pioneiros com TPL envolveram pássaros: o Peneireiro-vulgar (Falco
tinnunculus) apresenta atividade antecipatória ao horário de disponibilidade de alimento
(Rijnsdorp et al. 1981; Wilkie et al. 1996). Em condição de luz constante, estorninhos
(Sturnus vulgaris) mostraram aprendizagem espaço-temporal baseada em alimentação,
mesmo em livre curso (τ<24h), por quase uma semana, demonstrando a presença de um
relógio circadiano endógeno como mecanismo de temporização para aprendizagem
espaço-temporal (Wenger et al. 1991).
Apesar do conhecimento sobre TPL ser bastante limitado (Heydarnejadi &
Purser 2008), experimentos com resultados positivos já foram realizados em roedores
(Means et al. 2000; Thorpe & Wilkie 2002), aves (Wilkie et al. 1996; Boulos &
Logothetis 1990), e insetos (Breed et al. 2002). Por exemplo, foi observado que a
proporção de ratos que discriminam espaço-tempo em labirinto vertical aumenta com a
altura do, labirinto, ou seja, com o custo de resposta (Widman et al. 2000). Em outro
19
estudo, ratos Long Evans mostram antecipação de diferentes localizações de alimentos
em diferentes horas do dia (Boulos & Logothetis 1990).
Em peixes, a aprendizagem espaço-temporal foi observada em dourado
(Notemigonus crysoleucas) (Reebs 1996), Inanga (Galaxias maculatus) (Reebs 1999),
acará (Pterophyllum scalare) (Barreto et al. 2006), truta ártica (Salvelinus alpinus)
(Brännäs 2014), entre outros. Todos estes estudos utilizaram alimento como estímulo
para aprendizagem espaço-temporal, e embora alguns desses animais apresentem
comportamento social marcante, não há relatos de TPL usando estímulo social. Em
nosso estudo, utilizamos a presença de um grupo de coespecíficos como estímulo para
aprendizagem espaço-temporal no peixe paulistinha (Danio rerio). Dentro de nossa
hipótese, o estímulo social pode ser utilizado como elemento sincronizador, permitindo
tanto ritmo relacionado à oferta do sinal social, quanto aprendizagem espaço-temporal.
O peixe paulistinha é considerado um modelo animal promissor, tanto pela sua
alta praticidade de estocagem e manutenção em ambiente restrito quanto à elevada
semelhança fisiológica e comportamental com mamíferos (Ingham 2009), que permite
estudos translacionais. Além disso, diante da importância do fotoperíodo para aquisição
de informações ambientais e percepção de tempo nos organismos (Dunlap 1999), nosso
estudo se propõe a testar a aprendizagem-espaço temporal no peixe paulistinha em três
condições de limunosidade: claro-escuro (CE), claro constante (CC) e escuro constante
(EE).
20
APRENDIZAGEM ESPAÇO-TEMPORAL E EFEITOS DAS CONDIÇÕES
LUMINOSAS NO APRENDIZADO DO PEIXE PAULISTINHA
OBJETIVOS
Objetivo Geral
Verificar se há aprendizagem espaço-temporal no peixe paulistinha (Danio rerio)
em diferentes condições luminosas.
Objetivos específicos
Verificar se em ciclo claro-escuro (12:12 CE) o peixe paulistinha apresenta TPL;
Verificar se em condições de claro constante (CC) ou escuro constante (EE), ou
seja, sem sinalizadores luminosos, o peixe paulistinha apresenta aprendizagem espaço-
temporal;
Verificar se além de aprender a associar estímulos espaço-temporalmente, o peixe
paulistinha é capaz de se antecipar à chegada do estímulo;
Caracterizar o ritmo de atividade do peixe paulistinha nas condições de TPL em:
ciclo claro-escuro, claro constante e escuro constante;
21
Artigo 1: aceito para publicação pela revista Behavioural Processes
Time-place learning in the zebrafish (Danio rerio)
Clarissa de Almeida Moura, Ana Carolina Luchiari*
Departamento de Fisiologia, Centro de Biociências, Universidade Federal do Rio
Grande do Norte, Natal, RN, Brazil.
*Corresponding Author: Departamento de Fisiologia, Centro de Biociências,
Universidade Federal do Rio Grande do Norte, PO BOX 1511, 59078-970 Natal, Rio
Grande do Norte, Brazil. Phone: +55 84 32153409, Fax: +55 84 32119206, E-mail:
Abstract
Animals exhibit activity cycles that repeat over days. The most noteworthy
cyclical behaviors are related to forraging, which generally occur at the same times and
locations. The synchronization of animal activities via the association of different places
at different times for the occurrence of relevant biological events is known as time-place
learning (TPL). In the present study, we used zebrafish (Danio rerio) to test time-place
learning based on a different stimulus: social reinforcement. Fish were not only able to
associate time and place of the social stimulus, but also displayed anticipatory activity
prior to the arrival of the stimulus. Furthermore, we show that the group of conspecifics
is an relevant stimulus for time-place learning tasks, while other studies have only used
food.
Keywords: learning; social stimulus; conspecifics; time-place learning.
22
1. Introduction
Animals exhibit activity cycles that repeat over days, seasons, and years.
Among the most noticeable cyclical behaviors are forraging and resting, which
generally appear at established times and places (Carr and Wilkie, 1997; Dunlap, 1999).
The processes by which the animals synchronize its activities to occur at different
locations and different times is known as time-place learning (TPL) (Wilkie, 1995;
Widman et al., 2000). For this type of learning to occur, the time domain and spatial
awareness are the most important aspects. Moreover, the ability of TPL can be
considered adaptive, since it provides advantages for exploiting resources that are not
continuously available (Enright, 1970).
Groundbreaking TPL studies were conducted with bees, suggesting their ability
to flexibilize foraging to find food at specific times and locations (Wahl, 1932; Finke,
1958; Koltermann, 1974). After bees, TPL was also observed in other insects (Breed et
al., 2002), birds (Wilkie et al., 1996), and rodents (Means et al., 2000; Thorpe and
Wilkie, 2002). In fish, this cognitive ability has received little attention, and the
available studies focus on the time-place learning of social animals as a response to food
resources that vary over time and in space (Reebs, 1993, 1996; 1999; Gomez-Laplaza
and Morgan, 2005, Barreto et al., 2006; Brannas, 2014). Considering that food
availability changes daily, seasonally and annually, it is believed that TPL is functional
in optimizing the location and exploitation of the resource, as well as in avoiding
predators in an environment of predictable cyclical change, allowing a decrease in
energy costs and consequent increase in survival (Mulder et al., 2013).
Although studies on fish reveal the ability of food-related TPL, learning in
groups of animals may have been masked by the copy phenomenon, whereby leaders of
a group make the decisions while other individuals follow their behavioral patterns
23
(Reebs, 2000). Indeed, for social animals, grouping has advantages in terms of locating
food resources, foraging time and protection against predators (Pitcher et al., 1982;
Krause and Ruxton, 2002; Brown and Laland, 2003; McRobert, 2004; Luchiari and
Freire, 2009;). In addition, the possibility of grouping is understood as a reward (Al-
Imari and Gerlai, 2008), promoting a sensation of well-being and increasing dopamine
secretion in the brain (Saif et al., 2013). In this respect, conspecific groups may act as a
stimulus for markedly social animals, favoring time-place learning.
Therefore, the highly social zebrafish (Engeszer et al., 2007; Pritchard, et al.,
2001; Spence et al., 2008) can be considered a study model for TPL based on social
reinforcement. Social behaviour in zebrafish is innate and appers immediately after
hatching (Engeszer et al., 2007). If held isolated, zebrafish shows a fast regrouping
when placed together (Kerr, 1963), indicating the social group has a positive effect on
motivation to shoal. Thus, this study aimed at assessing whether the zebrafish (Danio
rerio), when tested individually, is capable of associating place with specific times of
the day in which a group of conspecifics is presented. First, the results of this study
contribute to increasing knowledge regarding the cognitive ability of fish, primarily
TPL, increasing the variety of fish tested for this phenomenon, as well as adding new
tasks to the behavioral investigation of this species. Furthermore, since it is a strong
stimulus for the species, the social stimulus used indicates that other events relevant for
the species can be considered during learning, in addition to those closely associated
with survival (for example, food).
2. Material and methods
2.1. Animals and procedures
Zebrafish Danio rerio (Hamilton, 1822) were obtained from a fish farm (Natal,
24
Rio Grande do Norte state) and kept in stocking tanks (2 fish/L) with aired and filtered
water. Four 50L tanks make up one stocking unit in the closed water circulation system,
with mechanical, biological and chemical filtration, in addition to UV disinfection.
Water was maintained at 28±1°C, with pH 7.2 and low levels of ammonium and nitrite.
The light cycle (fluorescent light, 150 Lux) was fixed at light-dark (LD), with the start
of the light phase at 7 am. The fish were fed commercial pellets twice a day (38%
protein, 4% lipids, Nutricom Pet) and Artemia salina.
Ten zebrafish (adults of both sexes) from the aforementioned stock were used to
test time-place learning. All the procedures with the animals were authorized by the
Animal Ethics Committee of Universidade Federal do Rio Grande do Norte (CEUA
039/2015).
2.2. Experimental design
The experimental animals were individually transferred to test tanks (100 x 25 x
25cm; length x width x height), divided horizontally into three same-size compartments
(33 cm long): one central and two lateral (Fig. 1). The compartments were separated by
opaque dividers, each with an 8 cm-diameter circular passage that allowed the fish to
swim between the compartments. The passage was located on the right of the right side
divider and on the left of the left side divider, such that the fish could not visualize more
than two compartments at the same time (Fig. 1), thereby preventing the stimulus placed
in one of the side compartments from being seen when the animal was in the opposite
side compartment. A cylindrical open-front receptacle (10 cm in diameter and 10 cm
high) was fixed to the upper part of the wall, and used to offer the stimulus (conspecific
group) at specific times. The side compartments were denominated morning
compartment and afternoon compartment. Each tank was constantly aerated through an
25
external filter (JEBO 50, 250L/h) located in the central compartment and porous rocks
in the side compartments.
Animals were kept for 5 days in experimental tanks for acclimatization and for a
further 30 days of the experimental phase. A 12 hour light-dark (LD) cycle (7 am-7 pm)
was used. A group of 5 zebrafish (same size and age) were introduced every day into
the receptacle located in the morning compartment at 8 am and removed at 9 am, and
into the receptacle of the afternoon compartment at 5 pm and removed at 6 pm, acting
as a stimulus for the experimental fish to occupy the compartment where the group was
placed. The group was introduced through a receptacle (500ml) connected to a handle
(2m) so that the experimenter could not be seen by the animals, which were separated
by an opaque curtain. The stimulus was introduced 1 hour after the onset of the morning
light and 2 hour before the end of the light phase. Food (artemia) was offered daily
(once a day) at random times, always in the central compartment so they would not be
associated with any other stimulus. The fish was also checked from behind the curtain
many times a day, thus providing cues of the experimenter presence to the fish that
could not be associated to any other cues.
On days 15 and 30 of the experimental period, the behavior of the animals was
recorded on video for 1h and 15 min, starting at 7:45 am and 4:45 pm, in order to
observe animals for 15 minutes before the arrival of the group (stimulus) and during
their entire presence. Behavior on day 15 was recorded in the presence of the group to
estimate the strength of this stimulus. However, on day 30, the animals were filmed
without the presence of the group, in order to assess time-place learning in the zebrafish.
Video records were made with Sony DCR-SX45 Digital Video Camera
Recorders placed in front of the tanks. Analysis of behavioral records was conducted
using the ZebTrack tracking program, developed at MatLab. The following parameters
26
were assessed: residence time and entry frequency in the morning and afternoon
compartments, average swimming speed, and total distance travelled. The distance of
the centre point of the fish from the previous sample frame to the following sample
frame indicated the distance travelled between frames. The ZebTrack calculates the total
distance travelled by summing the distances from the previous and current frames along
the total period of recording. The average speed takes into account the distance travelled
by the time spent to cover that specific distance, also calculated by the ZebTrack
software.
2.3. Statistical analysis
Behavioral data for time spent in the compartments and frequency of entry into
each compartment were compared in the morning and afternoon, on days 15 and 30,
using the paired Student’s T-test. We also assessed speed and total distance travelled in
the compartments with respect to the presence of the stimulus. These data were
compared for the period prior to group arrival (15 min before) and that related to the
presence of this stimulus, on days 15 and 30. The same parameters were also compared
between the morning and afternoon in both windows of time (15 min before the group
and 60 min with the group). The paired Student’s t-test was used for both comparisons,
in order to determine behavioral changes caused by the presence/absence of the
stimulus, and at different times of the day. We disregarded all data from the central
compartment, since it was a passage area and feeding site at random times. Thus, the
animal could visit this area to pass from the right compartment to the left or to search
for food.
3. Results
3.1. Responsiveness on day 15
27
During the 15 min before the group of conspecifics arrived, there was no
significant difference in residence time between the compartments in the morning
(Student’s t-test, df=9 t=1.29 p=0.23) or afternoon (Student’s t-test, df=9 t=0.72 p=0.49)
(Fig. 2a). The frequency of entry into the morning compartment was higher in the
morning (Student’s t-test, df=9 t=2.46 p<0.001; Fig. 2c), but did not differ in the
afternoon (Student’s t-test, df=9 t=0.15 p=0.88).
During the presence of the group, residence time in the morning compartment
was higher in the morning (Student’s t-test, df=9 t=5.19 p<0.001), and in the afternoon
compartment in the afternoon (Student’s t-test, df=9t=-3.18 p=0.01) (Fig. 2b). We
obtained a similar result for frequency of entry into the compartments in the morning
(Student’s t-test,df=9 t=5.22 p<0.001), and afternoon (Student’s t-test, df=9 t=-2.93
p=0.02) (Fig. 2d).
The average speed recorded was lower in the 60 min in which the group was
present, when compared to the 15 min before their arrival, in both the morning
(Student’s t-test, df=9t=8.18 p<0.001) and the afternoon (Student’s t-test, df=9 t=2.33
p=0.04; Fig. 3a). The total distance travelled by the individuals was greater during
exposure to the group, only in the afternoon (Student’s t-test, morning df=9 t=-0.72
p=0.49; afternoon df=9 t=-2.36 p=0.04; Fig. 3b). When the parameters were compared
between the morning and afternoon for each time period (15 min before the group and
60 min with the group), there was no significant difference in average speed (Student’s
t-test, df=9 t=0.17 p=0.87; Fig. 3a) or total distance travelled (Student’s t-test, df=9
t=0.59 p=0.57; Fig. 3b) in the 15 min before the group arrived, or during the 60 min of
exposure to them (Student’s t-test, average speed df=9t=-1.65 p=0.14; total distance
travelled df=9 t=0.48 p=0.64; Fig. 3).
3.2. Determination of learning on day 30
28
In the 15 min before the group was introduced into the tank, the fish remained
mostly in the morning compartment in the morning (Student’s t-test, df=9 t=6.68
p<0.001; Fig. 4a), but in the afternoon there was no difference between the time spent in
each compartment (Student’s t-test, df=9 t=-1.95 p=0.08). The frequency of entry was
also higher in the morning compartment in the morning (Student’s t-test, df=9 t=2.55
p=0.03) and the afternoon compartment in the afternoon (Student’s t-test, df=9 t=-2.18
p=0.05) (Fig. 4c).
During the 60 min that the group was expected to arrive, even in the absence of a
stimulus, the fish remained for a longer time in the morning compartment in the
morning (Student’s t-test, df=9 t=3.41 p=0.01) and the afternoon compartment in the
afternoon (Student’s t-test, df=9 t=-3.28 p=0.01) (Fig. 4b). The frequency of entry into
the compartments was higher in the morning (Student’s t-test, df=9 t=5.83 p<0.001) and
afternoon (Student’s t-test, df=9 t=-3.73 p=0.01) (Fig. 4d).
There was no significant difference in average speed between the 15 min preceding the
group’s arrival and the 60 min that the group was expected to arrive, in both the
morning and afternoon (Student’s t-test, morning df=9 t=-0.31 p=0.77, afternoon df=9
t=-0.59 p=0.57; Fig. 5a). The total distance travelled also did not differ between the 15
min prior to the group’s arrival and the time it was usually in the tank, in the morning
(Student’s t-test, df=9 t=-0.97 p=0.36), or the afternoon (Student’s t-test, df=9 t=0.67
p=0.52) (Fig. 5b). The parameters also did not differ when the morning and afternoon
were compared for each time period (15 min before the arrival of the group and 60 min
without it), both during the 15 previous minutes (Student’s t-test, average speed df=9
t=-1.31 p=0.22; total distance travelled df=9 t=0.09 p=0.93), and in the 60 min without
the presence of the group (Student’s t-test, average speed t=-1.64 p=0.13; total distance
travelled df=9 t=0.53 p=0.61) (Fig. 5).
29
4. Discussion
Our results show that zebrafish not only stayed longer where conspecifics were
present at predetermined times, but also exhibited anticipatory ability, remaining longer
and visiting more frequently the compartment of the tank where the group of
conspecifics was present, for 15 min before the stimulus itself was offered. These
results are relevant, since they indicate that zebrafish are not only able to associate
environmental cues (time and space) with important events (social stimulus), but also
suggests the ability to anticipate relevant signs.
Few studies have discussed the ability of time-place association in fish, and to
the best of our knowledge, there are only 10 studies in the literature on the issue (Reebs,
1993, 1996, 1999; Gomez-Laplaza and Morgan, 2005; Delicio et al., 2006; Barreto et
al., 2006; Delício and Barreto, 2008; Heydarnejad and Purser, 2008; Ebrahimi et al.,
2013; Brannas, 2014), most of which use groups of fish and all of them use food as the
learning stimulus.
In our study, we used a group of 5 zebrafish as a time-place learning stimulus. In
the 15 minutes before the group arrived, the zebrafish entered the correct compartment
more frequently only in the morning. Although the time spent in the compartments did
not differ between the morning and afternoon (Fig. 2a), the greater frequency observed
in the morning (Fig. 2c) indicates anticipation of the stimulus. Anticipatory activity has
previously been demonstrated in groups of Arctic char (Salvelinus alpinus) when faced
with a food stimulus (Brannas et al., 2005). Anticipating a temporal event may be
advantageous, providing benefits in terms of food, partners, or refuge acquisition (Davis
and Bardach, 1965; Reebs and Gallant, 1997). However, this did not occur in the
afternoon, possibly because a 15-day period may not have been sufficient for
individuals to memorize the exact time of stimulus presentation. Moreover, light may
30
have been used to predict the group’s arrival, favoring the perception of the time in the
morning that this took place, since the stimulus occurred 1 hour after the onset of the
light phase (7 am). In the afternoon, the stimulus occurred 1 hour before lights off (7
pm), such that the animals must have had more difficulty in associating the group’s
arrival with the subsequent light cue.
On day 15 of behavioral records, the experimental animals remained
significantly longer near the group of conspecifics during the exposure time in the
morning and afternoon (Fig. 2a and b). This reinforced the importance of conspecifics
for zebrafish, confirming the social behavior reported in other studies (Wright et al.,
2003; McRobert, 2004; Ruhl and McRobert, 2005; Engeszer et al., 2007; Paciorek and
MCRobert, 2012). With respect to the average speed, it was higher in the 15 min before
the group’s arrival, both during the morning and the afternoon (Fig. 3a), indicating fish
decreased swimming speed and stayed longer with the group during both presentation
time periods.
Studies on time-place learning usually used long testing times (Reebs, 1996;
Widman et al., 2000; Delicio et al., 2006; Ebrahimi et al., 2013), since they report on
learning that involves not only entrainment of the internal oscillator, but also the daily
routine of time and place association (Biebach, 1989). Thus, after 30 days of the
experiment we observed that in the 15 min before the presence of the stimulus, the
animals entered more frequently (Fig. 4c) and spent more time in the correct
compartments (Fig. 4a), in both the morning and afternoon. These results corroborate
the hypothesis of a circadian strategy in the time-place learning process. According to
Carr and Wilkie (1999), animals that use circadian strategy learn that the time of the
event has fixed periodicity, which is associated with different stages of endogenous
circadian phases. Thus, animals can use this information to predict the exact time that an
31
event must occur. Indeed, studies with rats show anticipation of time-place learning in a
T-maze with food reward (Deibel and Thorpe, 2013).
How some animals learn to associate time-place stimuli and others do not,
remains largely unknown. Many animals live in environments in which food resources,
sexual partners and predators vary periodically and predictably over time (Rijnsdorp et
al., 1981; Silver and Bittman, 1984; Becker et al., 1993; Wilkie et al., 1996). When
individuals predict events and learn to associate them with the place and time of
occurrence, they may obtain advantages, which are likely linked to the evolution of
cognitive and circadian systems (Enright, 1970; Daan, 1981; Aschoff, 1989; Reebs,
1996; Carr et al., 1999). Evidence that time-place learning is associated with
endogenous circadian rhythm was proposed by Van der Zee et al. (2008). These authors
showed that Cry1 and Cry2 knockout mice were unable to learn time-place association,
while wild mice learned in both circadian cycle and under constant light. The biological
clock includes an auto-regulated and oscillating temporizing system with defined
anatomical structures and DNA coded oscillators (Marques and Menna-Barreto, 2003).
Even though only few studies approach the system structure in zebrafish, it is known
that this species presents a biological clock (Cahill et al., 1998).
One could argue that instead of time-place learning based on the circadian
timing, fish may have shown win-stay, lose-shift strategy. This possibility, however,
may be discarded in the present study because win-stay, lose-shift strategy use to occur
within a shorter than 8h period of time and the absence of the shoal in the morning
compartment in the morning period would lead to an increase in the time spent in the
afternoon compartment, or, at least a higher frequency of entries in the afternoon
compartment, indicating the fish were checking the afternoon compartment. However,
32
we observed fish stayed longer in the morning compartment during the morninga and in
the afternoon compartment during the afternoon (Fig. 4b and d).
Therefore, our results regarding the time zebrafish spent on day 30 in each
compartment as a function of the stimulus (Fig. 4b), in which learning was tested
without the presence of a social stimulus, shows the time-place learning ability of this
animal. Thus, we corroborated earlier studies with fish (Reebs, 1993, 1996, 1999;
Gomez-Laplaza and Morgan, 2005; Barreto et al., 2006; Delicio et al., 2006; Delício
and Barreto, 2008; Heydarnejad and Purser, 2008; Ebrahimi et al., 2013; Brannas, 2014)
and found that time-place learning ability may be involved with relevant stimuli for the
species under study, in this case, the social signal. We did not found significant
differences in both average speed and total distance travelled before and during the 60
minutes of test on the 30º day, possibly due the absence of the stimulus fish did not
change swimming behaviour pattern on this probe day.
Finally, it is important to emphasize that in the present study, the sight of a
group of conspecifics was effective as a stimulus for the learning task proposed. A
social stimulus can be effectively used due to the highly social characteristic of
zebrafish (Wright et al., 2003; Engeszer et al., 2004; Luchiari et al., 2015). Other studies
with social signals as a learning stimulus indicate the validity of this signal, such as the
study by Karnik and Gerlai (2012) using a conspecific image for conditioning of place,
Silveira et al. (2015) using conspecifics in associative conditioning tasks, and Luchiari
et al. (2015) using a group of conspecifics in a latent learning task.
Due to the high sociability of zebrafish, future studies on time-place learning in
groups submitted to a stimulus, whether social or food, may contribute to a better
understanding of the animal’s behavior and learning copy strategies. Furthermore, since
our study does not discuss the day-to-day development of the time-place association,
33
additional studies are needed to follow the behavior patterns throughout the entire
experimental period. Another point for future investigation that could not be covered in
the present study is the offer of food in a different part of the tank, which forced us to
eliminate the central compartment from TPL analyses. Despite not using this
compartment or following the animal for the 30 days of the test, our results are robust in
that they show the time-place association ability of zebrafish, as demonstrated by data
on day 30 of the test (Fig. 4).
In our study, Danio rerio exhibited time-place learning, as well as anticipated
the arrival of the stimulus. Moreover, we showed that the group of conspecifics is an
revevant stimulus for time-place learning tasks, in which there are no records of studies
using this type of stimulus. Here, we used a circadian photoperiod (LD), but time-place
learning studies in the absence of light zeitgebers are needed to investigate TPL without
light signals to indicate time. Studies with molecular analyses must also be conducted to
indicate the association between the biological clock and this type of task, essential for
understanding the endogenous mechanisms involved in TPL.
Acknowledgements
We thank Ms Silveira, V.A.M. and Santos, T.S. for help in collecting data for this
article.
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40
Figure 1
Fig. 1. Schematic diagram of the tank used to test time-place learning. On the right, the
morning compartment, on the left, the afternoon compartment (sides were randomized
between subjects). The central compartment allows passage through the two 8 cm-
diameter round windows, one located on the right, the other on the left, preventing the
animal from seeing all the compartments at the same time. The values refer to the areas
of the tank in cm.
41
Figure 2
Fig. 2. Residence time (a and b) and frequency of entry (c and d) in the morning and
afternoon compartments on day 15 of the experiment with zebrafish (n=10) in a TPL
test. Observations were made between 7:45 and 9:00 am, and 4:45 and 6:00 pm. The
first 15 minutes of observation indicate their ability of anticipating the arrival of the
social stimulus (a and c). During the following 60 minutes, the social stimulus (group
with 5 conspecifics) was maintained in the experimental tank (b and d). * indicates
statistical significance (Student’s t-test, p<0.05) between the compartments
corresponding to each experimental period.
0
700
1400
2100
2800
3500
Morning Afternoon
Res
iden
ce t
ime
(s)
Morning
compartiment
Afternoon
compartiment
a) Anticipation
Morning Afternoon
b) Presence of the group
* *
0
20
40
60
80
100
Morning Afternoon
Fre
qu
ency
of
entr
y
Time of the day
c) Anticipation
*
Morning Afternoon
Time of the day
d) Presence of the group
*
*
42
Figure 3
Fig. 3. Mean velocity (a) and total distance travelled (b) by the zebrafish (n=10) on day
15 of the experimental test for TPL. The animals were observed for 15 minutes before
presentation of the social stimulus (from 7:45 to 8:00 am, and from 4:45 and 6:00 pm)
and for the 60 minutes that the stimulus was present (from 8:00 to 9:00 am, and from
5:00 to 6:00 pm). * indicates statistical significance (Student’s t-test, p<0.05) between
the period before presentation of the stimulus and during the presence of the social
stimulus.
0
5
10
15
20
Anticipation Presence of the stimulus
Aver
age
spee
d (
cm/s
)
a)
0
800
1600
2400
3200
4000
Anticipation Presence of the stimulus
To
tal
dis
tan
ce t
ravel
led
(cm
)
Morning Afternoonb)
*
43
Figure 4
Fig. 4: Residence time (a and b) and frequency of entry (c and d) in the morning and
afternoon compartments on day 30 of the experiment with zebrafish (n=10) in the TPL
test. Observations were made from 7:45 to 9:00 am, and from 4:45 to 6:00 pm. The first
15 minutes of observation indicate the ability to anticipate the arrival of the social
stimulus (a and c). During the next 60 minutes, the social stimulus was not introduced
into the experimental tank (b and d). * indicates statistical significance (Student’s t-test,
p<0.05) between the compartments corresponds to each experimental period.
0
500
1000
1500
2000
2500
3000
Morning Afternoon
Res
iden
ce t
ime
(s)
Morning
compartiment
Afternoon
compartiment
a) Anticipation
*
Morning Afternoon
b) Test
0
20
40
60
80
100
120
140
Morning Afternoon
freq
uen
cy o
f en
try
Time of the day
c) Anticipation
Morning Afternoon
Time of the day
d) Test
44
Figure 5
Fig. 5. Mean velocity (a) and total distance travelled (b) by the zebrafish (n=10) on day
30 of the TPL experimental test. The animals were observed for 15 minutes before
presentation of the social stimulus (from 7:45 to 8:00 am, and from 4:45 to 6:00 pm)
and during the period in which the stimulus was present, but without the presence of
social stimulus (from 8:00 to 9:00 am, and from 5:00 to 6:00 pm). * indicates statistical
significance (Student’s t-test, p<0.05) between the periods prior to presentation of the
stimulation and the presence of the stimulus.
0
5
10
15
20
Anticipation Test
Aver
age
spee
d (
cm/s
)
a)
0
1000
2000
3000
4000
Anticipation Test
To
tal
dis
tan
ce t
ravel
led
(cm
)
Morning
Afternoon
d)
45
Artigo 2: a ser submetido para publicação na revista Behavioural Processes
Time-place learning and activity profile under constant light and constant dark in
zebrafish (Danio rerio)
Clarissa de Almeida Moura, Jéssica Polyana da Silva Lima, Vanessa Augusta
Magalhães Silveira, Mário André Leocadio Miguel, Ana Carolina Luchiari*
Departamento de Fisiologia, Centro de Biociências, Universidade Federal do Rio
Grande do Norte, Natal, RN, Brazil.
*Corresponding Author: Departamento de Fisiologia, Centro de Biociências,
Universidade Federal do Rio Grande do Norte, PO BOX 1511, 59078-970 Natal, Rio
Grande do Norte, Brazil. Phone: +55 84 32153409, Fax: +55 84 32119206, E-mail:
Abstract
The ability to learn about the signs of variability in space and time is known as time-
place learning (TPL). To adjust the circadian rhythm, animals use stimuli that varies
regularly, such as light, temperature, food or even social stimuli. Because light is the
most important environmental cue, we asked how a diurnal animal would perform TPL
if this cue was removed. Zebrafish has been extensively studied in the chronobiology
area due to it diurnal chronotype, thus, we studied the effects of constant light and
constant dark on the time-place learning and acitivity profile in zebrafish. Our data
show that under constant light and dark condition, zebrafish was not able of TPL.
Moreover, even after 30 days under the constant conditions, it does not show free run,
but constant light leaded to higher activity and less rhythmicity.
Keywords: time-place learning, zebrafish, constant light, constant dark, circadian
rhythm.
46
1. Introduction
The availability of food, sexual partners, predators and other biologically
relevant stimuli varies both in time and in space (Carr and Wilkie, 1997). To process
temporal and spatial information, the animals use external cues such as light and
temperature to adjust the internal clock to the rhythm of daily variation (Dunlap, 1999).
Thus, a stimulus which varies regularly can be exploited effectively, providing adaptive
advantages (Reebs, 2002). The ability to learn about the variability of signs in the space
and time is known as space-time learning (TPL). According Gallistel (1990), the
occurrence of a biologically significant event promotes the formation of a memory code
that includes the type of event, the time and place of the occurrence. This ability is
related to the conection between the internal circadian system and associative memory
(Mulder et al., 2013).
Due to the 24h duration of the sidereal day, the vast majority of living beings are
subject to predict light and thermal fluctuations within this period (Kuhlman et al.,
2015) and use this information to estimate time. To adjust the circadian rhythm, the
animals use environmental cues (zeitgebers), which can be light, temperature (Aschoff,
1954) and even social stimuli (Erkert et al., 1986). However, light is the most
remarkable zeitgeber, because the majority of the animals present photoreceptive cells,
and thus can perceive light fluctuations throughout the day (Bell-Pedersen et al., 2005).
However, under constant light conditions (24h light or 24h dark), the organisms still
maintain rhythmicity, guided by endogenous regulators of the biological cycle (Johnson
et al., 2004; Weger et al, 2013). Therefore, even in free running, a set of self-regulated
molecular mechanisms generates the circadian rhythm throught gene expression
(Amaral et al., 2014), and allows organisms to predict and anticipate events that occur
within a period of 24 hours (Koukkari and Sothern, 2006).
47
Among the studies on learning related to the circadian rhythm, bees were the
pioneers to show TPL (Wahl, 1932; Finke, 1958), suggesting they possess a circadian
oscillator that allows for monitoring time (Pittendrigh et al, 1958 ). After these studies,
several others have found signs of both temporal and spatial learning linked to the
endogenous clocks. Starlings (Sturnus vulgaris) use internal oscillators to get
orientation regarding the position of an artificial sun (Kramer, 1950), Long-Evans rats
show the ability to locate food in different sites depending on the time of day (Boulos
and Logothetis, 1990) and various fish species (Inanga Galaxias maculatus, angelfish
Pterophyllum scalare, rainbow trout Oncorhynchus mykiss) present TPL based on food
(Reebs, 1999; Gomez-Laplaza and Morgan, 2005; Heydarnejad, 2008).
In addition to abiotic zeitgebers that favor ritimicity and learning, recurrent
visual, olfactory, auditory or tactile signals from one individual to another can entrain
an animal rhythm, and thus be considered a social synchronizer (Rajaratnam and
Redman, 1999). Several social species have their rhythms influenced by social cues,
such as rodents (Crowley and Bovet, 1980; Mrosovsky, 1988) and primates (Erkert and
Schardt, 1991). A study on common marmosets (Callithrix jacchus) living under light-
dark cycle showed that the circadian activity profile of these animals is modulated by
social interaction (Melo et al., 2013). Although the social stimuli can act as a zeitgeber,
there is no evidence of its role in the temporal and spatial learning. Knowing that
zebrafish (Danio rerio) is a highly social species that prefers swimming in groups
(Pritchard et al., 2001; Larson et al, 2006; Gerlai, 2014) and that presents TPL response
based on social reward (Moura and Luchiari, 2015), the aim of this study was to test the
time-place learning ability of zebrafish in the absence of light signals (constant light and
constant dark).
48
Zebrafish is considered a promising animal model, both for its high practicality
of storage and maintenance as the high physiological and behavioral similarity to
mammals (Ingham, 2009), allowing translational studies. In addition to these
advantages, the zebrafish has been extensively studied in the chronobiology (Vatine et
al., 2011) because of it diurnal activity pattern (Paciorek and McRobert, 2012), which
favors its translational research, opposite to rodents that are nocturnal. In this sense, we
use the zebrafish to study the effects of constant light or constant dark for the time-place
learning, offering social stimulus as reward.We hypothetized that if social stimulus
could be used as a synchronizer element, allowing rhythm, fish will increase and
decrease activity according to the stimulus presentation and TPL will also occur.
2. Material and methods
2.1. Animals and procedures
Zebrafish Danio rerio (Hamilton, 1822) were obtained from a local fish farm
(Natal, Rio Grande do Norte state) and kept in stocking tanks (2 fish/L) with aired and
filtered water. Four 50L tanks make up one stocking unit in the closed water circulation
system, with mechanical, biological and chemical filtration, in addition to UV
disinfection. Water was maintained at 28±1°C, with pH 7.2 and low levels of
ammonium and nitrite. The light cycle (fluorescent light, 150 Lux) was fixed at light-
dark (12:12 LD), with the start of the light phase at 7 am. The fish were fed commercial
pellets twice a day (38% protein, 4% lipids, Nutricom Pet) and Artemia salina.
Eighteen adult zebrafish of both sexes from the afore mentioned stock were used
to test time-place learning. All the procedures with the animals were authorized by the
Animal Ethics Committee of Universidade Federal do Rio Grande do Norte (CEUA
039/2015).
2.2. Experimental design
49
The experimental animals were individually transferred to testing tanks (100 x
25 x 25cm; length x width x height), divided horizontally into three same-size
compartments (33 cm long): one central and two lateral (same procedure of Moura and
Luchiari 2016). The compartments were separated by opaque dividers, each with an 8
cm-diameter circular passage that allowed the fish to swim between the compartments.
The passage was located on the right of the right side divider and on the left of the left
side divider, such that the fish could not visualize more than two compartments at the
same time, thereby preventing the stimulus placed in one of the side compartments from
being seen when the animal was in the opposite side compartment. A cylindrical open-
front receptacle (10 cm in diameter and 10 cm high) was fixed to the upper part of the
lateral walls, and used to offer the stimulus (conspecific group) at specific times. The
side compartments were denominated morning compartment and afternoon
compartment. Each tank was constantly aerated through an external filter (JEBO 50,
250L/h) located in the central compartment and air stones in each side compartment.
Animals were kept for 30 days under the above experimental conditions. A
group of 5 zebrafish (same size and age) were introduced every day into the receptacle
located in the morning compartment at 8 am and removed at 9 am, and into the
receptacle of the afternoon compartment at 5 pm and removed at 6 pm, acting as a
stimulus for the experimental fish to occupy the compartment where the group was
placed. The group was introduced through a receptacle (500ml) connected to a handle
(2m) so that the experimenter could not be seen by the animals, which were separated
by an opaque curtain. Food (artemia) was offered daily (once a day) at random times,
always in the central compartment so food would not be associated with any stimulus or
time.
50
To verify if the time-place learning occurs in constant light conditions, two
groups were tested: constant light group (LL; n=8) and constant dark group (DD; n=10).
In constant light group, the animals were exposed to constant light (150 lux) during the
30-day experiment and behavioral was record. The constant dark group followed the
same protocol, but with animals exposed to the total absence of light during the 30 days.
On days 15 and 30 of the experimental period, the behavior of the animals was
recorded on video for 1h and 15 min, starting at 7:45 am and 4:45 pm, in order to
observe animals for 15 minutes before the arrival of the group (stimulus) and during
their entire presence. Behavior on day 15 was recorded in the presence of the group to
estimate the strength of this stimulus. However, on day 30, the animals were recorded
without the presence of the group, in order to assess time-place learning in the
experimental zebrafish.
For the video records, we used a handcam (Sony DCR-SX45 Digital Video
Camera Recorders) placed 1.5m away and in front of the tanks. The behavioral analyses
were conducted using the ZebTrack software, developed in MatLab. The following
parameters were assessed: residence time and frequency of entry in the morning and
afternoon compartments.
2.3 Activity registry
From the 8 fish under the LL condition and 10 fish under the DD condition, 4
fish of each group were also recorded during the 6 last days of the TPL experiment.
Another 4 zebrafish from the stock condition were used to compose the LD condition,
in order to have a control group. These 12 zebrafish were used to evaluate the effects of
constant light conditions on the activity pattern. Fish held under light-dark (12:12 LD;
n=4) were also submitted to the TPL test, in order to have the same conditions of the
other groups. The activity of each fish was recorded using Sony Kit infrared security
51
cameras CCD, coupled to the DVR unit, for 144h (the last 6 consecutive days of the 30
TPL days). The behavior records were analyzed using the ZebTrack software. We
considered the average speed (cm/s) of each fish every 15 min of the 144h. The data
were plotted on diagrams of actogram, cosinor and waveform.
Actogram is a graphical representation of activity (average speed, y axes) along
24 h length of each plot line (x axes), and successive cycles are ploted below each other.
The cosinor (Halberg et al. 1967) is a model to analyse the biological rhythm consisting
of cosine curves with known periods (in our study, 24 h) to estimate the pattern of the
smooth rhythm. Each point of a cosinusoidal curve of a cosinor is a function of the
average value of the variable of interest. These variables are: MESOR (M, Midline
Estimating Statistic of Rhythm: the rhythm-adjusted mean that differs from the
arithmetic mean when the data are not equidistant and/or do not cover an integer
number of cycles), the amplitude of the oscillation (A), and the acrophase (φ, time at
which the peak of a rhythm occurs) (Refinetti et al., 2007). The waveform is the
prototypical cycle of a rhythm, defined by the amplitude (acrophase pairs of all
harmonic terms included in the model to account for the non-sinusoidality of the signal).
Then, the waveform can be considered an extended cosinor analysis in inferential
statistical chronobiology (Refinetti et al., 2007).
2.4. Statistical analysis
Behavioral data for residence time in the compartments and frequency of entry
in each compartment were compared in the morning and afternoon, on days 15 and 30,
using the student’s T-test. We excluded the data from the central compartment, because
it was a passage area and feeding site at random times, thus, the animal could visit this
area to pass from the morning compartment to the afternoon or to search for food.The
average speed data for activity registry for the lasts 6 days of TPL experiment were
52
compared between the groups LD, LL and DD using the one-way ANOVA. To verify
the acrophase of each experimental group the Rayleigh test was used (circular statistical
analisys).
3. Results
3.1. Constant light (LL)
On the day 15, during the 15 min before the group of conspecifics arrived, there
was no significant difference in residence time between the morning and afternoon
compartments in the morning (Student’s t-test, t=1.20 p=0.27) or afternoon (Student’s t-
test, t=-0.28 p=0.79), respectively (Fig. 1a). The frequency of entry into the
compartments did not differ in the morning (Student’s t-test, t=1.21 p=0.26; fig. 1c),
neither in the afternoon (Student’s t-test, t=-0.61 p=0.56; fig. 1c).
During presentation of the group, residence time in the morning compartment
was higher in the morning (Student’s t-test, t=8.82 p<0.001), and in the afternoon it was
higher in the afternoon compartment (Student’s t-test t=-4.99 p=0.002) (Fig. 1b). It was
found higher frequency of entry in the morning compartment during the morning
(Student’s t-test, t=4.32 p=0.003), but it did not differ in the afternoon (Student’s t=-
0.36 p=0.73) (Fig. 1d).
On the day 30, in the 15 min before the group was introduced into the tank, there
was no difference between the time spent in each compartment in both the morning
(Student’s t-test, t=-1.22 p=0.26) and the afternoon (Student’s t-test, t=-0.01 p=0.99; fig.
2a). The frequency of entries was higher in the morning compartment in the morning
(Student’s t-test, t=3.75 p=0.007), but it did not differ in the afternoon (Student’s t-test,
t=-1.22 p=0.26; fig. 2c).
53
During the 60 min that the group should be presented (absence of the stimulus),
the fish remained for a longer time in the afternoon compartment both in the morning
(Student’s t-test, t=-3.41 p=0.01) and afternoon (Student’s t-test, t=-2.37 p=0.05; fig.
2b) times. The frequency of entry did not differ in the morning (Student’s t-test, t=0.97
p=0.36) or the afternoon (Student’s t-test, t=-1.87 p=0.10; fig. 2d).
3.2. Constant dark (DD)
On the day 15, during the 15 min before the group of conspecifics arrived, there
was no significant difference in residence time between the morning and afternoon
compartments in the morning (Student’s t-test, t=0.40 p=0.70) or afternoon (Student’s t-
test, t=-0.05 p=0.96; fig. 3a. The frequency of entry into the right compartments did not
differ in the morning (Student’s t-test, t=-1.51 p=0.15), neither in the afternoon
(Student’s t-test, t=0.52p=0.61; fig. 3c).
During presentation of the group, residence time in the compartments did not
differ in the morning (Student’s t-test, t=-1.84 p=0.08), but it was higher in the
afternoon compartment in the afternoon (Student’s t-test t=2.85 p=0.01; fig. 3b). With
respect to the frequency of entry into the compartments, it was higher in the morning
compartment in the morning (Student’s t-test, t=2,54 p=0,02), but in the afternoon it did
not differ (Student’s t-test, t=-0,80 p=0,43; fig. 3d).
On the day 30, in the 15 min before the group presence into the tank, there was
no difference between the time spent in each compartment both in the morning
(Student’s t-test, t=-1.03 p=0.31) and in the afternoon (Student’s t-test, t=-1.40 p=0.18;
Fig. 4a). The frequency did not differ in the compartments in the morning (Student’s t-
test, t=-0.21 p=0.83), or afternoon (Student’s t-test, t=0.02 p=0.98) (Fig. 4c).
During the 60 min that the group was expected (absence of the stimulus), the fish
remained for a longer time in the morning compartment both in the morning (Student’s
54
t-test, t=-2.33 p=0.03) and afternoon (Student’s t-test, t=-2.07 p=0.05; fig. 4b) times.
However, there were no differences in the frequency of entries in the compartiments in
the morning (Student’s t-test, t=-0.31 p=0.76) or the afternoon (Student’s t-test, t=0.10
p=0.93; fig. 4d).
3.3. Activity registry
During the last 6 days of the experimental period for TPL, the animals under
constant light (LL), constant dark (DD) and light-dark cycle (LD) showed circadian
rhythm of 1440 min and their activity profile is represented by thea ctogram in fig. 5.
The activity (average speed) mean values statiscallydiffered between the three tested
conditions (One way ANOVA F=4.35 p=0.04; fig. 6a, b and c; table 1): animals under
LL showed higher activity than the animals under DD, but none of them differed from
LD. The animals under LD (Rayleigh r=0.997 p=0.007) and DD (Rayleigh r=0.938
p=0.017) groups showed significant acrophase, the same did not happen for LL group
(Rayleigh r=0.477 p=0.427; Fig. 6d, e and f), however the acrophase did not differ
between the groups (One way ANOVA F=0.979 p=0.412) (Table 2).The center of
gravity was also significant different between the groups (One way ANOVA F=4.83
p=0.04; table 1). There was no significant difference between the groups regarding the
total area under the curve (One way ANOVA F=3.92 p=0.06).
Regarding the subjective light phase (7am to7pm), both the mean interval (I-m
(w)) (One way ANOVA F=3.96 p=0.06) and the area under the curve (I-a (w)) (One
way ANOVA F=3.94 p=0.06) did not differ between the groups (Table 1). The
percentage of activity, as measured by percentage of the total area (I-a (w)%), showed
significant difference between the conditions (One way ANOVA F=10,60 p=0.004;
table 1).
55
On the last day of the experiment (probe day), in which the stimulus was not
presented to the experimental animals, the activity of the groups was significantly
different (One way ANOVA F=29.98 p<0.001). The animals under LL and LD showed
higher activity than the animals under DD (Fig. 7).
4. Discussion
According to our results, after 30 days under constant light or dark conditions,
zebrafish lost the ability to show TPL based on social stimulus (Figs. 2 and 4), however
it maintains 24h rhythm in both conditions (Table 1). Although zebrafish trained for 30
days to find a conspecific shoal at different time and place have remained longer in
only one of the places during the afternoon and the afternoon, fish from the dark
condition searched for the group on the opposite side of the fish from the light
condition. Furthermore, fish in constant dark decreased overall activity, while the fish in
constant light did not change activity level but it was homogeneously distributed
throughout the 24h-day period.
On the 15th
day of behavior registry, neither groups (LL or DD) showed
differences in time spent or frequency of entry in the compartments 15 min before the
stimulus presentation (Figs. 1a, c and 3a, c). These results suggest that fish could not
anticipate the social stimulus arrival. Under 12:12 LD cycle, zebrafish shows a weak
behavior of anticipation on the 15th
training day (Moura and Luchiari, 2016), and thus
one would expect that after only 15 days of constant light conditions fish would present
much difficulty to forecast the stimulus event. During the 60 min of the conspecific
shoal presence, zebrafish under LL remained significantly longer near the group both in
the morning and afternoon (Fig. 1b), but animals under DD only shoal with the
conspecific group in the afternoon (Fig. 3b). Due to the social nature of zebrafish
56
(Pritchard et al., 2001; Larson et al, 2006; Gerlai, 2014; Luchiari et al., 2015), the
presence of a shoal is a strong stimulus to drive a single fish towards it. Thus, we
expected the fish to join the group upon its presence, what did not happen in the DD
condition probably because the animals had no visual cues, but chemical and
mechanical cues to locate the group into the tank. Zebrafish is highly responsive to light
(Tamai et al., 2007; Moore and Whitmore, 2014) and its visual system is a very accurate
sense, presumably the most efficient in terms of stimuli detection (Fleisch and
Neuhauss, 2006). While chemical cues quickly disperse into the water and mechanical
cues may not have passed through the compartments, we believe the experimental
zebrafish struggle finding the stimulus in the dark.
Many other studies have already demonstrated TPL in fish (Reebs, 1993, 1996,
1999; Gomez-Laplaza and Morgan, 2005; Delicio et al., 2006; Barreto et al., 2006;
Delício and Barreto, 2008; Heidarnejad and Puser, 2008; Ebrahimi et al., 2013;
Brannas, 2014), all of them using food as the reward. The TPL protocol used here in
was a learning test based on social reward. In a previous study, we (Moura and Luchiari,
2016) have shown that live conspecifics were effective to induce robust TPL behavior
in zebrafish. However, recurrent lack of luminosity signals to indicate day and night
may have TPL implications: zebrafish under LL and DD did not seek for the correct
place in the morning and in the afternoon in order to get the social reward.
On the probe day (day 30; Figs. 2 and 4), both time spent and frequency of
entry in the correct compartments in the 15 min before the stimulus did not differ in the
LL and DD groups, showing the animals could not anticipate the event even after 30
days of training. During the 60 min that the group was expected to be present, zebrafish
under LL remained in the afternoon compartment at both testing times (Fig. 2b), while
fish under DD settle in the morning compartment at both testing times (Fig. 4b).
57
It is possible that in the absence of the LD cycle, which function as cue to
predict time, the ability of orientation had been impaired, since light is one of most
relevant zeitgebers for the guidance of individuals (Hastings 1991). However, it worth
to notice that fish seem to show some temporal association because it spent most time in
a specific compartment at both tested times, but did not discern the correct side in the
correct time, in other words, there was no time-place association. Tasks involving
appetitive/aversive events, in which the individual needs temporal perception, implicate
on interval timing and circadian rhythm as well as associative learning of predictive
cues (Ralph et al. 2013). According to Cain et al. (2004), time memory can be explained
by the circadian oscillator action, which is modulated by significant experiences. Thus,
in the absence of light zeitgebers (strong cue), the organisms are dependent on weaker
cues (such as temperature and social cue), and the endogenous clock. While our
zebrafish seem not to display free-running conditions (t=1440; Table 1 and 2), interval
timing to predict time and place was not observed. Indeed, learning to associate time
with spatial location is not an easy task (Biebach, 1989), and depending on the specie it
may require a significantly strong zeitgeber to show TPL, for instance the LD cycle.
Despite constant light conditions (LL and DD), the activity registry on the last
6 days of the 30-days test showed that zebrafish maintained circadian rhythm (t=1440).
It may have occurred due to the presence of daily and fixed times of stimulus
presentation, reinforcing the strength of the social cue to circadian rhythm species
(Mrosovsky, 1988). The LL group showed higher activity (average speed) than the DD
group, but similar to LD held fish (Fig. 6 and 7, Table 1). We believe this pattern was
related to the diurnal chronotype of the zebrafish (Hurd et al., 1998) that may have
induced the LL group to maintain light responsiveness. It is in accordance with Elbaz et
al. (2013) studyin which zebrafish kept under LL cycle became more active and lost
58
rhythm, although our fish under LL showed higher speed, activity was more distributed
over the day time (not concentrated in the interval of the subjective day) and they have
maintained the circadian rhythm probably due to the social cue presented.
According to Yokogawa et al. (2007), under prolonged constant conditions,
adult zebrafish sleep overnight in both LD and DD cycle, but sleep-wake rhythm is
deleted under LL and only returns after about seven days in this condition. Under
constant dark, zebrafish display rhythmic activity and increase it during the subjective
day (Cahill et al., 1998; Hurd et al., 1998). Following the same path, we showed that
only animals under LD and DD had significant acrophase with higher activity occurring
between 12am and 4pm (Figure 6).
Although zebrafish has been recently used as an effective model in cognitive
studies, no data associating LD cycles influence on learning has been provided to date.
In this paper we applied a previously validated protocol to test TPL under constant light
conditions, reaching negative results both for constant light or constant dark, thus
refuting our hypothesis. To demonstrate TPL, an animal must learn to associate
different times of the day at different locations of an event (Reebs, 1996). We also
observed that constant dark leads to decreased but more concentrated activity of the
animals than constant light condition.
Behavioral studies represent an important method to identify neurofunctional
changes. The finding that constant light conditions impair TPL implies that light is more
than only an environmental cue to adjust life rhythm. Moreover, the zebrafish represents
a useful vertebrate model to fulfill many scientific gaps regarding the learning
processes, leading an opportunity to research about the molecular mechanisms involved
in the maintenance of the circadian rhythm. However, our study presents some faults,
such as the need for more observation days beyond 15th
and 30th
days, a longer period of
59
24h activity registry in order to find out changes in behavior due to the imposition of an
altered light regime, and other LD cycles to test TPL (e. g. 16:08 and 18:06). Even
though other studies in this area are still needed to the better understanding of light
cycle hole on learning, we presented here robust results in respect to the negative effects
constant light conditions to TPL. Furthermore, this paper recommends zebrafish as an
appropriate model for chronobiology, as well as suggests further investments on the
relation between light cycles, some clock genes expression and learning.
Acknowledgements
We thank Ms Tavares, C.P.M., Ms Coutinho, J.R.S. and Mr Canejo, F.W.G. for help in
collecting data for this article.
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Figure 1
Fig. 1. Zebrafish residence time (a and b) and frequency of entry (c and d) in the
morning and afternoon compartments on day 15 of the TPL test (n=8) under constant
light. Observations were made between 7:45 and 9:00 am, and 4:45 and 6:00 pm. The
first 15 min of observation indicate their ability to anticipate the arrival of the social
stimulus (a and c). During the following 60 min, the social stimulus (group with 5
conspecifics) was maintained inside the experimental tank (b and d). * indicates
statistical significance (Student’s t-test, p<0.05) between the compartments
corresponding to each experimental period.
0
500
1000
1500
2000
2500
3000
Morning Afternoon
Res
iden
ce t
ime
(s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' with shoal
0
10
20
30
40
50
60
70
80
90
100
Morning Afternoon
Fre
qu
ency
of
entr
y
Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' with shoal
67
Figure 2
Fig. 2. Zebrafish residence time (a and b) and frequency of entry (c and d) in the
morning and afternoon compartments on day 30 of the TPL experiment (n=10) under
constant light. Observations were made from 7:45 to 9:00 am, and from 4:45 to 6:00
pm. The first 15 minutes of observation indicates the ability to anticipate the arrival of
the social stimulus (a and c), while the next 60 min indicates the ability to learn time
and place of the stimulus presentation (b and d). * indicates statistical significance
(Student’s t-test, p<0.05) between the compartments in each experimental period.
0
500
1000
1500
2000
2500
3000
Morning Afternoon
Res
iden
ce t
ime
(s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' without shoal - TPL
0
10
20
30
40
50
60
70
80
90
100
Morning Afternoon
Fre
qu
ency
of
entr
y
Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' without shoal - TPL
68
Figure 3
Fig. 3. Zebrafish residence time (a and b) and frequency of entry (c and d) in the
morning and afternoon compartments on day 15 of the experiment (n=8) in constant
dark. Observations were made between 7:45 and 9:00 am, and 4:45 and 6:00 pm. a and
c represent the 15 min before shoal arrival, c and d represent the 60 min in which the
social stimulus was present. * indicates statistical significance (Student’s t-test, p<0.05)
between the compartments corresponding to each experimental period.
0
500
1000
1500
2000
2500
3000
Morning Afternoon
Res
iden
ce t
ime
(s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' with shoal
0
10
20
30
40
50
60
70
80
90
100
Morning Afternoon
Fre
qu
ency
of
entr
y
Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' with shoal
69
Figure 4
Fig. 4.Zebrafish residence time (a and b) and frequency of entry (c and d) in the
morning and afternoon compartments on day 30 of the experiment (n=10) in constant
dark. Observations were made from 7:45 to 9:00 am, and from 4:45 to 6:00 pm. The
first 15 min of observation indicates the ability to anticipate the arrival of the social
stimulus (a and c) and the following 60 min indicates time and place association with
the reward (b and d). * indicates statistical significance (Student’s t-test, p<0.05)
between the compartments corresponds to each experimental period.
0
500
1000
1500
2000
2500
3000
Morning Afternoon
Res
iden
ce t
ime
(s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' without shoal - TPL
0
10
20
30
40
50
60
Morning Afternoon
Fre
qu
ency
of
entr
y
Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' without shoal - TPL
70
Figure 5
Fig. 5: Representative actogram (average speed) ofzebrafish submitted to LD: light dark
cycle, LL: constant light, DD: constant dark during the lasts 6 days of the 30-day TPL
experiment.
71
Figura 6
Fig. 7: Waveform of the activity (a, b, c) for each group, and representative cosinor
showing the acrophase (d, e, f) of the animals under LD (light-dark cycle), LL (constant
light) and DD (constant dark) conditions on the last 6 days of the TPL test. LD group
showed significant differencein acrophase between 12am and 2pm (Rayleigh, p<0.05).
DD group showed significant difference in acrophase between 2am and 4 am (Rayleigh,
p<0.05). LL group had no significant difference in acrophase(Rayleigh, p>0.05).
72
Figura 7
Fig. 8: Activity (average speed) of the animals under LD (light-dark cycle), LL
(constant light) and DD (constant darkness) on the probe day (30th
day) of the test for
TPL test. LL and LD groups showed significant higher activity than DD group (One
way ANOVA, p<0.05).
73
Table 1. Activity variables measured in zebrafish submitted to light-dark cycle (LD),
constant light (LL) and constant dark (DD).
Different letters indicate statistical differences between the groups in the same variable
evaluated (One way ANOVA, p<0.05).
Mean
activity
Center
of
gravity
Total
area
under
the
curve
Mean
interval
Area
under
the
curve
Percentage
of total
area
LD 3.94±0,11ᵃᵇ 740.90ᵃᵇ
364.63 5.00 245.35 64.29ᵃ
LL 4.17±0,39ᵃ
724.45ᵇ
401.07 4.35 213.45 52.66ᶜ
DD 3.20±0,12ᵇ
761.82ᵃ 307.41 3.76 184.62 59.45ᵇ
74
Anexo 1
Table 2. Cosinor summary of the zebrafish submitted to light-dark cycle (LD),
constant light (LL) and constant dark (DD).
Animals Mesor Amplitude Acrophase %Ve(total)
LD 1 4.11: 4.05~4.17 1.83: 1.72~1.93 797.23:
783.94~810.53
97.20
2 4.04: 3.98~4.10 1.52: 1.41~1.63 807.38:
791.02~823.74
96.88
3 4.027:
3.96~4.090
1.46: 1.35~1.57 823.06:
805.42~840.70
96.65
4 3.60: 3.51~3.70 1.36: 1.19~1.54 783.08:
754.07~812.09
90.81
LL 1 3.75: 3.68~3.82 0.65: 0.53~0.77 908.43:
865.19~951.67
95.25
2 3.96: 3.90~4.020 0.309: 0.20~0.41 784.82:
706.22~863.43
96.83
3 5.32: 5.25~5.40 0.60: 0.47~0.73 970.31:
921.28~1019.34
97.37
4 3.67: 3.58~3.75 0.30: 0.15~0.464 241.75:
118.32~365.18
91.98
DD 1 3.52: 3.35~3.68 1.19: 0.90~1.48 920.57:
864.73~976.4
76.94
2 3.01: 2.88~3.15 0.89: 0.64~1.14 884.12:
818.77~949.47
76.24
3 3.27: 3.12~3.42 1.23: 0.96~1.50 832.051:
781.49~882.61
76.83
4 2.99: 2.88~3.12 0.35: 0.14~0.57 1051.76:
902.03~1201.5
80.28
Period analyzed: 1440 minutes.
75
CONCLUSÃO GERAL
Nossos estudos nos levam a concluir que o peixe paulistinha é capaz de apresentar
aprendizagem espaço-temporal (TPL) em ciclo claro-escuro (12:12 CE), bem como
antecipar à chegada do estímulo. O grupo de coespecíficos, usado como estímulo para a
aprendizagem, se mostrou um excelente modelo de oferta para testar TPL, corroborando
os estudos sobre o marcado comportamento social de Danio rerio. Os animais expostos
à condições de claro constante e escuro constante não demonstraram TPL, com ambos
os grupos associando apenas um dos lados nos dois horários de teste. No que diz
respeito ao registro de atividade, podemos observar que após 30 dias nas diferentes
condições luminosas testadas, o peixe paulistinha se manteve mais ativo e com
atividade homogeneamente distribuída ao longo do dia. O grupo escuro constante,
apesar de ter apresentado menor atividade, manteve o padrão característico do cronotipo
diurno da espécie, ou seja, maior atividade concentrada na subjetiva fase clara. O peixe
paulistinha manteve ritmo circadiano (t=24h) em todas as condições testadas,
provavelmente não entrando em livre curso devido à presença, em horários diários
fixos, do grupo de coespecíficos que foi utilizado como estímulo. Dessa forma, os
resultados nos levam concluir que as condições prolongadas de luz constante
influenciam marcadamente o padrão de atividade do peixe paulistinha, bem como seu
comportamento padrão de maior atividade diurna, tornando-o mais arrítmico em relação
aos animais expostos ao ciclo claro-escuro e escuro constante. Ademais, é de se destacar
a relevância da pista luminosa, oferecida pelo ciclo-claro escuro, na orientação e
aprendizagem espaço-temporal do peixe paulistinha, o qual apesar de possuir relógio
biológico, necessita desse zeitgeber para apresentar esse tipo de aprendizagem
considerada complexa e adaptativa.
76
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UFRN – Campus Universitário – Centro de Biociências Celular Institucional (Claro): 9229-6491 Av. Salgado Filho, S/N – CEP: 59072-970 – Natal/RN e-mail: [email protected]
Universidade Federal do Rio Grande do Norte
COMISSÃO DE ÉTICA NO USO DE ANIMAIS - CEUA
PROTOCOLO N.º 039/2015
Professor/Pesquisador: ANA CAROLINA LUCHIARI
Natal (RN), 17 de setembro 2015.
Certificamos que o projeto intitulado “Aprendizagem espaço temporal do peixe
paulistinha (Danio rerio) sob diferentes fotoperíodos”, protocolo 039/2015, sob a
responsabilidade de ANA CAROLINA LUCHIARI, que envolve a produção, manutenção e/ou
utilização de animais pertencentes ao filo Chordata, subfilo Vertebrata (exceto o homem), para
fins de pesquisa científica encontra-se de acordo com os preceitos da Lei n.º 11.794, de 8 de
outubro de 2008, do Decreto n.º 6.899, de 15 de julho de 2009, e com as normas editadas pelo
Conselho Nacional de Controle da Experimentação Animal (CONCEA), e foi aprovado pela
COMISSÃO DE ÉTICA NO USO DE ANIMAIS da Universidade Federal do Rio Grande do
Norte – CEUA/UFRN.
Vigência do Projeto ABRIL 2016
Número de Animais 30
Espécie/Linhagem Peixes (Danio rerio)
Peso/Idade 0,5g / 6 meses
Sexo Machos e Fêmeas
Origem Adquiridos em Fazendo de Cultivo em Natal-RN
Informamos ainda que, segundo o Cap. 2, Art. 13 do Regimento, é função do
professor/pesquisador responsável pelo projeto a elaboração de relatório de
acompanhamento que deverá ser entregue tão logo a pesquisa for concluída.
Josy Carolina Covan Pontes Coordenadora da CEUA
