1 THIEZA GRAZIELLA ARAUJO DA SILVA GOES DE MELO
ANTIDEPRESSIVOS MODIFICAM A EXTINÇÃO DE UMA MEMÓRIA AVERSIVA EM RATAS
Dissertação apresentada à Universidade Federal do Rio Grande do Norte, para obtenção do título de Mestre em Psicobiologia.
2 THIEZA GRAZIELLA ARAUJO DA SILVA GOES DE MELO
ANTIDEPRESSIVOS MODIFICAM A EXTINÇÃO DE UMA MEMÓRIA AVERSIVA EM RATAS
Dissertação apresentada à Universidade Federal do Rio Grande do Norte, para obtenção do título de Mestre em Psicobiologia.
Orientadora: Profª. Drª. Regina Helena da Silva Co-Orientador: MSc PhD Geison de Souza Izídio
3 Título: ANTIDEPRESSIVOS MODIFICAM A EXTINÇÃO DE UMA MEMÓRIA AVERSIVA EM RATAS
Autor: Thieza Graziella Araújo da Silva Góes de Melo
Data da defesa: 16/05/2011
Banca Examinadora:
___________________________________ Profª. Drª. Flávia Teixeira da Silva Universidade Federal de Sergipe, SE
___________________________________ Profª. Drª. Elaine Cristina Gavioli
Universidade Federal do Rio Grande do Norte, RN
___________________________________ Profª. Drª. Regina Helena da Silva
4 AGRADECIMENTOS
À minha família e amigos, pela compreensão. Especialmente minha mãe, por estar sempre presente e ter sempre me dado o melhor exemplo a ser seguido. As minhas conquistas são tão minhas quanto dela e, algumas vezes, talvez até mais dela que minhas. Ao meu noivo, Tiago, também sempre presente, inclusive nos finais de semana, em experimento, me dando suporte. Ao meu padrasto, Pai-Tio Guilherme que tem, com toda certeza, ―direito‖ a pelo menos 1 cm² do certificado. À minha avó, meu exemplo de vida, e que me mostrou que tudo pode ser feito com paciência, boa vontade e um pouquinho de ousadia.
À professora Regina que me acompanha desde a iniciação científica, dando suporte acadêmico/científico e que verdadeiramente me ensinou a fazer ciência, mesmo sob as circunstâncias mais adversas.
Ao LEME, no corpo de seus ICs, mestrandos, doutorandos, pós-docs e professores pela ajuda durante os experimentos e dos quais recebi não menos que amizade. Dentro do LEME agradeço especialmente às duas pessoas que participaram mais diretamente nos experimentos e revisões, Luane e Geison.
Aos dois psiquiatras que me forneceram suporte técnico e informações clínicas Drª Nádira Hazboun e Drº Francisco Márcio Pinheiro.
À UFRN, CAPES, CNPq, FAPERN e PPG em Psicobiologia pelo apoio financeiro e estrutural.
A todos os professores e colegas do programa de pós-graduação em Psicobiologia.
5 Resumo
Os tratamentos de depressão, transtorno de estresse pós-traumático e outras psicopatologias que se utilizam de antidepressivos podem estar associados à melhora de déficits cognitivos relacionados a esses transtornos. Embora os mecanismos pelos quais a melhora nos déficits cognitivos ocorre não estejam totalmente esclarecidos, alterações na extinção de memórias aversivas podem estar presentes nestas psicopatologias. Além disso, pesquisas com animais de laboratório geralmente são realizadas com indivíduos do sexo masculino, e recentemente verificamos que a extinção de uma tarefa aversiva é diminuída em ratas quando comparada ao desempenho de ratos. No presente estudo, ratas Wistar foram tratadas prolongadamente com antidepressivos utilizados na clínica (nortriptilina, fluoxetina ou mirtazapina) e testadas na esquiva discriminativa em labirinto em cruz elevado e no teste do nado forçado, a fim de avaliar a aprendizagem, a memória, a extinção, a ansiedade e comportamentos relacionados à depressão. A exploração do braço aversivo na sessão de treino foi semelhante em todos os grupos, mostrando que todos os grupos aprenderam a tarefa, havendo, porém, uma melhora no desempenho dos grupos tratados com nortriptilina e mirtazapina. Na sessão teste todos os animais evocaram a tarefa. O tratamento prolongado com a fluoxetina, mas não com os outros antidepressivos, promoveu uma melhora na extinção da memória aversiva da EDLC. No teste de nado forçado, os animais tratados com fluoxetina e mirtazapina apresentaram diminuição na duração da imobilidade, comparados ao veículo. Em conclusão, os antidepressivos podem interferir no aprendizado, mas não na evocação de memórias aversivas. Além disso, ratas tratadas com fluoxetina apresentam um aumento da extinção da tarefa aversiva, em comparação ao veículo, enquanto os demais tratamentos impediram a extinção dessa tarefa. Além disso, tanto a fluoxetina como a mirtazapina foram eficazes no teste de nado forçado, sugerindo dissociação entre os efeitos antidepressivos e extinção de memórias aversivas.
6 Abstract
Treatment of major depression, posttraumatic stress disorder and other psychopathologies with antidepressants can be associated with improvement of the cognitive deficits related to these disorders. Although the mechanisms of these effects are not completely elucidated, alterations in extinction of aversive memories are believed to be present in these psychopathologies. Moreover, researches with laboratory animals usually focus on male subjects, and we have recently verified that extinction of an aversive task is reduced in female rats when compared to males. In the present study, female rats were long-term treated with clinically used antidepressants (fluoxetine, nortriptyline or mirtazapine) and tested in the plus-maze discriminative avoidance and forced swimming tests in order to evaluate learning, memory, extinction, anxiety and depression-related behaviors. All groups learned the task, but learning was somewhat faster in nortriptyline and mirtazapine-treated animals . Task retrieval was also showed by all experimental groups. Chronic treatment with fluoxetine, but not with the other antidepressants, increased extinction of the discriminative task. In the forced swimming test, animals treated with fluoxetine and mirtazapine showed decreased immobility duration. In conclusion, antidepressants interfere with learning and female rats treated with fluoxetine presented increased extinction of the aversive memory task. On the other hand, both fluoxetine and mirtazapine were effective in the forced swimming test, suggesting dissociation between the antidepressant effects and the extinction of aversive memories.
7 Sumário
I. INTRODUÇÃO ... 8
II. OBJETIVOS ... 20
III. ARTIGO PARA SUBMISSÃO... 21
IV. CONCLUSÃO E CONSIDERAÇÕES FINAIS... 55
8 I. Introdução
O uso de antidepressivos, sozinhos ou em combinação com a psicoterapia, é largamente difundido. Além da grande indicação para o tratamento de psicopatologias como transtornos de humor e ansiedade (Marks et al. 1998; Brunello et al. 2001; Nandam et al. 2007), eles são também indicados como uma opção alternativa para doenças debilitantes funcionais como, por exemplo, a síndrome da fadiga crônica, a fibromialgia, enxaquecas ou dores de cabeça crônicas e dores faciais atípicas (Marks et al. 2008; Mao et al. 2010).
A depressão é uma psicopatologia já devidamente classificada no Manual de Diagnóstico e Estatística de Doenças Mentais (em inglês Diagnostic and Statistical Manual of Mental Disorders), com suas últimas modificações na classificação para
9 psicomotora; diminuição da energia; sentimentos de desvalia ou culpa; dificuldades para pensar, concentrar-se ou tomar decisões, ou pensamentos recorrentes sobre morte ou ideação suicida; planos ou tentativas de suicídio. Sintomas físicos são comumente encontrados no episódio depressivo maior: propensão ao choro, irritabilidade, ruminação obsessiva, ansiedade, fobias, preocupação com a saúde física e queixas de dores, tanto de cabeça, quanto no corpo (APA 2000; Fava & Kendler 2000). Alguns indivíduos podem apresentar ataques de pânico que ocorrem segundo um padrão que satisfaz os critérios para transtorno de pânico. Em crianças pode ocorrer ansiedade de separação. Alguns indivíduos observam dificuldade nos relacionamentos íntimos, diminuição das interações sociais satisfatórias ou dificuldades no funcionamento sexual (APA 2000). A manifestação de episódios depressivos pode ainda vir como um transtorno depressivo maior, o qual é caracterizado pela ocorrência de um ou mais episódios depressivos, sem história de episódios maníacos, mistos ou hipomaníacos.
10 uso de antidepressivos também ao tratamento de outros transtornos como os da ansiedade (APA 2000; Marques 2001).
A incidência desses demais transtornos também pode ser relacionada à comorbidade deles com a depressão. Os transtornos de ansiedade mais comuns em mulheres, em sua grande maioria (Bekker & van Mens-Verhulst 2007) são os de maiores índices de comorbidade com a depressão. Dessa forma é imperativa a necessidade de uma maior atenção à sintomatologia diferenciada apresentada pelos indivíduos do sexo feminino. Mulheres e crianças podem apresentar mais comumente o humor irritado (APA 2000) e essa alteração no humor pode ser sintoma apenas da depressão e não ter nenhuma relação com outros transtornos de humor ou ansiedade. Paralelamente, a perda de peso pode estar associada mais facilmente aos transtornos alimentares, que podem ser a causa da depressão (APA 2000; Mischoulon et al. 2010; Modrzejewska 2010).
O tratamento de transtornos do humor é geralmente complexo, prolongado e adequado a cada indivíduo. Estudos com indivíduos saudáveis sugerem que a percepção de faces com expressões negativas começam a aparecer após o 7º dia de tratamento (Harmer et al. 2010), diminuição de perceptção essa, associada aos efeitos terapêuticos do uso contínuo de antidepressivos. Existem várias classes de drogas antidepressivas disponíveis na atualidade, com propriedades farmacodinâmicas distintas. De forma geral, tais fármacos aumentam as concentrações de monoaminas na fenda sináptica, mas apenas esse aumento, embora necessário, não é suficiente para explicar a remissão dos sintomas (Arantes-Gonçalves & Coelho 2006).
11 noradrenalina (NA) na fenda sináptica, visto que drogas que depletam as reservas desses neurotransmissores são indutoras de quadros depressivos (Freis 1954; Guay 2010; Tian et al 2010; Aia et al 2011; Ghia et al 2011), e o mecanismo de ação dos agentes depressivos mais utilizados envolve aumento da disponibilidade desses neurotransmissores (Kent 2000). Contudo, uma hipótese mais recente sobre a fisiopatologia e tratamento da depressão envolve também adaptação e plasticidade neural. De acordo com essa hipótese, a depressão seria resultado de uma falha na execução de respostas adaptativas apropriadas ao estresse e a estímulos aversivos, ou seja, perda da plasticidade sináptica. A isso pode se associar o fato de que a exposição crônica a situações de estresse induz um quadro semelhante à depressão, com redução dos níveis hipocampais de fator neurotrófico derivado do encéfalo (BDNF – do inglês brain-derived neutrophic factor) (Gould et al. 1999; Sen et al. 2003; Arantes-Gonçalves & Coelho 2006), componente da família das neurotrofinas, que, entre várias outras moléculas, apresenta um papel reconhecidamente importante na plasticidade sináptica (Arantes-Gonçalves & Coelho 2006; Monfils 2007; Kalueff 2007).
12 Dentre as várias opções de antidepressivos os mais prescritos são os inibidores seletivos de recaptação de serotonina (ISRS) tanto para adultos como para crianças (Dunlop & Davis 2008; Denizot et al. 2009). Introduzidos na clínica no final da década de 80 (Kent 2000), os ISRS agem bloqueando a ação do transportador de serotonina. Dessa forma eles impedem a recaptação do neurotransmissor, o que prolonga a sua exposição ao receptor, (Vázquez-Palacios 2004) e aumentando a atividade do sistema monoaminérgico no cérebro (Tsai et al. 2009). Dentre os antidepressivos dessa classe a fluoxetina é o mais prescrito (Dunlop & Davis 2008). É importante ressaltar que a serotonina (5-HT) possui também um papel regulador na divisão celular e um papel crítico no controle da proliferação das células adultas neurais, fato que explicaria sua eficácia na melhora dos sintomas depressivos considerando o papel da neurogênese hipocampal na fisiopatologia da depressão (Santarelli et al. 2003; Paizanis et al. 2007)
13 colateral importante decorrente do tratamento com ISRS (Dagtekin et al. 2010; Diaz & Maroteaux 2011).
Os antidepressivos tetracíclicos são uma nova classe de drogas utilizadas no tratamento da depressão, também chamados de atípicos, introduzidos depois da década de 80 (Kent 2000). Dentre estes, o antidepressivo atípico mirtazapina atua especificamente sobre as transmissões noradrenérgica e serotonérgica (noradrenergic and serotonergic specific antidepressants, NaSSA, em inglês) e é usado para tratar transtornos de humor e ansiedade (Gambi et al. 2005; Rauggi et al. 2005). A mirtazapina aumenta as transmissões noradrenérgica e serotonérgica central através da inibição dos autoreceptores α2 e heteroreceptores noradrenérgicos α2 em sinapses serotonérgicas (Bengtsson et al. 2000; Gambi et al. 2005). Além disso, é um agonista do receptor 5-HT1A (Rogóz et al. 2005) e também age bloqueando receptores, 5-HT2A, 5-HT2C e 5-HT3 e histaminérgicos H1 (Haddjeri 1998; Davis & Wilde 1996; Arnone 2009; de Boer 1995; Rauggi et al. 2005). A mirtazapina não possui efeitos na recaptação de monoaminas, além de apresentar baixa afinidade para receptores dopaminérgicos e alguns subtipos de receptores serotonérgicos.
14 tricíclico, enquanto machos respondem positivamente ao mesmo tratamento e com a mesma dose (Barros & Ferigolo 1998).
Assim como em grande parte dos casos de diversos transtornos psicopatológicos, pacientes com diagnóstico de depressão, não raramente apresentam prejuízos cognitivos, além de todo quadro afetivo característico (Yaffe 1999; Ravnkilde 2002; Pardo 2006). Dentre esses prejuízos incluem-se alterações nas funções de memória e aprendizado (Burt 1995; Weiland-Fiedler 2004). Esses prejuízos cognitivos seriam revertidos através do tratamento com antidepressivos (Austin et al. 2001; Castaneda et al. 2007) e evidências sugerem que essa melhora está associada ao aumento de neurogênese (Duman & Monteggia 2006; Dranovsky & Hen 2006; Nandam et al. 2007; Paizanis et al. 2007; Sahay & Hen 2007; Pittenger & Duman 2008) e a modificações no sistema monoaminérgico (Lee et al. 2010).
15 al. 2006; Krystal & Neumeister 2009), a participação de processos relacionados à memória emocional e a fisiopatologia da depressão não está bem estabelecida (Blaney 1986; Newman & Sweet 1992; Ilsley et al. 1995; Veiel 1997; Fava & Kendler 2000; Ravnkilde 2002). Apesar de prejuízos na memória serem comuns em diversos transtornos de ansiedade e relatados na depressão, a conexão entre antidepressivos e memória permanece ainda não completamente elucidada (Austin et al. 2001). Os resultados dos estudos prévios são bastante controversos, mesmo em machos. Estudos que avaliam a extinção de memórias aversivas após a administração de drogas com efeito antidepressivo mostram resultados contraditórios. Dentre estes estudos, alguns, utilizando machos, verificam os efeitos do rolipram, uma droga com efeitos ansiolíticos e antidepressivos. Essa droga diminuiu a extinção em um teste de medo condicionado e em um paradigma de resposta de sobressalto potencializada pelo medo (Monti et al. 2006; Mueller et al., 2010). Além disso, este fármaco melhorou o desempenho no labirinto aquático de Morris, aumentando a distância e o tempo em que os animais nadam no quadrante no qual previamente havia uma plataforma submersa; e ainda impediu a extinção relacionada a uma tarefa de esquiva passiva (Cheng et al, 2010). Um outro estudo foi realizado com um modelo animal para transtorno de estresse pós-traumático, característico pela ausência de extinção da memória do evento traumático (APA 2000). Nesse estudo, a cicloserina-D (DCS), droga utilizada como auxiliar em tratamentos antidepressivos, melhorou a extinção em testes de condicionamento de medo em machos (Yamamoto et al. 2008).
16 pelas doenças debilitantes funcionais e em grande parte dos transtornos psicopatológicos a proporção dos indivíduos atingidos é de duas mulheres para cada homem (Mahedran & Yap 2005; Bekker & van Mens-Verhulst 2007). Apesar de não se descartar fatores culturais e sociais, diversas evidências indicam que a principal origem dessas diferenças está relacionada aos hormônios esteróides gonadais (Toufexis et al. 2006; Solomon & Herman 2009), que podem ter efeitos nas vias neuroquímicas (Kelly et al. 1999). Além disso, aspectos do dimorfismo sexual no funcionamento do sistema nervoso também podem estar relacionados a estas diferenças (Bruder et al. 2001; Esel et al. 2005; Monteggia et al 2007; Elaković et al. 2011).
17 pode ter relação com a predominância de psicopatologias em seres humanos do sexo feminino.
No presente estudo, nós utilizamos o modelo utilizado por Ribeiro et al. (2010): a esquiva discriminativa em labirinto em cruz elevado (EDLC). A EDLC é um método adequado para estudos concomitantes de memória/aprendizado e medo/ansiedade em modelos animais. Consiste em um labirinto em cruz elevado modificado, onde um dos braços fechados possui dois estímulos aversivos (sendo um sonoro de intensidade equivalente a 80dB e um luminoso de 100W). Os animais são submetidos a uma sessão de treino, na qual uma vez colocados individualmente no centro do labirinto por 10 min, poderão explorar os braços, recebendo os estímulos aversivos cada vez que o animal entrar com as quatro patas no braço aversivo, até a saída do braço em questão. O tempo de permanência e o total de entradas nos braços abertos e em cada braço fechado são quantificados. Decorridas 24 horas da sessão de treino é efetuada uma sessão de teste, na qual não se aplica o estímulo aversivo. Dessa forma, ao longo da sessão pode-se também avaliar a extinção da tarefa previamente aprendida, uma vez que os animais agora aprendem que não há mais estímulos aversivos naquela localização. Este modelo foi inicialmente descrito por Silva (1997) e Silva & Frussa-Filho (2000), que padronizaram o modelo com machos e averiguaram que, usualmente os três minutos iniciais são referentes à evocação da memória adquirida e os demais à extinção.
18 de ansiedade a exploração desses braços poderá aumentar ou diminuir. Considerando que alterações nos níveis de ansiedade podem afetar o perfil comportamental dos animais em modelos animais de estudo de memória, o modelo da EDLC apresenta a grande vantagem de permitir a avaliação simultânea destes dois parâmetros (Calzavara et al. 2004; Silva & Frussa-Filho 2000). A diferenciação entre os braços fechados (aversivo e não-aversivo) serve como parâmetro para avaliação de memória e aprendizado. Esse modelo permite ainda a avaliação da atividade locomotora que pode ser realizada por meio da exploração de todos os braços e do centro, pelo total de passagens entre os braços (Silva et al. 2002) ou, ainda, quantificando-se a distância total percorrida (Ribeiro et al., 2010).
19 tentativa de fuga de situações aversivas, entre machos e fêmeas (Ribeiro et al. 2010), embora em outro tipo de tarefa comportamental. A diminuição da duração da imobilidade e o aumento do tempo de escalada podem estar relacionados a uma melhora dos sintomas tipo depressivos através da transmissão noradrenérgica (Detke et al. 1995, 1997; Reneric & Lucki 1998; Page et al. 1999; Cryan & Lucki 2000; Lopez-Rubalcava & Lucki 2000; Cryan et al. 2002a; Reneric et al. 2002; Consoni et al. 2006).
20 II. Objetivos
Objetivo Geral
O presente estudo propôs-se a averiguar os efeitos de três antidepressivos provenientes de classes farmacológicas distintas no aprendizado, memória, extinção, ansiedade/emocionalidade e comportamento de desamparo aprendido em ratas. Tendo em vista a prevalência dos transtornos relacionados à depressão e ansiedade em mulheres e a pequena quantidade de pesquisas relacionando os efeitos desses fármacos em processos cognitivos em indivíduos do sexo feminino, a nossa proposta pretende contribuir no avanço da compreensão dos efeitos destas classes de drogas em fêmeas.
Objetivos específicos
(a) Avaliar as diferentes etapas de memória (aquisição, consolidação, evocação e extinção) em fêmeas tratadas com os antidepressivos na EDLC;
(b) Avaliar os comportamentos relacionados à ansiedade em fêmeas tratadas com os antidepressivos na EDLC;
(c) Avaliar possíveis efeitos locomotores dos fármacos utilizados;
21 III. Artigo para submissão
Date of submission:
ANTIDEPRESSANTS FLUOXETINE, NORTRIPTYLINE AND MIRTAZAPINE MODIFIES THE EXTINCTION OF AN AVERSIVE MEMORY AND THE ANXIETY IN
FEMALE RATS
Thieza G. Melo, Geison S. Izídio, Luane S. Ferreira, Diego S. Silveira, Priscila T. Macedo, Alícia Cabral, Alessandra M. Ribeiro, Regina H. Silva
Memory Studies Laboratory Department of Physiology
Universidade Federal do Rio Grande do Norte 59.078-970, Natal, RN, Brazil·.
*Corresponding author: Regina H. Silva Laboratório de Estudos da Memória
Departamento de Fisiologia - Centro de Biociências Universidade Federal do Rio Grande do Norte
Av. Salgado Filho, s/n - Caixa Postal 1511 - CEP 59078-970 - Natal, RN, Brazil fax: (55) 84 3211 9206 e-mail: reginahsilva@gmail.com
Progress In Neuropsychopharmacology & Biological Psychiatry (Classificação Qualis A2, em Fevereiro de 2011)
22 Abstract
23 1. Introduction
Psychopathological conditions such mood and anxiety disorders are frequently related to aversive emotional experiences (APA, 2000) that could benefit from a reframing of the aversive memory formatted, which is usually attempted in psychotherapeutic procedures (Freud, 1914). It has also been suggested that the cognitive deficits present in these disorders are reversed by treatment with antidepressants (Austin et al., 2001; Castaneda et al., 2007), which are largely indicated for both anxiety and mood disorders (Marks et al., 1998; Brunello et al., 2001; Nandam et al., 2007). Several studies indicate that the improvement of these cognitive deficits is associated with hippocampal neurogenesis (Santarelli et al., 2003; Duman and Monteggia, 2006; Dranovsky and Hen, 2006; Nandam et al., 2007; Paizanis et al., 2007; Sahay and Hen, 2007; Pittenger and Duman, 2008), which would explain the delay of amelioration of depressive symptoms after the beginning of clinical treatment. Despite memory deficits being common in many anxiety and depression- related disorders the relationship between the improvement of depression or anxiety symptoms by antidepressants treatment, as well as their effect on memory, remains to be elucidated (Austin et al., 2001).
25 In the present study, we investigated the effects of three antidepressants (fluoxetine, nortriptyline and mirtazapine) of different pharmacological classes on learning, memory and extinction of an aversive task in female rats, as well as anxiety and depression-related behaviors in the same subjects. Fluoxetine is a selective serotonin reuptake inhibitor (SSRI), the most prescribed class of antidepressants (Dunlop and Davis, 2008). Nortriptyline is a tricyclic antidepressant, an older class of drugs, and act by nonselective inhibition of monoaminergic reuptake. Although these compounds are no longer largely prescribed, they are still an option to individuals which are non-responsive to other antidepressants and to subjects susceptible to the serotonin syndrome (Dagtekin et al., 2010). Finally, the atypical antidepressant mirtazapine is used to treat both mood and anxiety disorders (Rauggi et al., 2005; Gambi et al., 2005) and enhances central noradrenergic and serotonergic neurotransmission through inhibition of the noradrenergic α2-autoreceptor and α2 -heteroreceptor in serotonergic synapses (Bengtsson et al., 2000; Gambi et al., 2005).
We used the plus-maze discriminative avoidance task (PMDAT) to evaluate concomitantly memory and anxiety-related behaviors. Several studies performed with this task have shown the effects of memory-enhancing or amnestic drugs, procedures that modify anxiety-like behaviors and/or locomotor activity (Silva et al., 1997; Silva and Frussa-Filho, 2000; Silva et al., 2002a; Silva et al., 2002b). It also fits for the evaluation of learning and extinction processes, by the evaluation of aversive arm avoidance across the behavioral sessions (Silva et al., 2004; Ribeiro et al., 2010; see Methods). Additionally, female rats were also tested in the forced swimming test (FST) in order to evaluate depression-related behaviors.
26 2.1 Animals
Three-month-old female Wistar rats (120–230g), from our own colony were housed in groups of 4 animals in plastic cages (30 x 37 x 16 cm) in a room with acoustic isolation, airflow and controlled conditions of temperature (24 – 26 °C), humidity and luminosity (12h light: 12h dark, lights on 06h30). Food and water were available ad libitum throughout the experiments. Animal care was according to Brazilian law nº 11.794/2008 for the use of animals in research, and all experiments were approved by the local ethical committee (CEUA-UFRN). All efforts were made to minimize animal pain, suffering or discomfort as well as the number of animals used.
2.2 Procedures and drugs
27 predominance of any stage among the experimental groups during the experiments (data not shown). Rats were handled for the whole injection period, for 5 minutes per day. From the 17th day of treatment onwards, behavioral tests started at 1:30 p.m. and injections and cycle control continued at 6 p.m., after the behavioral procedure.
2.3 Plus-maze discriminative avoidance task (PMDAT)
28 (Rodgers et al., 1997). A decrease in risk assessment behavior and an increase in head dipping indicate less anxious behavior (Rodgers and Dalvi, 1997; Rodgers et al., 1997). Distance traveled in the apparatus, percent time spent in the aversive enclosed arm (time spent in aversive enclosed arm / time spent in both enclosed arms) and percent time spent in open arms (time spent in open arms / time spent in both open and enclosed arms) were registered min by min throughout the sessions and used to evaluate motor activity, learning/memory/extinction and anxiety, respectively. The behavior of the animals was monitored and analyzed by the video-tracking software ANY-maze, Stoelting, USA.
2.4 Forced Swimming Test (FST)
For evaluation of the depression-related behaviors we used the FST (Porsolt et al., 1977). This test consists in placing the animal in a cylinder (40 cm high, 25 cm diameter) with water (30 cm deep) in a temperature of 24 to 27°C, for two consecutive days. On the first day, animals were submitted to 15 min of forced exposure (pre-test session) with no behavioral observation. In the second day, 24 h after the pre-test session, rats were placed once again in the water tank (test session) in the same conditions described above and total immobility duration, climbing behavior and the latency to engage in immobility were registered for 5 min.
2.5 Statistical Analyses
29 total length of the sessions in the PMDAT, behavioral sessions were divided in 5 blocks of 2 minutes each. Comparison among these blocks was used to evaluate learning (training) or extinction (test) of the task. ANOVAs with repeated measures were used to compare the percent time spent in the aversive and open arm throughout PMDAT sessions and pairwise comparisons was used. We used the software SPSS (version 17) to perform the statistical analysis. Differences were considered significant at p<0.05.
3. Results
Plus-maze discriminative avoidance task:
30 the aversive arm during the first session block (paired samples t-test) (Fig. 1B). The ANOVA for the 2 minutes session blocks showed no difference between the groups for each session (for the first block [F (3,34) = 0.58; p = 0.62], second [F (3,34) = 1.00; p = 0.40], third [F (3,34) = 1.04; p = 0.38], fourth [F (3,34) = 1.30; p = 0.29] and fifth [F (3,34) = 2.03; p = 0.13]).
Comparison of the exploration of the aversive arm was marginally significant in the test session [F (3,34) = 2.52; p = 0.07], and pos hoc analysis with Bonferroni‘s
31 Percentage of time spent in the open arms did not differ between different treatments, suggesting that animals did not present anxiety-related differences [training session: F (3,34) = 0.060; p = 0.98; test session: F (3,34) = 0.50; p = 0.68] (Fig. 2). Similarly, no differences were found in the frequency of head dipping [training session: F (3,34) = 1.352; p = 0.276; test session: F (3,34) = 0.060; p = 0.98] (Fig.3C and D). A treatment effect was found for time spent in risk assessment in the test [F (3,34) = 3.876; p = 0.018] (Fig. 3B), but not in the training session [F (3,34) = 0.278; p = 0.841] (Fig. 3A). In the test session, the post hoc with Bonferroni‘s test
showed a significant difference between vehicle and fluoxetine (p = 0.04), while the comparison between vehicle and nortriptyline almost reached significance (p = 0.05).
When the whole sessions were considered for analysis, ANOVA revealed a treatment effect in the distance traveled in the apparatus [F (3,34) = 4.56; p < 0.01] and [F (3,34) = 3.86; p < 0.05] in training and test, respectively. Post hoc with Bonferroni‘s test showed that animals treated with mirtazapine exhibited a
significantly decreased locomotor activity compared to the other groups (Table 1). In the forced swimming test, ANOVA revealed a treatment effect in immobility duration [F (3,33) = 13.64; p < 0.001]. Post hoc with Bonferroni‘s test showed that
animals treated with fluoxetine and mirtazapine had decreased immobility duration compared with the control and nortriptyline groups (Fig. 4A). The latency to start immobility was not different among the groups (data not shown). Moreover, ANOVA revealed a treatment effect on climbing behavior [F (3,33) = 4.17; p = 0.014]. Post hoc with Bonferroni‘s test showed that fluoxetine group had an increased time in
38 Table 1. Distance travelled in meters in both training and test session by female rats repeatedly treated with vehicle, 20mg/kg fluoxetine, 20 mg/kg nortriptyline or 10 mg/kg mirtazapine; *p<0.05 compared to all other groups (ANOVA followed by Bonferroni‘s test).
4. Discussion
In summary, our data showed that some antidepressants could interfere on both learning and memory processes, more specifically on the acquisition and, more importantly, extinction of an aversive task. The nortriptyline- and mirtazapine-treated groups learned the task faster than the vehicle and fluoxetine counterparts, as demonstrated by a significant decrease in the aversive arm exploration earlier in the training session (see figure 1B). During the test session, vehicle and fluoxetine groups have shown extinction of the task, i.e., significant increases in aversive arm exploration across the session blocks, what did not occur in the other groups (see figure 1D). Moreover, the extinction was anticipated by treatment with fluoxetine. The treatment with fluoxetine and nortriptyline also reduced one of the anxiety-related behaviors in the elevated plus-maze discriminative avoidance task (risk assessment behavior, see figure 3B). Results obtained in the forced swimming test showed that
Treatment Training Test
Vehicle 19,0±2,4 21,1±1,9
Fluoxetine 20,0±3,0 21,6±4,4
Nortriptyline 22,9±3,1 16,8±4,4
39 treatment with fluoxetine and mirtazapine reduced the immobility time, which is consistent with the antidepressant-like effects of these drugs (see figure 4A).
As mentioned above, the analysis of the aversive arm exploration throughout the test session of the PMDAT provides indication of both task retrieval (beginning of session) and extinction (subsequent session blocks) (Ribeiro et al., 2010). Indeed, with the aversive stimuli no longer present, the animals avoid the aversive arm at first, but eventually realize the arm is now safe and increase its exploration, reaching the amount they would explore a regular enclosed arm by chance (or even more, since this arm is almost novel for them in the test session). This pattern is usually observed in males, but in a previous study we have shown that female rats keep avoiding the aversive arm until the end of the test, or even at a subsequent retest (Ribeiro et al., 2010). The lack of extinction by females points out to stronger consolidation and/or impaired extinction of aversive memories, which is in line with the better emotional memory usually reported for women (Canli et al., 2002; Hamann, 2005). In particularly, this lack of extinction is similar to what occur in some anxiety disorders, as for example, the posttraumatic stress disorder (PTSD), and it could be related to the greater prevalence of depression and anxiety disorders among female gender (Kendler et al., 2001; Mahedran, 2005; Rauch et al., 2006; Bekker and van Mens-Verhulst, 2007).
40 raise the hypothesis that fluoxetine could exert its therapeutic action by modulating the extinction of aversive memories. In line with this reasoning, this drug has been shown potential benefits in the treatment of PTSD, which has a close relationship with deficits in extinction of traumatic memories (APA, 2000; Quirk et al., 2006; Yamamoto et al., 2008; 2009). Moreover, despite the effects of fluoxetine treatment on males in the same test has not been investigated yet, these results, taking together with previous extinction studies performed with males (see Introduction) suggest an important role of gender in the ability of antidepressants in modifying extinction.
41 fluoxetine administered chronically to adult rats reduces, in the amygdala, the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a molecule involved in the synaptogenesis (Varea et al. 2007; Homberg et al., 2011). It has also been proposed that some types of antidepressants could elicit an increase on the perception of the threat cues and improve the trace of the formatted memory avoiding or decreasing its extinction (Rawlings et al., 2010). In summary, it seems plausible that the clinical action of antidepressants could be related to a modulation of plastic processes related to extinction of aversive memories.
42 aversive memory formatted could appear (Morgan and LeDoux, 1995; Lebrón et al., 2004; Quirk et al., 2006).
The hypolocomotor effects observed here in females treated with mirtazapine have already been found in other studies in this same dose range (Reneric et al., 2002) or not (Rauggi et al., 2005). It is interesting to note that, in the present study, the hypolocomotor effects were specifically found in the plus-maze discriminative avoidance task, i.e. they were not present in the forced swimming test. It is known that mirtazapine enhances both 5-HT and NA neurotransmissions, yet differently from monoamine reuptake inhibitors since its antidepressant effects are mediated through the direct antagonism of both α2 and 5-HT2C receptors (de Boer et al., 1996;
Haddjeri et al., 1996). It has been shown that this mediation can cause hypolocomotor effects (Franowicz et al., 2002). Mirtazapine also induces blockage of histamine-H1 receptors, other possible mechanism that may produce sedation and decrease the exploratory activity (Schüle et al., 2003; 2006; Gambi et al., 2005).
43 a better evaluation of the parameters. On the other hand, some authors argue that the evaluation of anxiety-related behaviors in a re-exposition to the conventional elevated plus-maze is not adequate, because the reasoning of the paradigm implies novelty (File, 1990).
44 al. 1999; Mirza et al., 2007) while others showed anxiogenic effects (File et al., 1999; Robert et al., 2011).
The forced swimming test is normally used in the screening of antidepressant drugs, with immobility duration levels being used to measure indices of ―behavioral despair‖ (Borsini and Meli, 1988). Moreover, the immobility presented by the animal
is reversed by repeated antidepressant treatment and for this reason it is used as an index of ‗depressive-like‘ state (Cryan et al., 2005; Porsolt et al., 1978). In the present study, animals treated with fluoxetine and mirtazapine, as expected, showed decreased time of immobility duration in the forced swimming test. However, females treated with nortriptyline did not exhibit this decrease, at a dose that was previously shown to be effective in a study with males (Consoni et al., 2006). They also showed less climbing behavior than fluoxetine–treated animals, suggesting that nortriptyline group were less active in this behavioral test. However, the possibility that these effects were only due to alterations in locomotion seems unlikely, because in the plus-maze discriminative avoidance task there were no significant decreases in ambulation of nortriptyline-treated animals.
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PROGRESS IN NEUROPSYCHOPHARMACOLOGY & BIOLOGICAL PSYCHIATRY
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DESCRIPTION .
56 experimental and clinical aspects of neuro-psychopharmacology and biological psychiatry. Another important aim of the journal is to supply pertinent information, provided by national and international bodies, that contributes to progress in the scientific and professional fields. Finally, the journal intends to foster and encourage communications between members of the communities of neuro-psychopharmacology and biological psychiatry.
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X-Y. Zhang, Houston, TX, USA
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INTRODUCTION
61 Studies on natural products The journal does not publish work on the actions of biological extracts unless the pharmacological active molecular substrate and/or specific receptor binding properties of the extract compounds are elucidated.
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