universidade federal do rio grande do norte … · graduações, um movimento que se articula e...
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
PRÓ-REITORIA DE PÓS-GRADUAÇÃO
CENTRO DE BIOCIÊNCIAS
PROGRAMA DE PÓS GRADUAÇÃO EM PSICOBIOLOGIA
EZEQUIEL BATISTA DO NASCIMENTO
PAPEL DOS HORMÔNIOS SEXUAIS FEMININOS SOBRE O EFEITO DO ISOLAMENTO SOCIAL EM RATAS: UM ESTUDO ACERCA DOS PERFIS
NEUROENDÓCRINOS RELACIONADOS ÀS ALTERAÇÕES EMOCIONAIS E COGNITIVAS.
.
NATAL, 17 DE MAIO DE 2018
EZEQUIEL BATISTA DO NASCIMENTO
PAPEL DOS HORMÔNIOS SEXUAIS FEMININOS SOBRE O EFEITO DO ISOLAMENTO SOCIAL EM RATAS: UM ESTUDO ACERCA DOS PERFIS
NEUROENDÓCRINOS RELACIONADOS ÀS ALTERAÇÕES EMOCIONAIS E COGNITIVAS.
NATAL-RN
2018
Tese apresentada ao Programa de Pós‐graduação
em Psicobiologia da Universidade Federal do Rio
Grande do Norte como requisito parcial para a
obtenção do título de Doutor em Psicobiologia
(Área Psicologia fisiológica)
Orientadora: Profa. Dra. Alessandra Mussi Ribeiro
Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI
Catalogação de Publicação na Fonte. UFRN - Biblioteca Central Zila Mamede
Nascimento, Ezequiel Batista do. Papel dos hormônios sexuais femininos sobre o efeito do isolamento social em ratas: um estudo acerca dos perfis neuroendócrinos relacionados às alterações emocionais e cognitivas / Ezequiel Batista do Nascimento. - 2018. 156 f.: il. Tese (doutorado) - Universidade Federal do Rio Grande do Norte, Centro de Biociências, Programa de Pós-Graduação em Psicobiologia. Natal, RN, 2018. Orientadora: Profª. Drª. Alessandra Mussi Ribeiro. 1. Hormônios - Tese. 2. Isolamento social - Tese. 3. Depressão - Tese. I. Ribeiro, Alessandra Mussi. II. Título. RN/UF/BCZM CDU 159.92:591.51
Elaborado por Ana Cristina Cavalcanti Tinôco - CRB-15/262
Tese de Doutorado defendida por EZEQUIEL BATISTA DO NASCIMENTO e aprovada em 17 de maio do ano de dois mil e dezoito pela banca examinadora constituída pelos doutores:
_______________________________________________________________ Dra. ALESSANDRA RIBEIRO MUSSI
ORIENTADORA DEPARTAMENTO DE BIOCIENCIAS/UNIFESP – SANTOS/SP
_______________________________________________________________ Profa. Dra. ROVENA CLARA JANUARIO GALVÃO ENGELBERTH (titular)
DEPARTAMENTO DE FISIOLOGIA/UFRN – NATAL/RN
_______________________________________________________________ Profa. Dra. MARIA BERNARDETE CORDEIRO DE SOUSA (titular)
INSTITUTO DO CÉREBRO/UFRN – NATAL/RN
_______________________________________________________________ Profa.Dra. DEBORAH SUCHECKI (titular)
DEPARTAMENTO DE PSICOBIOLOGIA/UNIFESP – SÃO PAULO/SP
_______________________________________________________________ Prof. Dr. JOSE RONALDO DOS SANTOS (titular)
DEPARTAMENTO DE BIOCIÊNCIAS/UFS
NATAL – RN 2018
Dedico toda minha trajetória
acadêmica e profissional à minha
mãe, que desde sempre não mediu
esforços para me ajudar alcançar
meus sonhos e objetivos, sem o seu
carinho e dedicação possivelmente
não chegaria até aqui.
“If I had to define a major depression in a
single sentence, I would describe it as a
genetic/neurochemical disorder requiring a
strong environmental trigger whose
characteristic manifestation is an inability to
appreciate sunsets”
(R.M. Sapolsky, 1982)
AGRADECIMENTOS
Aqui constará minha trajetória, meus agradecimentos e minha percepção de
tudo que vi e vivenciei nesses últimos 10 anos na graduação e pós-graduação.
Antes de tudo, gostaria de dizer que eu não deveria estar aqui. Em 2010 o Censo
Demográfico do IBGE junto à Secretaria Especial de Políticas de Promoção da
Igualdade Racial (Seppir) e organização educaAfro relataram que somos menos de
25% nas pós-graduações. O número é ainda mais triste quando consideramos a
porcentagem de docentes nas universidades federais, chega a ser menos que 5%.
Em contrapartida, O atlas da violência em 2017 revelou que 78,9% dos jovens
assassinados nos últimos 10 anos são jovens negros de regiões periférica.
Felizmente, faço parte do grupo privilegiado de negros fora dessa realidade, mas
não por meritocracia, eu nem acredito nisso, mas porque ao longo da minha vida
eu encontrei pessoas que confiaram em mim e me ajudaram a sair da nefasta
realidade do jovem negro no Brasil. Mesmo da periferia, da escola pública e
estigmatizado com um diagnóstico de TDAH aos 12, eu consegui avançar.
Primeiramente, agradeço a minha mãe que nunca mediu esforços no meu
processo de educação, embora sempre fomos uma família sem condições
financeiras, e a situação ficou mais grave logo após a morte do meu pai, ela sempre
acreditou que a educação seria a única maneira de ascensão social, e por ela ter
esse entendimento, sempre investiu na nossa formação, mesmo seus esforços
apenas terem sidos concretos a mim, único filho a ter uma graduação, mestrado e
doutorado. Eu dedico a ela dedico toda minha vida. Também agradeço aos meus
irmãos, aos mais próximos, e aqueles que tomaram outros rumos.
Sou ex-aluno bolsista, de uma universidade particular, vale salientar que isso
foi motivo de chacota e descrédito por colega e professores! Mas isso nunca me
atingiu, pois sempre fui ciente das minhas limitações e dos meus potenciais, para
chegar aqui tive de aprender a conviver com isso, além do mais, nunca desejei ser
o aluno exemplar, o melhor, a referência. Apenas desejei ser melhor do que ontem,
sempre desafiei minhas limitações no intuito de superá-las.
Ingressei no mestrado em psicobiologia na UFRN, eu simplesmente amo
esse lugar, apesar de novamente conviver com pessoas que achava estranho a
presença de um psicólogo nas aulas, na maioria das vezes alunos da biologia,
medicina, biomedicina, ecologia e afins. Até hoje fico na dúvida se eles sabem que
a psicobio é reconhecido na área base da CAPES como um programa em
Psicologia. Por favor, parem de falar que psicólogos não sabem de neurociência,
antes de falar algo do tipo, deve-se considerar que a psicologia no Brasil foi
construída no modelo clínico baseado na corrente social crítica europeia, algo
bastante distante da psicologia americana total experimental. Isso é uma grande
barreira, pois o aluno de psicologia que deseja estudar neurociência terá de estudar
por si só, algo que os demais recebem na sua formação, é muita desonestidade
fazer comparações desse tipo sem ponderar esses fatores. Além disso, uma boa
parte dos teóricos citados nessa tese são psicólogos, corrigindo, psicólogas, pois
boa parte dos estudos sobre hormônios sexuais são produzidos por mulheres. A
ciência é tão patriarcal que a padronização dos nomes dos pesquisadores nos
periódicos faz referência ao sobrenome, majoritariamente masculinos, você só
saberá que é uma mulher quando for pesquisar de forma mais detalhada.
Ainda sobre a Psicobiologia, gostaria de falar que tenho afeições
inimagináveis por alguns professores. Aprendi muito nas aulas das professoras
Bernadete, Fívia e Arrilton, esses dois últimos me fizeram morrer de amores pela
psicologia evolucionista e etologia. O fascínio foi tão grande que me fez prestar um
concurso para área de etologia na UFPA, não fiquei em primeiro, fiquei em quarto.
Vocês não sabem a sensação maravilhosa que senti, um doutorando tirar 3ª melhor
nota na prova teórica e melhor nota na prova didática numa seleção com doze
biólogos pós-doc, incluindo um português formado pelo Instituto Max Planck de
biologia. Bernadete e Deborah, vocês são minhas referências, grande parte do
interesse e gosto pela endocrinologia comportamental vieram de vocês.
Alessandra, destruímos barreiras juntos, ainda lembro da sua batalha no
concurso para departamento de fisiologia, te vi chorar na sala junto com a Regina,
mas também de vi chorar de alegria ao passar no concurso e me ligar falando da
aprovação, e mencionando que nosso trabalho na iniciação científica foi de extrema
importância.
Profª Regina, você sempre foi temida, com pulso forte ou como você mesmo
falou citando seu pai “geniosa”. Contudo, acho que todos do LEME reconhecem e
te dão os créditos pelas conquistas. Te ver chorar naquela fatídica última reunião
do laboratório me fez enxergar a pessoa sensível e compromissada.
Aos amigos queridos do laboratório, Dudu, Lady Di, Sarah, Isabela, Raí,
Anderson, Priscila, Nanda, Diego, André, Ramon e Ronaldo, simplesmente vivi os
melhores dias da minha vida com vocês, nossos risos, conversas, trocas de
experiências, estudos, trabalhos e farras ajudaram a torna essa caminhada
divertida.
Profº Jeferson e Profª Rovena, obrigado pelo acolhimento, reconheço e
sempre irei agradecer por ter dado oportunidade de trabalharmos em conjunto, O
Lenq foi fator importante nesses últimos anos.
Aline, Carlos, Sara e André, esse trabalho também é de vocês, obrigado pela
ajuda e por ter acreditado nessa ideia.
Claudinho, meu companheiro, tudo isso só foi possível porque tenho você,
obrigado por existir na minha vida.
Agradeço imensamente aos demais colegas de convivência, ao demais
professores, técnicos e demais funcionários da UFRN.
As agências de fomento e instâncias governamentais que possibilitaram os
recursos financeiros para produção do conhecimento e sobrevivência do aluno de
pós-graduação. Pude vivenciar um período produtivo e dignificante na ciência
brasileira, mas também vi tudo desmoronar nos últimos anos, esses dois últimos
anos sentimos na pele a falta de investimento na ciência, dificuldades para
insumos, ferramentas e animais dificultaram muitos dos nossos trabalhos, inclusivo
do meu. O cenário atual é TEMERoso e o futuro assustador. Mas há de vir bons
tempos.
E por falar em bons tempos, observo um movimento acontecendo nas pós-
graduações, um movimento que se articula e luta pelo bem-estar e qualidade de
vida do aluno. Hoje a pós-graduação é uma máquina de destruir saúde mental, um
modelo obsoleto, engessado, sádico e totalmente produtivista. Eu pensava que isso
era uma realidade apenas brasileira, mas ao ler um relatório da OMS, eu percebi
que o cenário é mundial! Os mestrandos e doutorando são 6x mais propensos a
desenvolverem quadros de ansiedade e depressão. Desde o estudo publicado na
Nature “Under a cloud: Depression is rife among graduate students and postdocs”
em 2012, a OMS vem pontuando em seu relatório de saúde mental. Esse
movimento levou o Reino Unido a contabilizar entre 2015 e 2017 mais de 16 mil
estudantes da pós-graduação afastados por problemas mentais. Só no ano
passado mais de 137 alunos cometeram suicídios. Ironicamente, eu estudei a
relação de estresse e depressão, e no final do doutorado eu vivencie todo esse
sofrimento e percebi como o acumulo dessas experiências aversivas na pós me fez
adoecer. Apenas um estudo da UFRGS pode demonstrar como se encontra a
realidade brasileira. Infelizmente, sabemos que grande parte dos docentes e
programas fingem que nada acontecem, o máximo de entendimento que eles
conseguem abstrair é “quando vocês forem orientadores vão entender”. Aqui na
UFRN já é possível discutir e direcionar projetos que visam saúde mental na pós.
Por fim, fecho mais um clico para começar outro. O último ano foi bem difícil
para mim, cenário político econômico difícil, sem perspectivas, me senti por vezes
sozinho, a ida de Alessandra para São Paulo tornou as orientações difíceis, nos
víamos rapidamente em congressos ou bancas de defesas que ela participava aqui
em Natal, os demais colegas também tinham as mesmas angústias com seus
trabalhos, alguns se disponibilizavam em ajudar, outros simplesmente ignoravam.
Em situações escutei coisas do tipo “deveria procurar Bernadete, ela é a pessoa
que poderia te ajudar”. A indiferença nutria em mim um sentimento de insuficiência
e de descrença no meu trabalho, nos meus dados, na minha pergunta científica.
Agosto de 2017 acabou meu prazo final e bolsa, meu mundo desmoronou
sem saber o que fazer, havia passado em segundo lugar para docente de psicologia
na federal do Vale do São Francisco, com chances de ser chamado, mas ainda só
possiblidades. Consegui emprego em uma universidade privada e pude sentir um
pouco de alívio, trabalhei 40 horas semanais para poder ter um pouco de conforto,
era horista.
Só agora fui reconhecido pela instituição e passo a ser professor integral
com apenas 20h de horas na docência e demais com extensão e pesquisa. E por
falar em pesquisa, hoje assumo na Universidade Potiguar um laboratório de
pesquisa em desenvolvimento infantil. Não faço mais pesquisa básica, apenas
aplicada. Atualmente eu e mais 6 alunas (no momento só oriento meninas) estamos
investigando papel das funções executivas no manejo e estratégias de
enfretamento ao estresse em adolescentes infratores. Um outro grupo investiga a
relação de estresse e alterações emocionais nas fases inicias e finais da fase lútea
(utilizando o modelo de TSST), e isso associado a um modelo de treino cognitivo
para manejo da resposta de estresse. Um projeto previsto para iniciar próximo
semestre será focado em “Training based program” para crianças com dificuldades
de aprendizagem e transtornos de aprendizagem como dislexia e discalculias. Um
mesmo modelo de treinamento cognitivo será lançado para idosos com demências.
Ainda nesse semestre iniciarei aulas e orientações em dois programas de lato
sensu nas áreas de terapia cognitiva comportamental e neuropsicologia.
No mais, fica aqui meu registro tanto em forma de agradecimentos, críticas
e percepções sobre essa minha trajetória. Muitos podem me enxergar pequeno,
mas olho para o meu passado e me sinto um gigante.
SUMÁRIO
AGRADECIMENTOS .......................................................................................................... 7
LISTA DE FIGURAS E ILUSTRAÇÕES ............................................................................ 15
LISTA DE ABREVIATURAS E SIGLAS ............................................................................ 17
RESUMO ........................................................................................................................... 19
ABSTRACT ....................................................................................................................... 20
1. APRESENTAÇÃO ........................................................................................................ 18
2. INTRODUÇÃO .............................................................................................................. 21
2.1. A resposta de estresse ......................................................................................................... 21
2.2. Hormônios adrenais: Alterações cognitivas e emocionais associadas ao estresse ... 27
2.2. Hormônios sexuais e alterações cognitivas e emocionais.......................................... 33
3. HIPÓTESES .................................................................................................................. 37
4. OBJETIVO GERAL ....................................................................................................... 37
4.1. Objetivos específicos .................................................................................................. 37
PARTE I ............................................................................................................................. 38
Introdução ........................................................................................................................ 39
Artigo 1 .............................................................................................................................. 43
Abstract ........................................................................................................................................... 44
1.Introduction ................................................................................................................... 45
2.Material and methods ................................................................................................... 48
2.1. Animals .................................................................................................................................... 48
2.2. Drugs........................................................................................................................................ 48
2.3. Estrous Cycle .......................................................................................................................... 48
2.4. Stress induction protocol ............................................................................................ 49
2.5. Sucrose Splash Test (SST) .................................................................................................. 49
2.6. Social Interaction Test (SIT) ................................................................................................. 49
2.7. Sucrose Preference Test (SPT) ........................................................................................... 50
2.8. Experimental design ................................................................................................... 50
2.8.1. Experiment I: Influence of estrous cycle on the depressive-like behavior induced by the social stress ................................................................................................................. 51
2.8.2. Experiment II: Effects of the estradiol and tamoxifen on the depressive-like behavior induced by the social stress ................................................................................ 51
2.8.3. Experiment III: Effects of progesterone and mifepristone on the depressive-like behavior induced by the social stress ................................................................................ 52
2.9. Statistical analysis ...................................................................................................... 52
3. Results .......................................................................................................................... 53
3.1. Experiment I: Influence of estrous cycle on the depressive-like behavior induced by the social stress. ................................................................................................................ 53
3.1.1. Sucrose splash test (SST) ....................................................................................... 53
3.1.2 Social interaction test (SIT) ...................................................................................... 54
Sniffing Behavior ............................................................................................................................ 54
Following Behavior ........................................................................................................................ 54
Avoidance Behavior ...................................................................................................................... 54
3.1.3 Sucrose preference test (SPT) ................................................................................. 55
3.2. Experiment II: Effects of the estradiol and tamoxifen on the depressive-like behavior induced by the social stress. .............................................................................................. 56
3.2.1 Sucrose splash test (SST) ........................................................................................ 56
3.2.2 Social interaction test (SIT) ...................................................................................... 56
Sniffing behavior ................................................................................................................ 56
Following behavior ............................................................................................................. 57
Avoidance behavior ........................................................................................................... 57
3.2.3 Sucrose preference test (STP) ................................................................................. 58
3.3 Experiment III: Effects of the progesterone and mifepristone on the depressive-like behavior induced by the social stress. ............................................................................... 59
3.3.1 Sucrose splash test (SST) ........................................................................................ 59
3.3.2 Social interaction test (SIT) ...................................................................................... 60
Sniffing behavior ................................................................................................................ 60
Following behavior ............................................................................................................. 60
Avoidance behavior ........................................................................................................... 60
3.3.3 Sucrose preference test (SPT) ................................................................................. 61
4. Discussion ................................................................................................................... 61
5. Conclusion ................................................................................................................... 65
Acknowledgements ......................................................................................................... 65
6. References ................................................................................................................... 66
PARTE II ............................................................................................................................ 76
Introdução ........................................................................................................................ 77
Artigo 2 .............................................................................................................................. 82
Abstract ............................................................................................................................. 83
1.Introduction ........................................................................................................................ 84
2. Materials & Methods ........................................................................................................ 86
2.1. Animals ....................................................................................................................... 86
2.2. Drugs ....................................................................................................................................... 86
2.3. Estrous cycle ............................................................................................................. 87
2.4. General procedures and stress condition ................................................................... 87
2.5. Open Field ............................................................................................................................... 88
2.6. Plus-maze discriminative avoidance task (PMDAT) ................................................... 88
2.7. Drug administration ..................................................................................................... 89
2.8. Statistical Analysis ...................................................................................................... 89
3. Results .......................................................................................................................... 90
3.1. Anxiety and exploratory behavior on OF .................................................................... 90
3.1.1. Open field test: 17β-estradiol treatment .................................................................. 90
3.1.2. Open field test: Tamoxifen treatment ...................................................................... 90
3.1.3. Open field test: Progesterone treatment .................................................................. 90
3.1.4. Open field test: Mifepristone treatment .................................................................... 90
3.2. Learning and memory on PMDAT .............................................................................. 92
3.2.1. Plus-maze discriminative avoidance task: 17β-estradiol treatment ......................... 92
3.2.2. Plus-maze discriminative avoidance task: Tamoxifen treatment ............................. 92
3.2.3. Plus-maze discriminative avoidance task: Progesterone treatment ........................ 94
3.2.4. Plus-maze discriminative avoidance task: Mifepristone treatment .......................... 94
3.3: Anxiety and exploratory behavior .............................................................................. 94
3.3.1. Plus-maze discriminative avoidance task: 17β-estradiol treatment ......................... 94
3.3.2. Plus-maze discriminative avoidance task: Tamoxifen treatment ............................. 95
3.3.3. Plus-maze discriminative avoidance task: Progesterone treatment ........................ 95
3.3.4. Plus-maze discriminative avoidance task: Mifepristone treatment .......................... 96
4.Discussion .................................................................................................................... 97
5.Conclusion .................................................................................................................. 101
Acknowledgements ......................................................................................................... 102
6. References ................................................................................................................. 102
4. CONSIDERAÇÕES FINAIS ........................................................................................ 111
5. CONCLUSÃO .............................................................................................................. 113
REFERENCIAS ............................................................................................................... 114
ANEXO……………………....……………………………………............………..……...…...126
LISTA DE FIGURAS E ILUSTRAÇÕES
Figura 1: Eixo Hipotálamo-pituitária-adrenal (HPA) ...............................................................................................................................23
Figura 2: Diagrama de ativação da resposta de GC nos receptores de MR e GR..........................................................................................................................27
Figuras do artigo 1
Figure 1: Schematic representation of the experimental design……………...…..51
Figure 2: Effect estrous cycle on grooming behavior of socially stressed female rats. total time of grooming behavior and latency to start the grooming behavior……………………………………………………………………………...…...53
Figure 3: Effects of social isolation and estrous cycle on sucrose consumption in female rats. Percentage of total sucrose consumed into 24h………….....…………55
Figure 4: Effects of treatment 17β-estradiol (17β), tamoxifen (TAM) and vehicles (VEH) on grooming behavior of socially isolated female rats...................................56
Figure 5: Percentage of sucrose consumption by socially isolated females treated with estradiol (17β), tamoxifen (TAM) or vehicle (VEH) in diestrus, proestrus and estrus phases………………………………………………………………………………….…59
Figure 6: Effects of estrous cycle and treatment with progesterone (PROG), mifepristone (MIF) and vehicles (VEH) in the latency to start the grooming behavior in sucrose splash test……………………………………………………………………59
Figure 7: Effects of estrous cycle and treatment of progesterone (PROG), mifepristone (MIF) and vehicles (VEH) on sucrose consumption of social isolated female rats………………………………………………………………………………..61
TABELAS
Table 1: Effects of estrous cycle and social isolation in the social interaction test in rats..……………………………………………………………………………………….58
Table 2: Effects of estrous cycle (DIE, PRO and EST) and pharmacological treatment of 17β-estradiol (17β), tamoxifen (TAM) or vehicle (VEH) on social behavior of isolated female rats. Total number, time and latency were registered for sniffing, following and avoidance behavior in social interaction test…………….…55
Table 3: Effects of estrous cycle (DIE, PRO and EST) and pharmacological treatment of progesterone (PROG), mifepristone (MIF) or vehicle (VEH) in the social behavior of isolated female rats in social interaction test. Total number, time and latency were registered for sniffing, following and avoidance behavior…………….60
Figuras artigo 2
Figure 1: Schematic diagram of the behavioral plus maze discriminative avoidance task - PMDAT…………………………………………………………………………...89
Figure 2: Effects of social isolation (SI), Control condition (CT) and treatment of Estradiol (EST, A), Tamoxifen (TAM, B), Progesterone (PROG, C) and Mifepristone treatment (MIF, D) on learning and retrieval of female rats submitted to the plus- maze discriminative avoidance task (PMDAT)……………………………………………………………………….…………93
Figure 3: Influence of social isolation in the treatment and hormonal manipulation on distance travelled, Control condition (CT) in the treatment of Estradiol (EST, A), Tamoxifen (TAM, B), Progesterone (PROG, C), Mifepristone treatment (MIF, D) and respective vehicle (VEH) on females tested in the plus-maze discriminative avoidance task……………………………………………………………………….…..95
Figure 4: Influence of social isolation and treatment of Estradiol (17β, A), Tamoxifen (TAM, B), Progesterone (PROG, C), Mifepristone treatment (MIF, D) and respective vehicle (VEH) on open arms exploration (%OA) by females tested in the plus-maze discriminative avoidance task…………………………………………………………..96
TABELAS
Tabela 1: Effect of social isolation and hormonal treatment in anxiety and exploratory behavior………………………………………………………………….....91
LISTA DE ABREVIATURAS E SIGLAS
11β-HSD: 11β-dehidrogenase hidroxiesteroide
17β: 17beta-estradiol
5-HT: Serotonina ou 5-Hidroxitriptamina
ACTH: adrenocorticotropic hormone ou hormônio adreno-corticotrófico
ALLO: Allopregnanolone
AMG: Amígdala
AMPA: ácido proprióico α-amino-3-hidroxi-5-metil-4-isoxazole
AVP: vasopressina
BDNF: Fator neurotrófico derivado do cérebro - inglês brain derived neurotrophic factor)
BNST: bed nucleos of stria terminalis ou núcleo leitoso da estria terminal
CA1: Cornus de Amon 1
CA3: Cornus de Amon 3
CMS: Chronic mild stress ou estresse cronico moderado
CRH: corticotrophin releasing hormone ou fator liberador de corticotrofina.
CT: Grupo controle
DIE: diestro/metaestro
DO: Dopamina
ERK: quinases reguladoras de sinalização extracelular - inglês extracellular-signal-regulated-kinases)
EST: estro ou estrus
GABA: GAMMA-aminobutyric Acid ou Ácido gama-aminobutírico
GR: receptores de glicocorticóides
GCs: glicocorticoides
HPA: hipotálamo-pituitaria-adrenal
HPC: Hipocampo;
ISRS: Inibidor seletivo de recaptação de serotonina
LTP: potenciação de longa duração
mGC: receptores de glicocorticóides de membrana
MIF: mifepristone
mPFC ou CPFm: Córtex pre-frontal medial;
MpPVN: núcleo medial dorsal do paraventricular
mPVN: núcleo medial paraventricular do hipotálamo
MRs: mineralocorticoides
NE: noradrenalina
NMDA: N-metil-D-aspartato
NST: nucleos of solitary tract ou núcleo do trato solitário
OP: open field
periPVN: periVentricular do hipotálamo
PMDAT: Plus-maze discriminative avoidance task ou tarefa esquiva discriminativa em labirinto em cruz elevado (EDLCE),
PMDS: Premenstrual dysphoric syndrome
PRO: proestro ou proestrus
PROG: Progesterone
PVN: paraventricular do hipotálamo
SGA: Síndrome Geral de Adaptação
SI: Social isolation
SIT: SOcial interaction Test
SNC: Sistema Nervoso Central
SPT: Sucrose Preference Test
SST: Sucrose Splash Test
TAG: transtorno de ansiedade generalizada
TAM: tamoxifen
TEPT: transtorno do estresse pós-traumático.
TSST: Trier Social Stress Test
RESUMO
O estresse compreende um conjunto de alterações psicofisiológicas eliciadas no organismo diante de demandas intrínsecas e extrínsecas. A exposição frequente a eventos estressantes promove alterações em processos cognitivos, emocionais e comportamentais do indivíduo, e tais alterações podem estar relacionadas à etiologia de diversos transtornos psiquiátricos. Os glicocorticoides (GCs) são os principais hormônios mediadores da resposta de estresse. Estes se ligam a receptores que estão extensamente distribuídos em diversas células do organismo, incluindo ampla distribuição em todo sistema nervoso central (SNC), especialmente em áreas cerebrais como hipocampo, amígdala e córtex pré-frontal. Contudo, ainda não está completamente esclarecido como o estresse pode prejudicar o funcionamento dessas estruturas e de que maneira isto pode provocar o surgimento de transtornos psiquiátricos como, por exemplo, transtornos de humor (depressão) e ansiedade (transtorno de ansiedade generalizada). Em contrapartida, oscilações de hormônios sexuais, como o estradiol e progesterona influenciam o SNC modulando alguns processos diante a ação deletéria dos GCs. Neste sentido, utilizamos um modelo de indução de estresse crônico – isolamento social – em ratas jovens em diferentes fases do ciclo estral para investigar possíveis alterações nas respostas cognitivas, emocionais e comportamentais em resposta a exposição ao estressor. No intuito de investigarmos a influência dos hormônios sexuais utilizamos agonistas e antagonistas para induzir ou bloquear efeitos desses hormônios nas diferentes fases do ciclo. Nossos achados reúnem um corpo de dados que demonstram que o isolamento social induz comportamentos do tipo depressivo e promove prejuízos mnemônicos fase dependentes. Estes resultados foram observados quando ratas foram submetidas à uma tarefa de memória aversiva e aos testes de autocuidado, preferência por sacarose e interação social. De maneira geral, animais em fases de baixo perfil hormonal (diestro/metaestro, DIE) apresentaram comportamentos do tipo depressivo além de déficits de memória. Por outro lado, ratas em proestro (PRO) e estro (EST) não demonstraram prejuízos nos testes, não mostrando os efeitos deletérios provocados pelo estresse. O tratamento farmacológico mostrou um efeito dissociativo do estradiol e progesterona sobre os aspectos comportamentais e mnemônicos. Assim, o tamoxifeno (antagonista do estradiol) quando administrado nas fases PRO e EST prejudicou o desempenho dos animais levando a comportamentos do tipo depressivo e déficits de memória, mas não o antagonismo feito pela mifepristona (antagonista da progesterona). Além disso, a administração de estradiol na fase DIE, mas não progesterona, foi capaz de melhorar os prejuízos observados nessa fase. Em conclusão, estes achados sugerem que o isolamento social induz alterações cognitivas e emocionais em ratas, dependente das oscilações hormonais do ciclo estral, e esta modulação ocorre principalmente pela ação do estradiol, que é capaz de amenizar prejuízos comportamentais observados ao longo do ciclo. Palavras-chave: estradiol, progesterona, memória, ansiedade, depressão.
ABSTRACT
Stress can be defined as a set of physical and psychological mechanisms to coping disturbances that threat the homeostasis. Long-term exposure to stressful events is associated with negatives effect on cognition, emotion and behavior, moreover, these alterations are suggested to be in onset of psychiatric disorders. Glucocorticoids (GCs) are the main hormones involved in stress response. The glucocorticoids receptor (GR) are extensile expressed in different types of tissues in the body, including the central nervous system (CNS), especially in hippocampus, amygdala, and prefrontal cortex. These structures are involved in mood regulation, anxiety and mnemonic processes and are targets of the stress. Although, it remains controversial how stress can impair functionality in these brain areas and how it correlates with disturbances in mood, anxiety and memory impairment. In contrast, oscillations of sex hormones, such as estradiol and progesterone, can profoundly influence the CNS, acting against deleterious effects of stress. In this regard, we used an animal model of chronic stress - social isolation - in female rats with intact estrous cycle to investigate modulatory effect of sex steroid on cognitive and emotional responses. Moreover, we investigated the dissociative effect of estradiol and progesterone, using agonist or antagonist. Our main results demonstrated that: (1) females in diestrus showed depressive-like behaviors and memory deficits when compared to estrus and proestrus phases; (2) depressive-like behaviors and memory deficits induced by social isolation was estrous-cycle dependent; (3) stressed females treated with estradiol (but not progesterone) showed less depressive-like behaviors and memory impairments; (4) tamoxifen (but not mifepristone) induced depressive-like behaviors in proestrus females; (5) There was a anxiolytic effect induced by progesterone. Taken together, these findings suggest that endogenous variations of sex hormones are important to modulate mood, anxiety and mnemonic process in socially isolated females.
Key words: estradiol, progesterone, memory, anxiety, depression.
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1. APRESENTAÇÃO
A descoberta dos fisiologistas Flerko and Szentagothai (1957) de receptores
para hormônios esteroides em regiões cerebrais (hipotalâmicas) que medeiam
alguns aspectos da função reprodutiva e de sobrevivência, ampliou a definição de
“neuroendocrinologia” para incluir a comunicação recíproca entre o cérebro e o
corpo, por meio de vias hormonais e neurais. O cérebro é o órgão central da
adaptação aos fatores endógenos e exógenos porque percebe o que é ameaçador
ou necessário, gerando as respostas comportamentais e fisiológicas para ajustes a
essas demandas (McEwen et al, 2015). Desde os memoráveis achados de Claude
Bernard sobre milieu intérieur ainda no século XIX, passando pelos conceito de
homeostase e mecanismo de fight-or-fight propostos por Walter Cannon (1929); e
pela definição de estresse representada pela Síndrome Geral da Adaptação (SGA)
elucidado pelo fisiologista Hans Selye (1950), o campo da neuroendocrinologia
moderna tem trazido importantes informações. Evidências apontam que alças de
regulações neuroendócrinas são capazes de produzir um conjunto de alterações
que se iniciam a nível de processamento molecular até vias finais de respostas
cognitivas, emocionais e comportamentais, que foram conservadas evolutivamente
nos vertebrados (Denver, 2009).
O estresse provoca uma cascata de reações emergenciais em vias de
circuitos neurais que podem alterar a expressão de comportamentos e emoções.
Esse conjunto de respostas emergenciais, por sua vez, afeta a fisiologia sistêmica
por meio de efetores neuroendócrinos, autonômicos, imunológicos e metabólicos.
Em curto prazo, estas respostas geram um aumento da excitabilidade e do estado
de atenção frente a um ambiente ameaçador, essas mudanças adotam um padrão
de resposta adaptativa de alto valor biológico para sobrevivência. Mas, na ausência
de fatores de risco, se há um prolongamento dessas reações associado ao estado
comportamental alterado, tal resposta adaptativa passa a eliciar uma sobrecarga
nos sistemas fisiológicos resultando em padrões anormais de funcionamento e
desorganização, que levam consequentemente a respostas mal adaptativas
(McEwen et al, 2015). Muitas dessas alterações estar correlacionadas à
manifestação de transtornos psiquiátricos, como transtornos de humor e ansiedade,
com importantes diferenças sexuais (McEwen et al, 2015; Luine, 2014).
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Desde as primeiras evidências sobre a importância dos estrogênios em
roedores descritas por Charles Stockard e George Papanicolau (1917) até
posteriormente, seu isolamento de ovários de humanos (Allen & Doisy, 1923) e a
descoberta dos seus receptores (O’Malley et al, 1968; Green et al, 1986; Kuiper et
al, 1996) um grande corpo de evidências fisiológicas e bioquímicas mostra os
hormônios sexuais como moduladores da resposta ao estresse.
Estudos epidemiológicos têm demonstrado diferenças sexuais na
prevalência de doenças psiquiátricas. Segundo a Organização Mundial de Saúde
(2008) estima-se que o risco de adquirir algum tipo de transtorno de ansiedade e
humor é duas vezes maior em mulheres do que para os homens. Ainda, em 2030
a depressão será a doença mais presente na vida das mulheres, impactando
diretamente a saúde e qualidade de vida. Embora seja reconhecido que os fatores
socioeconômicos, estilo de vida, dieta, cultura e educação influenciem no
surgimento dos transtornos de ansiedade e depressão. Uma possível explicação
pode estar associada ao período reprodutivo da mulher. De fato, antes da
puberdade meninos e meninas apresentam baixas taxas de sintomas do tipo
depressivo, contudo o risco aumenta logo após a menarca nas meninas
(Cyranowski et al, 2000).
O início do período reprodutivo parece estar envolvido com as primeiras
manifestações dos sintomas de transtorno de humor. As mulheres nesse período
vivenciam intensas alterações emocionais devido às flutuações hormonais cíclicas.
Desta forma, o transtorno disfórico menstrual, a depressão pós-parto, a depressão
pós-menopausa estão diretamente relacionados com essas oscilações hormonais
(Soares et al, 2007). É sugerido que as alterações no humor observadas possam
ocorrer devido a modulação que os hormônios exercem no cérebro feminino,
especialmente a progesterona e o estradiol. De fato, os hormônios sexuais, em
especial o estradiol, tem um efeito neuroprotetor bem descrito. E, as manifestações
de sintomas depressivos parecem surgir em períodos quando esses hormônios
estão baixos, tais evidências são observadas tanto em roedores quanto em
primatas (Cohen et al, 2006; Luine, 2014). A razão pela qual esses hormônios são
capazes de modular processos emocionais e cognitivos ainda não está esclarecida,
contudo, acredita-se que as vias de ativação sejam tanto genômica como não-
genômica. Prévios estudos vêm mostrando que experiências estressantes são
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capazes de modular processos cognitivos e emocionais através dessas mesmas
vias moduladas por hormônios sexuais (Luine, 2014; Autry & Monteggia, 2012).
Assim, especula-se que a ausência do estrógeno poderia tornar a atividade cerebral
vulnerável à ação dos glicocorticoides envolvidos na resposta do estresse o que
poderia estar associado ao risco de desenvolvimento dos transtornos de ansiedade
e depressão. Tais achados permanecem inconclusivos e tem sido foco de pesquisa
no campo da neuroendocrinologia.
Diante disso, compreender a relação existente entre a atividade
neuroendócrina, os perfis de risco aos transtornos de ansiedade e depressão, bem
como a modulação desses hormônios nas respostas psicofisiológicas diante de
fatores estressantes é importante e desafiador.
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2. INTRODUÇÃO
2.1. A resposta de estresse
Todos os seres vivos necessitam, para sua sobrevivência, manter certa
constância de seu meio interno em relação às demandas extrínsecas que incidem
sobre o organismo - esse conceito é denominado de homeostase (Canon, 1932).
Fatores intrínsecos e/ou extrínsecos afetam os seres vivos alterando os padrões
complexos e dinâmicos do equilíbrio homeostático, desencadeando alterações no
organismo. Baseado neste conceito de equilíbrio interno, alguns fisiologistas
estudaram como os organismos reagiriam a situações adversas que pudessem
desafiar a condição homeostática.
O pesquisador Hans Selye observou o comportamento de ratos que
apresentavam respostas fisiológicas inespecíficas a diferentes tipos de agentes
nocivos. Em seus experimentos foram observados que, independente dos
estímulos, os animais apresentavam aumento do tamanho das glândulas adrenais,
atrofia do timo dentre outros órgãos linfoides, aparecimento de úlceras no trato
gastrintestinal, grande perda de peso, retardo no crescimento, supressão da
atividade sexual, assim como redução e perda de tecido muscular (Selye, 1936).
Ainda, Selye observou que a exposição crônica a agentes nocivos podia levar à
exaustão e morte dos animais. Assim, foi considerado que o estresse seria uma
situação gerada por um desafio ao qual o organismo estaria submetido. Desta
forma, o agente causador (estímulo desencadeador) seria denominado de agente
nocivo, e a “Síndrome Geral da Adaptação (SGA) ” seria a condição provocada no
organismo na presença deste agente. Com o avanço de seus estudos, Selye
descreveu que a SAG estaria associada a três fases distintas. O primeiro momento
estaria relacionado com uma fase de alarme, na qual o organismo estaria em um
estado de alerta diante de um estressor; posteriormente, na fase de resistência ou
adaptação, o corpo começa a promover os ajustes necessários para compensar os
estímulos causadores de estresse. E, por fim, com a persistência do estressor o
organismo entra num estado de esgotamento, uma vez que a capacidade
adaptativa do organismo em promover os ajustes necessários para o enfretamento
do estressor se torna menos eficiente (Selye, 1950).
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O principal sistema hormonal de resposta ao estresse é o eixo Hipotálamo-
pituitária-adrenal (HPA) (Chrousos & Gold, 1992). Assim, numa situação de
estresse, neurônios parvocelulares do núcleo paraventricular do hipotálamo (PVN)
recebem inputs de vias sensoriais, nociceptores somáticos, aferentes viscerais,
vias sensoriais humorais e aferentes do tronco encefálico, bem como regiões do
sistema límbico, envolvidas com transmissão noradrenérgica e adrenérgica. Estes
estímulos promovem a liberação do neuropeptídio fator liberador de corticotrofina
(CRH) através de projeções axonais através da haste hipofisária, que se dirigem à
rede de capilares presentes na eminência mediana do hipotálamo, pertencente ao
sistema porta-hipofisário (revisado por, Herman & Cullinan, 1997). Assim, a maioria
dos neurônios neurossecretores do PVN são posicionados para desempenhar
atividade excitatória capaz de rápida ativação do eixo HPA.
Cabe ressaltar, que a ativação do eixo HPA também envolve a expressão da
vasopressina (AVP), esses neuropeptídios são produzidos em neurônios
parvocelulares co-localizados aos neurônios secretores de CRH, sendo atribuídos
à resposta de estresses físicos, enquanto que o CRH parece estar relacionado aos
estresses psicológicos (Ramos et al, 2006; Ramos et al, 2016). A ação do CRH
sobre os receptores do fator de liberação de corticortrofina, e do AVP no receptor
AVP1B, presente em células basófilas da porção anterior da hipófise, promovem a
secreção do hormônio adrenocorticotrófico (ACTH), que por sua vez atua nas
glândulas adrenais, estimulando as células da região cortical a liberar os
glicocorticoides (GCs, cortisol em humanos e corticosterona em roedores),
resultando na complexa resposta ao estresse (Herman et al, 2003; Ulrich Lai &
Herman, 2009; Joels & Baram, 2009).
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Figura 1. Eixo Hipotálamo-pituitária-adrenal (HPA). Ativação do núcleo paraventricular do hipotálamo induz liberação de AVP e CRH, que age sequencialmente na liberação do hormônio adrenocorticotrófico, este irá induzir a liberação de cortisol no organismo através das adrenais (adaptado de Lupien et al, 2009).
Os CGs também têm papel regulatório em relação ao estado de vigília e no
metabolismo catabólico de lipídios e proteínas (Elverson et al 2005; Christiansen et
al 2007).
A ativação do eixo HPA é inibida pela alça de retroalimentação negativa.
Assim, o aumento de glicocorticoides no sangue é rapidamente detectado por
neurônios neurossecretores do PVN no hipotálamo. Os GCs inibem a produção de
CRH através da supressão da atividade excitatória desses neurônios - resposta de
retroalimentação rápida. Através de mecanismo retroalimentação tardio via
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genômica de ativação de receptores de GRs no encéfalo (Herman & Cullinan,
1997). É importante ressaltar que o PVN do hipotálamo recebe diferentes
aferências excitatórias e inibitórias, sendo a aferência inibitória modulada pela
atividade do neurotransmissor ácido gama-aminobutírico (GABA) em diferentes
núcleos do hipotálamo envolvidos na regulação homeostática (Herman et al, 1993;
Roland e Sawchenko, 1993). Nesse sentido, é sugerido que essa ativação inibitória
gabaérgica contribui também para a regulação da expressão dos GCs no
organismo.
Embora a modulação da resposta do estresse seja feita pelo eixo HPA,
outras estruturas encefálicas têm papel fundamental na modulação desta resposta.
De fato, a amígdala, hipocampo e o córtex pré-frontal constituem três áreas extra-
hipotalâmicas envolvidas na regulação da resposta ao estresse (Lupien et al, 2009).
A amigdala (AMG) é uma estrutura integrante do sistema límbico
responsável pela produção da resposta emocional, estando relacionada a
ansiedade, estados de alerta e ativação simpática frente ao perigo. Essa estrutura
recebe aferentes de áreas do tronco encefálico como o núcleo do trato solitário
(nucleos of solitary tract, NST), relacionado com o monitoramento da atividade do
sistema nervoso autônomo (Kerfoot et al 2008), bem como do núcleo leito da estria
terminal (bed nucleos of stria terminalis, BNST), que projeta tanto vias excitatórias
e inibitórias para o PVN quanto para a AMG, sendo capaz de modular mecanismos
autônomos e comportamentais perante estímulos aversivos (Walker et al, 2002).
As vias eferentes do sistema límbico como da AMG têm como alvo neurônios
GABAérgicos em núcleos circunvizinhos ao PVN, como o periventricular (PeriPVN)
e medial dorsal (MpPVN). Assim, estes núcleos podem permanecer sobre inibição
tônica. A modulação da AMG pode ocorrer pela desinibição dos neurônios
neurosecretorres de CRH no PVN, sendo a ativação do HPA auxiliada pela
desinibição, tendo em vista que maioria da atividade modulatória da AMG (por
exemplo, dos núcleos central e medial) é mediada por projeções GABAérgicas
tanto nas áreas circunvizinhas do PVN (medial e periventricular) quanto no BNST
(revisado por Herman et al, 2006)
Algumas áreas corticais também participam da regulação do eixo HPA. A
área pré-frontal medial do córtex (mPFC) tem papel importante na redução da
ativação autonômica, hormonal e comportamental da resposta ao estresse
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(Herman & Cullinan, 1997). A modulação do mPFC sobre o PVN ocorre
indiretamente através de projeções para estruturas diencefálicas, como o BNST,
que por sua vez emite projeções para áreas límbicas e então partem em tratos que
se juntam às áreas de integração modais do tronco cerebral, possibilitando a
regulação da atividade dos núcleos hipotalâmicos (Spencer et al., 2005)
O hipocampo é uma estrutura límbica composta por três áreas principais: as
regiões conhecidas como Cornos de Amon, CA1, CA2 e CA3; e pelo giro denteado
(Amaral & Witter, 1989). Ademais, estruturas circunvizinhas (córtex entorrinal,
subículo, pré-subículo e para-subículo) compõem a formação hipocampal (Amaral
& Witter, 1989). Estas estruturas estão relacionadas com diversos processos
cognitivos como a memória declarativa. A parte ventral do hipocampo também se
conecta ao hipotálamo e amígdala, sugerindo que essa estrutura converge
informações que são necessárias para o processamento de memórias de conteúdo
emocional e resposta ao estresse (Revisado por Komorowski et al, 2013; Fanselow
& Dong 2009).
A capacidade de convergir e modular informações no hipocampo somente é
possível devido à intensa atividade de neuroplasticidade encontrada nessa
estrutura (Pittenger & Duman, 2008). De fato, diversos estudos demonstraram que
a interação ambiental e aquisição de novas informações promovem modificações
morfofuncionais em diversas regiões do hipocampo (Kumar, 2011; Carasatorre &
Cintra, 2016; Gomez-Pinilla & Kesslak, 2001). O fenômeno de neuroplasticidade
compreende a capacidade que o cérebro tem em reorganizar circuitos neuronais
envolvidos na aquisição de novas experiências e informações promovidas por
pressões ambientais (Pascual-Leone et al, 2005). No hipocampo, os processos de
neuroplasticidade parecem estar diretamente envolvidos nos mecanismos
moleculares de formação de novas memórias (Lynch, 2003; Alonso et al, 2005).
Dentre esses mecanismos, é sugerido que o fenômeno de potenciação de longa
duração (LTP) (Bliss & Lomo, 1973; Whitlock et al, 2006; Malenka & Bear, 2004).
Neste sentido, como já mencionado, é visto que modificações no hipocampo podem
comprometer as circuitarias e mecanismos moleculares mnemônicos, o que
acarretaria prejuízos cognitivos e alterações comportamentais (Lynch, 2003;
Driscoll et al, 2003).
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Por outro lado, a grande concentração de receptores de GRs e de
mineralocorticoides (MRs) no hipocampo sugere que essa região seja diretamente
susceptível aos efeitos destes hormônios (McEwen, 2000; Lupien et al, 2007; Kim
et al, 2007; Conrad, 2008; Leuner & Gould, 2010; Herman et al, 1989). os GCs se
ligam aos dois tipos de receptores, contudo o controle da ligação dos GCs ao
receptor MR é controlado pela atividade da enzima - 11β-dehidrogenase
hidroxiesteroide (11β-HSD) que converte o GC ativo em cortisona inativa, tal efeito
garante que a aldosterona se ligue ao receptor MR, pois as concentrações
sanguíneas dos GC são 2000 vezes maiores do que aldosterona. No hipocampo
ocorre um efeito de saturação, pois há uma expressão reduzida da proteína 11β-
HSD, sendo assim, GCs se ligam com uma afinidade 100 vezes maior aos
receptores do tipo MR, e só irá se ligar ao GCR quando saturar todos os receptores
de MR (John & Funder, 1997; De Kloet, 2004).
Consequentemente, os GRs requerem uma maior concentração de
hormônio para serem ativados, algo que ocorre durante a resposta ao estresse (Fig.
2). Assim, na exposição a uma situação de estresse agudo, os GCs mantêm a
ativação de seus receptores promovendo um efeito de excitabilidade nos neurônios
do giro denteado, seguido também de um aumento no disparo de neurônios na
região CA1 do hipocampo. Este efeito tem uma ação benéfica nos processos de
atenção, excitabilidade e memória, como por exemplo, promovendo uma melhora
no processo LTP (De Kloet, 2004).
Por outro lado, a exposição crônica a GCs promove um efeito deletério
devido à ativação de GCRs localizados em CA3, promovendo redução da
arborização dendrítica, alterações nas espinhas dendríticas, na formação de LTP e
induzindo padrões de depressão de longa duração – (LTD) (Conrad, 2008; Kim et
al, 2015). Ademais, a ação crônica dos GCs promove a morte de neurônios, e essa
ação parece contribuir para uma maior atividade do eixo HPA (Herman et al, 2003;
Ulrich Lai & Herman, 2009; Joels & Baram, 2009). Os GCRs nucleares distribuídos
no hipocampo são sensíveis a concentrações elevadas de GCs, e isto resulta numa
alça de retroalimentação negativa (Ulrich Lai & Herman, 2009; Lupien et al, 2009).
De fato, uma hipótese é que essa capacidade de inibir o eixo HPA poderia ser
explicada pelas projeções oriundas da região subicular do hipocampo, estimulando
neurônios GABAérgicos situados no núcleo do leito da estria terminal a inibir a
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secreção de CRH nos neurônios do núcleo paraventricular (Herman et al, 2003;
Forray & Gysling, 2004).
Figura 2. Diagrama de ativação da resposta de GC nos receptores de MR e GR (Adaptado de Yau & Seckl, 2012). À nível basal dos GC é possível observar a taxa de ligação aos MR (a coloração em cinza significa nível de saturação do receptor). Após exposição ao estresse é possível observar a saturação dos MR e começo da ativação dos GR (símbolo com barras verticais).
2.2. Hormônios adrenais: Alterações cognitivas e emocionais associadas ao
estresse
Experiências estressantes podem estar relacionadas ao surgimento de
sintomas depressivos ou agravar estados de humor alterados. Segundo DSM-V
(2013) os transtornos de humor ou ansiedade são constituídos de quadros
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nosográficos onde as respostas da expressão e regulação do humor (motivação,
interesse, euforia, distimia, etc.); e ansiedade (excitação, medo, neuroticismo,
alerta) estão disfuncionais, gerando padrões de respostas comportamentais
atípicas com comprometimento na adaptação funcional. Além disso, sabe-se que
os efeitos negativos dos estressores tem impacto direto em estruturas do sistema
límbico que regulam e controlam a expressão das emoções (Lojko et al, 2014).
Os GCs claramente desempenham um papel importante na manutenção da
resposta do estresse. No entanto, a resposta ao estresse é mais complexa,
envolvendo diferentes alças de regulação e crosstalk entre diferentes sistemas. As
respostas fisiológicas mais rápidas aos estressores envolvem a ativação do sistema
nervoso simpático e a liberação de adrenalina, que medeia as alterações
simpaticomiméticas, como as alterações cardiovasculares, broncodilatação e
midríase. Isto é seguido pela ativação do eixo HPA, sendo a alça neuroendócrina a
principal da resposta. À nível emocional, cognitivo e comportamental a ação da
atividade simpática e a resposta inicial ao estresse promovem melhorias no sentido
de aumentar o estado de alerta, vigilância, estimulação cognitiva e robustez na
resposta comportamental. De fato, evidências tem demonstrando uma curva de U
invertido, onde baixas doses de peptídeos como adrenalina, cortisol e
noradrenalina ou altas doses desses hormônios comprometem o desempenho de
humanos e roedores avaliados em tarefas de aprendizagem e memória, havendo
assim um nível ótimo necessário para o bom desempenho comportamental (Salehi
et al, 2010).
Há evidências de uma relação entre estresse e depressão que são relatadas
principalmente de duas linhas de investigação: primeiro, disfunções da ativação
simpática e do eixo HPA correlacionam com pacientes deprimidos e/ou transtornos
associados a ansiedade e adaptação ao estresse; segundo, a exposição à eventos
estressantes é correlacionado com a incidência de depressão tanto em modelos
animais como em relatos clínicas (revisado por Schneiderman et al, 2005) .
No que tange a resposta simpática na etiologia dos transtornos de humor e
ansiedade, é visto que o sistema nervoso autônomo é uma das principais vias
neurais hiperativadas pelo estresse. Estudos clínicos associam transtornos de
humor, como o transtorno depressivo maior, à ativação da resposta simpática, onde
é visto uma resposta contínua ativada sem a ativação do sistema nervoso
parassimpático (Revisado por Won & Kim, 2016). Em um estudo recente Mandy e
29
colaboradores (2016) descreveram o aumento da atividade simpática, visto na
variabilidade de frequência cardíaca, em pacientes diagnosticados com depressão
maior e transtorno de ansiedade generalizada. Esse estudo ainda mostrou a
correlação entre hiperatividade da resposta simpática e risco de doenças cardíacas.
Ademais, outros estudos corroboram esse achado ao demonstrar que transtornos
de ansiedade e depressão são fatores de risco para doenças cardiovasculares
(Dhar & Barton, 2016; Hare et al, 2014).
A desregulação do eixo HPA é observado em pacientes homens com
depressão maior, indicado por hipercortisolismo na resposta ao despertar, além de
demonstrar maior sensibilidade na ativação do eixo no modelo de indução de
estresse em humanos – Trier Social Stress Test (Brooks & Robles, 2009). Também
é visto um efeito similar da elevação dos GCs, adrenalina, noradrenalina, em
pacientes com depressão distímica (Anisman et al, 1999) e transtornos bipolar
(Manenschijn et al, 2012), ainda esse último estudo demonstrou o aumento do
cortisol ao longo da vida do paciente e a relação direta entre níveis elevados de
cortisol e o surgimento de comorbidades com outras condições psiquiátricas. A
desregulação do eixo HPA pode incluir alterações tanto na atividade basal quanto
na resposta do cortisol ao despertar (Para revisão, Dedovic & Ngiam, 2015), além
de falha na ativação do feedback negativo do eixo HPA, tais como falha no teste de
supressão do eixo com dexametasona (Konstantion et al, 2008). Alguns estudos
indicam que pacientes deprimidos têm níveis elevados na resposta basal do cortisol
(Bremmer et al 2007; Oldehinkel et al 2001), mas outro estudo não relata tais
alterações (Peeters et al, 2003). Kathryn & Brooks (2009) demonstraram não haver
correlação entre auto relatos de pacientes e sintomas depressivos e aumento do
cortisol salivar. Nesse sentido, observamos estudos com inclusão mais específicas
de grupos com condições mórbidas, estado de hospitalização, e idade avançada,
esses indivíduos diagnosticados com depressão que tem maiores incidências de
níveis médios de cortisol elevados em comparação com indivíduos recém
diagnosticados com depressão (Ahmed et al, 2015; Moica et al,2016; Poole et al,
2016). Em modelos animais os dados encontrados são consistentes com as
evidências clínicas. Um estudo demonstrou uma hipersensibilidade do eixo HPA
com aumento de corticosterona em camundongos submetidos ao estresse de
derrota social, e também foi observado um aumento de imobilidade na tarefa de
nado forçado e aumento da anedonia, bem como redução de motivação para uma
30
tarefa de estimulo apetitivo (Pérez-Tejada et al, 2013). Em outro estudo ratas
estressadas, após sucessivas exposições a um ruído durante 40 minutos, falharam
na inibição do eixo HPA com uma dose de dexametasona, por outro lado, ratas
expostas por uma única sessão eram capazes de reduzir atividade do eixo
(Andrews et al, 2012). Contudo, um estudo demonstrou que administração crônica
de corticosterona não eliciou comportamentos do tipo depressivos em
camundongos (Sturm et al, 2015). Além disso, outra evidência relaciona
hipoatividade do eixo HPA aos comportamentos do tipo depressivos em um modelo
de pressão atípica em ratos, onde apenas há alterações na ansiedade, fadiga,
redução atencional e aumento atividade psicomotora (Bowens et al, 2011)
Os níveis elevados de GCs parecem participar de quadros de depressão
profunda associadas à melancolia. A depressão melancólica é, na verdade, um
estado de hiperatividade e cronicidade patológica nos transtornos de humor. Suas
manifestações psicológicas são ansiedade intensa; sentimentos de inutilidade;
ruminação de pensamentos, memórias congruentes ao humor, desamparo
aprendido; estados de ideação suicida e embotamento emocional (Gold &
Chrousos, 1999). A depressão melancólica grave tem sido associada a níveis
frequentemente elevados de cortisol (Carrol, 1982; Davidson et al, 1984). Suas
manifestações fisiológicas incluem hiperatividade de resposta simpática,
hipercortisolismo, supressão do hormônio de crescimento e dos eixos reprodutivos,
alterações na ritmicidade circadiana. A melancolia é maior no início da manhã e
parece correlacionar a alteração na resposta do cortisol ao despertar (revisado por
Dedovic & Ngiam, 2015).
Embora evidencias apontem a relação dos transtornos de humor e
anormalidades do eixo HPA, sabe-se que o eixo também pode influenciar a
atividade neural através da modulação de vias de projeções envolvidas com outros
neuromoduladores, como os sistemas de neurotransmissores. O sistema
monoaminérgico é considerado o sistema mais importante na neurobiologia da
depressão. É visto que a resposta ao estresse envolve uma ativação coordenada
de sistemas fisiológicos, incluindo a ativação do eixo HPA e outros mecanismos
envolvidos na transmissão e sinalização de neurotransmissores (Revisado por Lai
et al, 2003).
A formação reticular através de vias noradrenérgicas tem um papel
importante nos processos de atenção, sono/vigília, aprendizagem/memória,
31
emoção, alerta e ativação sinérgica na resposta ao estresse (Berridge et al, 2012).
Estudos têm apoiado o envolvimento funcional do locus coeruleus, principal área
envolvida na produção de NA, na regulação do eixo de estresse da HPA, a
estimulação elétrica ou farmacológica dessa área aumenta os estados de alerta
associado a ameaça (Para revisão Sved et al, 2002). Além do mais, projeções
noradrenergicas que partem para áreas frontais promovem maior ativação dessas
áreas e participam da regulação inibitória da resposta emocional. Falhas nessas
projeções parecem aumentar os sintomas depressivos e comprometem estratégias
de enfrentamento do estresse (Revisado por Moret & Briley, 2011). Além disso, as
classes de antidepressivos de terceira geração utilizam os mecanismos de ação da
noradrenalina e dopamina para reduzir comportamentos depressivos.
A neurotransmissão serotoninérgica compreende um conjunto de projeções,
também pertencente a formação reticular, que se inicia no tronco cerebral e se
difundi para áreas do sistema límbico e sistemas corticais (Hensler, 2006; Strac et
al, 2016). Dada à dimensão de alcance dessas projeções no SNC é visto que as
mesmas participam do surgimento e manutenção da resposta emocional e
diferentes comportamentos (ansiedade, impulsividade, agressividade, motivação,
comportamento sexual, alimentação). Alterações na expressão da serotonina estão
associadas com transtornos neuropsiquiátricos (transtornos de humor, desordens
da personalidade, alimentares, ansiedade e esquizofrenia). Nos transtornos de
humor, a serotonina tem papel importante na etiologia devido à: (1) desregulação
da resposta de humor mediada pela serotonina; (2) grande parte das intervenções
farmacológicas nos transtornos de humor e ansiedade utilizam mecanismos de
ação da serotonina como no caso dos inibidores seletivos da receptação da
serotonina (ISRS).
Prévios estudos mostraram uma relação entre estresse e depressão via
transmissão glutamatérgica, onde falhas na recaptação do glutamato e redução da
expressão glial promovem aumento da resposta glutamatérgica com efeitos de
excitotoxicidade, gerando potencial risco de dano e morte neuronal em células
hipocampais (kaufer 2017; Pearson-Leary et al, 2016). Além disso, a exposição ao
estresse crônico alterações na transmissão glutamatérgica como falhas na
expressão dos receptores AMPA e subunidades do receptor NMDA, e isso diminui
aspectos de neuroplasticidade sináptica. De fato, os receptores de AMPA e NMDA
estão co-localizados em diferentes neurônios do hipocampo. A ativação de AMPA
32
possibilita a entrada de cálcio na célula através do receptor AMPA, que por sua vez
vai promover a síntese de fatores neurotróficos, como o BDNF. A ligação de BDNF
ao receptor TrKB possibilita a expressão de AMPA que vai ser responsável pela
regulação da plasticidade neuronal com efeitos nos processos sinápticos da
potenciação de longa duração. Além disso, a expressão de BDNF vai regular a
formação de dendritos e aumento da densidade de espinhas sinápticas,
possibilitando ganhos em termos de processamento sináptico. Já os receptores
mGlu participam na regulação da expressão de receptores AMPA e NMDA e na
regulação da liberação de outros neurotransmissores (Para revisão Popoli et al,
2011).
Vários estudos apoiam a “hipótese neurotrófica da depressão” que postula
que os níveis cerebrais reduzidos de BDNF podem contribuir com a atrofia e perda
neuronal, como observado no hipocampo de modelos animais e em indivíduos
deprimidos (Revisado por Duman & Nanxili, 2012). No entanto, o mecanismo
subjacente dos efeitos genômicas do estresse sobre atividade neurotrófica não foi
totalmente compreendido.
33
2.2. Hormônios sexuais e alterações cognitivas e emocionais
Muitas pesquisas têm investigado as diferenças sexuais na resposta ao
estresse (para revisão, Kudielka & Kirschbaum, 2005) e a influência dos hormônios
sexuais. Por exemplo, ratas sob influência de estrógenos demonstram melhora no
desempenho prejudicado devido à exposição a agentes estressores ou até mesmo
mostram respostas comportamentais diferentes quando comparadas com fêmeas
com baixos níveis de estrógenos (Conrad et al, 2004). Em geral, nos roedores o
ciclo estral tem duração de 4 a 5 dias e neste período há uma flutuação dos níveis
sanguíneos dos hormônios gonadais que prepara o organismo para a reprodução.
Entretanto, é sugerido que a ação destes hormônios vai além da atividade sexual e
podem modular respostas comportamentais em fêmeas, como por exemplo, a
adoção de diferentes estratégias para a realização de tarefas que envolvem
processos de memória e aprendizagem (Conrad et al, 2004; Blokland et al, 2006).
De fato, o estradiol parece ter um efeito principalmente nas tarefas dependentes do
hipocampo. Por exemplo, melhorias induzidas pelo estradiol parecem ser mais
robustas em tarefas espaciais, especialmente aquelas que requerem o uso de
pistas (Frye et al, 2005). Outras formas de memória também podem ser melhoradas
após o tratamento com estradiol. Por exemplo, durante a tarefa de memória de
trabalho em labirinto T, o animal precisa lembrar o braço numa tarefa de alternância,
uma de memória de trabalho (Daniel & Dohanich, 2011), como também lembra da
informação 24h depois no modelo de memória com recompensa apetitosa (Hussain
et al, 2013). Experimentos utilizando o labirinto aquático de Morris, o labirinto em T
e labirinto radial demonstraram melhorias no desempenho de ratos tratados com
estradiol (Frye et al., 2005; Daniel et al, 1997; Luine e Rodriguez, 1994; Mclure, et
al, 2013).
As regiões encefálicas apresentam uma grande distribuição de receptores
para hormônios esteroides. É sugerido que esses hormônios podem modificar
circuitos neurais, o que resulta em alterações na neuroplasticidade, influenciando
alterações morfológicas e sinaptogênese em regiões como hipocampo e córtex pré-
frontal em modelos animais e humanos (Para revisão, Srivastava et al, 2013;
Catenaccio et al, 2016). Ademais, também tem sido descrito que há diferenças
sexuais na ligação dos hormônios esteroides e seus receptores, mesmo porque
34
fêmeas tem maior concentração basal de corticosterona do que machos (Conrad
et al, 2004), bem como diferentes níveis de excitação de neurônios hipocampais e
da amígdala (Figueiredo et al, 2002).
Os efeitos prejudiciais da exposição a diversos tipos de estressores podem
ser modificados pela ação neuroprotetora dos estrogênos, resultando na melhora
no desempenho comportamental de fêmeas. Esta hipótese tem sido fortalecida
devido a estudos que demonstram que fêmeas tratadas com estrógenos
apresentam aumento da expressão de proteínas relacionadas à sinaptogênese e
maturação das espinhas dendríticas (Srivastava et al), além do fortalecimento de
LTP em processos mnemônicos (Kramar et al, 2009). Ainda, há estudos mostrando
que os estrógenos são capazes de promover ações benéficas e protetoras contra
diversos tipos doenças neurodegenerativas, inclusive melhorando danos em
diversas estruturas cerebrais e prejuízos nos processos cognitivos (Revisado,
Luine, 2014).
Nesse contexto, Beck e Luine (2002) observaram o efeito de neuroproteção
do estradiol em relação ao estresse por contenção em diferentes tipos de tarefas
de memória espacial em ratas. Outro estudo mostrou que ratas ovariectomizadas
e estressadas por contenção, ao receberem tratamento crônico de estradiol,
apresentaram um melhor desempenho no labirinto radial e aumento de
noradrenalina em células hipocampais, promovendo assim uma maior
excitabilidade nos neurônios da região CA3, sugerindo que o estradiol modula o
efeito do estresse nos processos de memória e ansiedade através de efeitos
ativacionais e organizacionais (Bowman et al, 2003).
Além do mais, as ações genômicas destes hormônios parecem contribuir
para o aumento na expressão de diversos fatores neurotróficos, especialmente o
BDNF. Estudos indicam que o aumento de BDNF promovido pelo estradiol exerce
função neuroprotetora em relação à ação dos glicocorticoides, estresse oxidativo e
de doenças neurodegenerativas, além de promover efeito neuroprotetor contra
déficits cognitivos induzidos por estresse (Brann et al, 2007; Herrera & Mather,
2015).
Alguns relatos apontam que a ação neuroprotetora de fatores neurotroficos,
como o BDNF, é maior durante as fases proestro e estro do ciclo estral de ratas
(Scharfman et al, 2003, 2007). Ainda, como já mencionado, diversos estudos
35
apontam que prejuízos cognitivos induzidos pelo estresse estejam relacionados
com a diminuição de fatores neurotróficos, neurogênese e morte celular, mas que
durante as fases proestro e estro esses prejuízos cognitivos seriam ausentes ( Para
revisão, Frick et al, 2015; Horst et al, 2012).
A ação dos estrógenos pode promover alterações emocionais e sintomas do
tipo depressivo (Douma et al, 2005). Já foi reportado que a administração aguda de
tamoxifeno, um antagonista seletivo de receptor de estrógeno bastante conhecido,
pode promover o surgimento de sintomas depressivos em mulheres, mas a
administração de um antidepressivo pode amenizar esses sintomas (Bourque et al,
2009). Ainda, a redução dos níveis de estradiol em fases distintas do ciclo
reprodutivo da mulher é acompanhada com alterações no humor e sintomas
depressivos, como visto nos períodos de menopausa, peri-menopausa, pós-parto
e logo após o período de ovulação (Douma et al, 2005).
Alguns transtornos emocionais em mulheres estão diretamente associados
com alterações nos níveis de estradiol. Na síndrome pré-menstrual (SPM),
mudanças no ciclo menstrual são relatadas por alterações emocionais e cognitivas
reportadas por mulheres em idade reprodutiva. A SPM compreende uma variedade
de sintomas físicos, emocionais e psicológicos que inicia após a ovulação e termina
no começo da fase menstrual. Embora a causa da SPM ainda permaneça
desconhecida, algumas evidências têm impulsionado a redução abrupta dos níveis
de estradiol e ausência de progesterona, que pode ocorrer num intervalo entre o
final da fase folicular e início na fase lútea (Potter et al, 2009; Nillni et al, 2011; Tolga
& Young, 2015).
Em modelos animais para investigar depressão, alguns estudos mostram
que a administração aguda de estradiol é capaz de reduzir comportamento do tipo
ansioso e depressivo em camundongos idosos e que durante as fases de baixa
circulação de estradiol (fases metadiestro e diestro) do ciclo estral induz
manifestações de comportamentos do tipo depressivos (Horst et al, 2012; Walf &
Frye, 2010). Recentemente, em nosso laboratório investigamos o papel do ciclo
estral de fêmeas estressadas sobre uma tarefa de memória hipocampo-
dependente, a esquiva discriminativa em labirinto em cruz elevado. Neste estudo,
nós demonstramos que a indução crônica de estresse por contenção e isolamento
social promove prejuízos no processo de consolidação/evocação de uma nova
memória. Contudo, tal déficit não ocorre quando as fêmeas aprendem a tarefa
36
durante as fases proestro e estro, ou seja, na presença de altas concentrações de
hormônios sexuais (Dados não publicados).
37
3. HIPOTÉSES
Diante do exposto, esse trabalho baseia-se na investigação da influência dos
hormônios sexuais femininos sobre os aspectos emocionais, mnemônicos e
comportamentais de ratas socialmente isoladas, considerando as seguintes
hipóteses:
O modelo crônico de isolamento social pode induzir comportamentos do tipo
depressivo e déficits de aprendizagem/memória em ratas.
As oscilações hormonais ao longo do ciclo estral de ratas diminuem ou
agravam os efeitos deletérios eliciados pelo isolamento social.
4. OBJETIVO GERAL
O objetivo deste estudo foi investigar a influência dos hormônios sexuais
durante o ciclo estral de ratas socialmente isoladas.
4.1. Objetivos específicos
a) Avaliar a resposta comportamental de ratas estressadas por isolamento
social nas diferentes fases do ciclo estral.
b) Avaliar os efeitos do ciclo estral de ratas sobre a resposta emocional,
cognitiva e comportamental.
c) Avaliar o efeito da manipulação das concentrações de estradiol e
progesterona em tarefas de comportamentos do tipo depressivo.
d) Avaliar o efeito da manipulação das concentrações de estradiol e
progesterona nas tarefas de aprendizage, memória e ansiedade.
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PARTE I
Papel dos hormônios sexuais femininos sobre as respostas comportamentais e emocionais de
ratas socialmente isoladas.
39
Introdução
A ativação do eixo HPA em resposta a um estímulo aversivo é emergencial
e temporária, necessária para garantir os ajustes psicofisiológicos e
comportamentais necessários para o enfrentamento, permitindo posteriormente
que o organismo retorne à homeostase por meio de mecanismos de
retroalimentação mediados pela alça sensível aos níveis de GCs (McEwen et al,
2015). No entanto, esse mecanismo pode ser mal adaptativo com a ativação
prolongada do eixo HPA, levando a um estado de exaustão e padrões
disfuncionais. McEwen (2003) descreve que a resposta de estresse ativa diferentes
sistemas com um custo energético ao organismo, ele define esse custo como carga
alostática. A carga alostática promove efeitos negativos cumulativos forçando o
organismo a adaptar-se, sobrecarregando o sistema por hiperatividade. Nesse
sentido, o desequilíbrio da resposta do eixo HPA pode levar a um padrão de
alterações que, na maioria das vezes, está relacionado a anomalias funcionais,
como por exemplo, alteração na morfologia e funcionamento de estruturas límbicas
responsáveis por regular e produzir as respostas emocionais levando um perfil não
adaptativo da resposta de medo ou humor deprimido (McEwen et al, ,2015). Estas
consequências estariam baseadas no modelo da diátese-estresse que descreve a
somatória das características genéticas individuais e das diferentes situações de
estresse que resultariam em endofenótipos associados aos transtornos
psiquiátricos. Diversas condições psiquiátricas relacionadas ao estresse, tais como
ansiedade generalizada (TAG), transtorno do estresse pós-traumático (TEPT),
transtornos do humor e transtornos de adaptação ao estresse tem uma prevalência
maior em mulheres do que em homens (Bangasser & Valentino, 2014.). E isto
parece estar associado a preparação para atividade reprodutiva (Pinkerton et al,
2010; Romans et al, 2012). De fato, mulheres são suscetíveis a diferentes formas
de transtornos de humor, como a depressão pós-parto, síndrome disfórica pré-
menstrual clássica, transtorno disfórico da fase lútea tardia, depressão associada a
menopausa ou devido a procedimentos cirúrgicos de ovariectomia, e todas essas
condições estão associadas a eventos modulados por hormônios sexuais
(Revisado por Albert, 2015).
40
Estudos prévios mostram que os hormônios sexuais agem sinergicamente
com os GCs para alterar o comportamento tanto em modelos animais e humanos
(Figueiredo et al 2006; Herrera & Mather, 2016). Em animais, o estresse aumenta
os níveis de estradiol e progesterona, sugerindo que o aumento desses hormônios
possa ser uma resposta compensatória ao estresse (Suzuki & Handa 2004; Kalasz
et al 2014). É visto que o estradiol e progesterona também modulam a atividade de
neurotransmissores implicados no TEPT, como a noradrenalina (NA, Shansky et al,
2009), e ainda no transtorno de depressão maior, envolvendo a serotonina (5HT,
Epperson et al, 2012), NE (Vega-Rivera et al, 2013) e dopamina (DA, Jacobs &
D'Esposito, 2011). Além disso, os hormônios sexuais femininos parecem modular
funções cognitivas e os níveis de ansiedade através de efeitos sobre a
neurotransmissão glutamatérgica (Oberlander & Woolley, 2016) e gabaérgica
(Luscher et al, 2011).
Portanto, estudos sugerem que a atividade hormonal presente no ciclo estral
de roedores ou menstrual em mulheres tem efeitos benéficos sobre a ansiedade e
alterações no humor (Luine, 2014). Em linha com essa ideia, o padrão oscilatório
da resposta emocional aliada à resposta crônica do estresse pode predizer perfis
neuroendócrinos de responsividade e vulnerabilidade aos transtornos de
depressão, ansiedade e adaptação ao estresse (Luine, 2014). Assim, embora a
hipótese sobre as oscilações de hormônios sexuais como fatores de risco para uma
maior vulnerabilidade ao desenvolvimento de transtornos neuropsiquiátricos em
mulheres seja bem aceita, ainda não há um consenso sobre quais hormônios são
responsáveis por esta modulação. O estradiol (o estrogênio circulante
predominante) é o candidato mais forte, principalmente porque pode agir nos
diferentes sistemas de neurotransmissores, modulando os processos cognitivos e
de regulação do humor (Luine, 2014).
Uma das hipóteses mais aceita é sobre as bases biológicas da depressão
considera que esta patologia ocorre devido à vulnerabilidade biológica combinada
com o desencadeamento de eventos estressantes ao longo da vida (Patten, 2013).
A função alterada das regiões cerebrais importantes para a resposta ao estresse
tem sido consistentemente encontrada em indivíduos com depressão e inclui
hiperatividade do eixo HPA e hiporesponsividade do sistema de retroalimentação
negativa do cortisol (revisado por Pechtel et al ,2011). A liberação dos GCs durante
41
uma situação estressante mostra um padrão totalmente dependente nas
concentrações de esteroides sexuais. Em modelos animais com fêmeas e estudos
com mulheres é visto que a resposta de ativação de estresse é mais sensível se a
exposição ao estressor ocorrer na fase do ciclo com baixa circulação de hormônios
(Vanderlei et al, 1996; D’souza et al, 2017). Assim, estudos mostram que fêmeas
podem apresentar uma variação de humor após exposição a estressores,
principalmente devido a influências dos hormônios ovarianos no eixo HPA e nos
circuitos cerebrais relacionados a reatividade desse eixo.
Estudos epidemiológicos multiétnicos e de longos períodos de
acompanhamento apoiam a hipótese de que a mudança nos níveis hormonais está
associada ao aumento do risco de sintomas depressivos, independentemente de
outros fatores (Bromgerger et al, 2011; Shakeel et a, 2015). Essas evidências
apontam que alterações nas concentrações de estradiol estão associadas a
alterações cognitivas e de humor e que a menopausa precoce (ou gonadectomia)
pode estar intrinsecamente associada ao risco de depressão. A transição de uma
expressão hormonal para outra fase específica pode apresentar um período de
vulnerabilidade para desregulação emocional, maior responsividade ao estresse e
fragilidade cognitiva, essas alterações são sempre observadas nos períodos pré-
menstrual, pós-parto, menopausa, etc. Em modelos animais, disfunções nas
respostas emocionais, bem como aumento da resposta de medo e sintomas do tipo
depressivo são característicos em fases de baixa presença do estradiol e
progesterona (Cover et al 2014; Graham & Daher). À vista disso, se a queda
abrupta desses hormônios durante o período de transição entre fases ou perda do
padrão cíclico hormonal aumenta o risco de depressão e / ou reatividade do eixo
HPA, é esperado que a reposição desses hormônios possa amenizar as alterações
observadas. De fato, estudos em modelos animais que investigaram a retirada
abrupta, da progesterona e estradiol, demonstraram perfis de vulnerabilidade para
as alterações emocionais e cognitivas (Gibbs, 2000: Maayan et al, 2005; Chen et
al, 2001), bem como alterações ao longo do ciclo estral (Chen et al, 2009; Horst et
al, 2012). Aliado a isso, evidências demonstram um efeito benéfico da
administração exógena de hormônio em ratos, ou ainda em mulheres a reposição
hormonal, visto pela melhora os sintomas depressivos, prejuízos cognitivos e
emocionais (Rodgers et al, 2010; Fischer et al,2014).
42
No entanto, como mencionado muitos desses estudos mostram resultados
controversos, alguns estudos indicando que o estradiol aumenta a ansiedade e
outros mostrando diminuição (revisado por Luine, 2014). Além disso, outras
evidências sustentam que modulação da ansiedade ocorra especificamente pela
ação da progesterona (para revisão, Wirth, 2011), e o estradiol regularia aspectos
cognitivos e da resposta de humor (Hendersen, 2008), ou os dois hormônios
(Berent-Spillson et al, 2015).
Essas inconsistências podem surgir devido às diferenças nos protocolos
utilizados, que incluem desde diferentes tipos de manipulações do estradiol e
progesterona, além das tarefas comportamentais empregadas. Diante disso, a
primeira parte dessa tese objetivou investigar a influência do ciclo estral sobre o
comportamento de ratas socialmente isoladas. Nesse mesmo estudo,
posteriormente, investigamos o efeito dissociativo do estradiol e progesterona
sobre essas alterações, utilizando agonistas e antagonistas nas diferentes fases do
ciclo.
43
Artigo 1
Dissociation of sex steroid modulatory effects on depressive-like behavior in socially stressed female rats
Ezequiel Batista do Nascimento1,2, Aline Lima Dierschnabel1,2, Antônio Carlos
Queiroz2, Sara Sophia Guedes2, André de Macêdo Medeiros3,4, Ramon Hipólito
Lima1,2, Deborah Suchecki5, Regina Helena Silva1,3, Jeferson de Souza
Calvacante2, Alessandra Mussi Ribeiro1,6*
1Memory Studies Laboratory, Department of Physiology, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
2Laboratory of Neurochemical Studies, Department of Physiology, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
3Laboratory of Behavioral Neuroscience, Department of Pharmacology, Universidade Federal de São Paulo, SP, Brazil
4Center of Health and Biological Sciences, Universidade Federal Rural do Semiárido, Mossoró, Brazil
5Department of Psychobiology, Universidade Federal de São Paulo, SP, Brazil 6Laboratory of Neuroscience and Bioprospecting of Natural Products, Department of Biosciences,
Universidade Federal de São Paulo, Santos, SP, Brazil
*Corresponding author: Alessandra M. Ribeiro
Departamento de Biociências
Universidade Federal de São Paulo
Rua Silva Jardim 136 – CEP: 11015-020, Santos/SP
Tel: (55) 13 3229-0137
E-mail: [email protected]
44
Abstract
The role of sex steroids in mammals is beyond the classical effect reported
on reproductive system. Evidence accumulated over the last decades show sex
hormones as modulatory factors on the cognitive function and emotionality in animal
models and humans. Herein, we investigated differences of the estrous cycle
phases on depressive-like behavior using social interaction test, sucrose splash test
and sucrose preference test. In addition, we used a model of social stress to induce
depressive-like behavior in females at all phases of the estrous cycle. These
females were treated with estradiol, progesterone and the antagonists of both
hormones tamoxifen and mifepristone, respectively. Our main results demonstrated
that: (1) depressive-like behavior induced by social isolation was dependent on the
estrous cycle; (2) females in diestrus had higher depressive-like behavior; (3)
stressed females treated with estradiol (but not progesterone) showed less
depressive-like behavior; (4) tamoxifen (but not mifepristone) induced depressive-
like behavior in proestrus females. Taken together, these findings suggest that
endogenous variations of sex hormones are important to modulate depressive-like
behavior in female stressed by social isolation. Interestingly, the dissociative effect
between estradiol and progesterone based on the distinct actions of the antagonists
could enlighten the differential relevance of these sex hormones on mood disorders
such as depression.
Key words: Estrous cycle. Social isolation. Depression. Estradiol. Progesterone.
Tamoxifen. Mifepristone.
45
1. Introduction
Sex steroids such as estradiol and progesterone have unquestionable
influence on reproductive processes and sexual behaviors. In addition, both
estradiol and progesterone receptors are largely distributed throughout the
mammalian brain, especially in the hippocampus, striatum, amygdala,
hypothalamus and cortex – all areas related to cognition and emotional responses
(Bossé & Di Paolo, 1996; Zeidan et al, 2011; Luine & Frankfurt, 2013). Studies have
also demonstrated the importance of these sex steroids on other physiological
processes (Cui et al, 2013). For example, in the central nervous system, estrogens
affect brain functions, including changes in neuronal excitability, hippocampal
synaptic plasticity, synaptic transmission and glucose metabolism in rodents and
humans (Zadran et al, 2009; Foy, 2001; Toffoletto et al, 2014; Brinton, 2008; Barth
et al, 2016).
In the hippocampus, estradiol – the main circulating estrogen – modifies the
morphology and activity of the pyramidal neurons (Gould et al, 1991; Garcia-Segura
et al, 1995). Estradiol also exerts protective effects on the hippocampal circuitry
against injuries and abnormalities, such damage and impairments resulting from
transient global ischemia, cortical lesion induced by glutamate excitotoxicity and
oxidative stress (Durham et al, 2012; Mendelowitsch et al, 2001; Behl et al, 1995).
Although the mechanisms related to neuroprotection are not completely elucidated,
several studies have reported that during adulthood estradiol increases
synaptogenesis and dendritic branching of the CA1 pyramidal neurons (Murakami
et al 2006; Phan et al, 2010; McEwen & Gianaros, 2011). Furthermore, it seems that
the increase of estradiol during the estrous and menstrual cycle can improve the
excitatory input on the dopaminergic transmission n the pre-frontal cortex (PFC) in
both female rats and women (Khan et al, 2013; Rey et al, 2014 Jacobs & D’esposito,
2011). These morphological and functional changes are related to a better
performance in cognitive tasks in rodents and human (Ooishi et al, 2012; Luine,
2014). Indeed, estradiol facilitates acquisition and consolidation of the memory in
rodents and human (Luine & Frankfurt, 2013; Luine, 2014; Mordecai et al, 2008;
Maki et al, 2011).
46
Estradiol also acts on amygdala depedent fear extinction process (Zeidan et
al, 2011). Graham and Milad (2013) showed, in a translational study, that women
using hormonal contraceptive –estradiol inhibitor – exhibited decreased fear
extinction, and female rats treated with combined hormonal contraceptives had fear
extinction impairment. Moreover, this condition was abolished with a single dose of
estradiol.
Regarding the estrous cycle, animals in proestrus and estrus display a better
consolidation of the fear extinction (Milad et al, 2009). Concerning this modulatory
effect of estradiol may be closely related to the vulnerability of depressive symptoms
increased after puberty, especially in women (Williams et al, 2009; Sisk & Zehr,
2005; Douma et al, 2005). Likewise, low levels of estradiol induce cognitive decline
and impairment of emotional responses in postmenopausal woman (Henderson,
2008; Morrison et al, 2003). Thus, these findings suggest that low estradiol levels
are associated with increased expression of fear and higher vulnerability to anxiety
and depression disorders.
Similarly, progesterone has been reported to modulate cognitive, emotional
and motivational responses in female rats (Barros et al, 2014; Frye & Walf, 2002).
Behavioral changes across estrous cycle may be linked to expression of
progesterone, especially in the estrus phase. Nevertheless, the progesterone
modulatory effects over emotional responses remain controversial. In female rats,
evidence reported that progesterone increase mobility in water maze test, improved
novel object recognition and anxiety (Casas et al, 2011; Frye & Walf, 2009). In
addition, previous studies showed that rats in proestrus and estrus phases have
less depressive-like behaviors, however these studies did not discriminate the role
of the hormones in each phase (Gouveia jr et al, 2008; Jenkins et al 2001; D’souza
& Sadananda, 2017). Further, there is effect of the progesterone in anxiety
expression in rats, when in diestrus phase was observed anxiogenic behavior
whereas in proestrus and estrus phase was demonstrated anxiolytic behavior (Hiroi
& Neumaier 2006; Gangitano et al 2009). In human, it seems no correlation of
depression and anxiety to estrogen and progesterone, but the findings are still
controversial (Hsiao et al, 2004). Herrera and coworkers (2016) demonstrated
increased cortisol circulations and cognitive deficits in luteal phase, the stage which
progesterone is higher in women.
47
Depression disorders are also associated with alterations in stress response
of humans and animal models (Vreebug et al, 2010). For instance, about 40-60 %
of patients with major depression have high levels of cortisol and HPA axis
hyperactivity (Zunszain et al, 2011; Juruena et al, 2006). There is a reduction in
hippocampus volume and brain-derived neurothophic factor (BDNF) levels in
depressive patients (Sheline, 1996; Frodl et al, 2007) causing changes on mood
and cognition (reviewed in, Alkadhi, 2013). Furthermore, it is well discussed in the
literature that chronic release of corticosterone elicits deleterious effects on rodent’s,
especially in the hippocampus (Pariante, 2006). In another study, animals submitted
to chronic stress demonstrated atrophy of CA3 pyramidal cells (Lakshminarasimhan
& Chattarji, 2012). Likewise, evidence has linked stressful situations to decreased
BDNF in animal models of mental disorders (reviewed in, Autry & Monteggia, 2012).
Indeed, BDNF levels decreased in response to genomic effects of glucocorticoids,
triggering morphophysiological changes (Parient, 2006). Neurotrophic factors are
important to hippocampal integrity consequently for cognition and emotional
responses, moreover, it seems that this structure has an important role on negative
feedback of HPA axis (Gillies & Mcarthur, 2010; Nestler et al, 2002).
In view of what was mentioned, hormones related to the HPA and the HPG
axis can interact with each other and influence affective-like behaviors. However,
the neurobiological basis of these hormones pathways on depressive disorders
induced by chronic stress remain controversial in part because of the paucity of
studies using females in their experimental protocols. Thus, the aim of the present
study was to investigate whether modulation of the sex steroid hormones during the
estrous cycle employing receptor antagonists or by increasing their endogenous
levels alter the depressive-like behavior in rats stressed by social isolation.
48
2. Material and methods
2.1. Animals
One hundred and thirty female Wistar rats (3-4 month-old) were housed in
polypropylene cages (42 cm length, 34 cm width and 18 cm height), under controlled
conditions of temperature (22±1°C) and light-dark cycle (12h/12 h, lights on 6:00
a.m.) with water and food ad libitum. The local ethical committee (CEUA-UFRN
#006/2014-2) approved all animal procedures and protocols in according to
Brazilian law for the use of animals in scientific research (Law Number 11.794). All
efforts were made to minimize potential pain, suffering, or discomfort that the animal
might suffer.
2.2. Drugs
17β-estradiol soluble water (17β-EST, 0.2 mg/kg, Sigma-Aldrich, St. Louis,
MO, USA) was diluted in physiological saline solution 0.9 %. Tamoxifen citrate
(TAM, 1 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in vehicle (saline
solution containing Tween 80 10 %). Progesterone (PROG, 0.5 mg/kg, Tocris
Bioscience, Ellisville, MO, USA) was diluted in sesame oil. Mifepristone (MIF, 10
mg/kg, Tocris Bioscience, Ellisville, MO, USA) was diluted in a solution containing
absolute ethanol, sesame oil and Tween 80. The doses were chosen based on
previous studies (Sandstrom & Williams, 2001, Sharma et al, 2007, Aisa et al.,
2006). The 17β-EST, Tamoxifen and mifepristone were administrated
subcutaneously.
2.3. Estrous Cycle
Five days prior the beginning of the stress protocol and experimental
procedures to determine the intact estrous cycle, vaginal washes were obtained
daily from females rats through plastic pipettes filled with distilled water in a volume
of 0.2 ml. The washes were stained with methylene blue (5 mg/ml) and analyzed by
optical microscopy. According to the morphological characteristics of the cells, the
phases of the estrous cycle were classified as: (1) Diestrus/metestrus (DIE) – few
nucleated and cornified cells and many leucocytes, it is conventional to consider
49
metestrus and diestrus (or Diestrus I and II) as the characterized phase of estrus
cycle where steroid hormones are low and vaginal cytology are quite similar
(Caligione, 2009; (2) Proestrus (PRO) – abundant nucleated epithelial cells and (3)
Estrus (EST) – abundant cornified cells (Paccola et al, 2013). The hormonal profile
throughout the estrous cycle comprise low levels of estradiol and progesterone
during the DIE, a transient increase in estradiol during the PRO followed by a
transient increase in progesterone levels during the EST (Aritonang et al, 2017).
2.4. Stress induction protocol
All animals were randomly assigned to two conditions: control and social
isolation. During seven consecutive days, control animals remained in their home
cages while other group of animals were separated and induced to stress through
social isolation. The stress protocol was performed as described previously by Yates
et al. (1990). Briefly, animals were housed in individual cages (length 31 cm, width
20 cm, high 22 cm) with acoustic isolation to avoid social vocalizations. The social
isolation condition occurred up to 24h before the behavioral tasks.
2.5. Sucrose Splash Test (SST)
The protocol of this test was described and validated previously (David et al,
2009). Animals were placed into small cages (length 31 cm, width 20 cm, high 22
cm) for 5 min. The task consists in squirting 10% sucrose solution on the dorsal coat
of the animal and quantify the time spent and latency to start the grooming behavior
when they start to clean the local where the solution was applied. At the end of the
session, animals returned to their home cages.
2.6. Social Interaction Test (SIT)
In this paradigm, each experimental animal was simultaneously placed with
an unfamiliar rat (same age and sex) in the center of the open field to interact with
each other for 10 min. At the end of the session, animals were returned to their
respective home cages. Social behaviors as sniffing (when the animals nose poke
each other), following (when there is motivation to follow each other), and social
avoidance (when the animal approaches to interact with the other animal, but there
50
is a social avoidance) were quantified by the time spent and latency to start the
interaction in seconds. All parameters are based on animal models of social
avoidance and social fear (revised by Toth & Neumann, 2013).
2.7. Sucrose Preference Test (SPT)
Prior the start of the test, animals were single housed with free access to food
and habituated to the presence of two bottles: one containing 2% of sucrose solution
and the other water during a 24 h period. At one point (12 h), the bottles were
switched to avoid spatial association. No food restriction was applied before of the
test. Consumption of water and sucrose was measured by the difference of the
bottle weigh before and after the test. Sucrose preference was calculated as the
percentage of sucrose consumption relative to the total amount of liquid consumed
[sucrose consumption/(sucrose consumption + water consumption) * 100], adapted
from Forbes et al, 1995.
2.8. Experimental design
A summary of the behavioral task and experimental design can be seen in
Figure 1. One day after the end of the stress induction protocol, all animals
performed the behavioral tasks beginning with the sucrose splash test (SST), after
30 minutes, animals were submitted to habituation of 5 minutes on open field (OF);
then, after 30 minutes animals returned to the open field in order to perform the
social interaction test (SIT). Immediately after SIT, animals weree placed individually
in homecage in order to perform the sucrose preference test (SPT) and were kept
during 24 h. The OFT, SPT and SIT tests were recorded by digital camera placed
above the apparatus. The computational monitoring of the behavioral sessions
occurred in a separate room to avoid any interference by the experimenter. All
apparatus used in the experiments were cleaned with a 5% alcohol solution between
the beginning of each session. Behavioral parameters were analyzed by Any-
maze® (Stoelting, EUA). The estrous cycle schedule stared at 10:00 and drug
administration and the occurred at 11 a.m. All behavioral tests started at 12:00 p.m.
51
Figure 2. Schematic representation of the experimental design. The behavioral tasks started with
Sucrose splash test (SST), followed by Social interaction test (SIT) and ends with Sucrose preference
test (SPT).
2.8.1. Experiment I: Influence of estrous cycle on the depressive-like behavior induced by the social stress
Female rats in each phase of the estrous cycle were randomly assigned to
one of six groups: DIE control (n = 19), DIE social isolation (n= 14), PRO control (n
= 7), PRO social isolation (n= 7), EST control (n =7) and EST social isolation (n =
7). In this experiment, we investigated whether fluctuations of ovarian hormones
during the estrous cycle would influence the expression of depressive-like behavior
induced by the social stress.
2.8.2. Experiment II: Effects of the estradiol and tamoxifen on the depressive-like behavior induced by the social stress
In this experiment, socially stressed females were assigned once it was
determined by estrous cycle phase to receive the drug treatment. Afterwards,
animals in DIE phase were treated with one single administration of saline (VEH,
n = 14), or 17β-estradiol (17β, n = 13). For animals in PRO and EST, tamoxifen
treatment was administered (TAM, n = 7) one hour prior behavioral tasks. We
treated animals with estradiol in DIE due evidence that suggests the low profile of
those ovarian hormones in both phases (Aritonang et al, 2017). We hypothesized
that estradiol administration mitigated depressive-like behaviors induced by social
isolation in DIE phase, whereas TAM treatment would inihibit the beneficial effect of
estrogen on depressive-like behavior.
52
2.8.3. Experiment III: Effects of progesterone and mifepristone on the depressive-like behavior induced by the social stress
Animals in DIE were treated with progesterone (PROG, n = 9) or vehicle
(VEH, n = 6), whereas animals in PRO or EST were treated with mifepristone (MIF,
n = 7) or vehicle (VEH, n = 6). PRO and EST were chosen to receive MIF due the
high profile of estradiol and progesterone, respectively (Aritonang et al, 2017). We
investigated the effect of progesterone to promote beneficial effect on the
depressive-like behavior induced by social stress in DIE phase. Moreover, whether
progesterone blockade impair beneficial effect of PRO and EST phases on the
depressive-like behavior induced by social isolation. The drugs and vehicles were
injected one hour before beginning of behavioral test (fig. 1)
.
2.9. Statistical analysis All data were tested for normality using the Kolmogorov-Smirnov’s test, and
for homogeneity of variances using Levene’s test. Comparisons were taken
considering condition effect (control X stressed groups), estrous cycle phases effect
(DIE X PRO x EST) and interaction condition x phase effect. Further, differences in
social interaction test, sucrose splash and sucrose preference tests were analyzed
by two-way analysis of variance (ANOVA) followed by unequal Tukey’s post hoc
test. For drug treatment in isolated females, comparisons were taken considering
estrous phases effect (DIE X PRO X EST), treatment effect (DIE: VEH x 17β x
PROG and PRO or EST: VEH x TAM X MIF) or estrous phases x treatment
interaction. All behavioral tasks were analyzed by two-way ANOVA followed by
unequal Tukey’s post hoc test. Results are expressed as mean ± SEM. Statistical
differences were considered significant for p ≤ 0.05. All analyses were performed
using STATISTICA statsoft®.
53
3. Results
3.1. Experiment I: Influence of estrous cycle on the depressive-like behavior induced by the social stress.
3.1.1. Sucrose splash test (SST) Considering the grooming behavior in SST, two-way ANOVA did not reveal
differences for frequency (data not shown), but for time were observed condition
effect [F (2,37) = 8.24; p = 0.006] and phase effect [F (2,37) = 13.85; p = 0.001].
Overall, stressed animals reduced time of the grooming behavior (Fig. 2A). Tukey’s
post hoc test revealed that control females in PRO and EST spent more time
displaying grooming behavior when compared to DIE. Likewise, stressed animals in
PRO increased grooming behavior when compared to stressed DIE. Moreover,
isolated females in DIE and EST were different from control groups in PRO. There
is no difference between control PRO and isolated PRO.
For latency to start the grooming behavior, two-way ANOVA revealed
condition [F (1,37) = 5.75; p = 0.02] and phase effects [F (1,37) = 73.09; p = 0.01].
Overall, social isolation increased latency for start the grooming behavior (Fig. 2B).
Tukey’s post hoc showed that latency is lower in PRO and EST control, but only
stressed animals in PRO had this effect (fig. 2B).
Fig. 3. Effect estrous cycle on grooming behavior of stressed female rats by social isolation. (A) total time spent in the grooming behavior and (B) latency to start the grooming behavior. Data are expressed as mean ± SEM; #p < 0.05 stressed female compared to control group, *p < 0.05 compared to DIE; **p < 0.05 isolated PRO compared to isolated DIE (Two-way ANOVA followed Tukey’s post-hoc test).
54
3.1.2 Social interaction test (SIT)
Sniffing Behavior
For sniffing behavior, two-way ANOVA revealed phase effect for frequency
[F (1,56) = 6.16; p = 0.01], time [F (2,56) = 21.62; p = 0.01] and latency [F (2,56) =
6.582; p = 0.01]. Moreover, condition effect was showed for time F (1,56) = 75.97;
p = 0.01] and latency [F (1,56) = 11.49; p = 0.01]. Overall, social stress impaired the
sniffing behavior by decreasing frequency and time as well as increasing latency for
this behavior (Table 1). Tukey’s post-hoc test for phase effect showed that control
rats in PRO increased sniffing behavior frequency compared to DIE in all conditions
(p < 0.05). Further, animals in PRO and EST increased sniffing time and decreased
latency when compared to DIE in both conditions (p < 0.05).
Following Behavior
For the following behavior, two-way ANOVA revealed of the phase and
condition for frequency [F (2,59) = 12.81; p = 0.009; F (1,56) = 3.96; p = 0.05], time
[F (2,59) = 25.15; 0.01; F (1,56) = 4.47; p = 0.03] and latency [F (1,56) = 11.495, p
= 0.01; F (1,56) = 6.582, p = 0.01], respectively. Tukey’s post-hoc test for frequency
showed that control in PRO displayed more following behavior than control and
isolated in DIE as well as isolated in EST (p < 0.05). Similarly, time spent in following
behavior for control in PRO phase was increased when compared to control and
isolated in DIE (table 1), same effect was observed in isolated in EST when
compared to DIE phase (p < 0.05). Considering latency to start this behavior, post-
hoc test showed that control in PRO decreased latency when compared to control
DIE and isolated DIE (p < 0.05). Likewise, stressed animals in PRO and EST
reduced latency when compared to stressed females in DIE (Table 1).
Avoidance Behavior
For the avoidance behavior, two-way ANOVA revealed phase effect for
frequency [F (1,56) = 5.465; p = 0.006] and time [F (1,56) = 13.08; p = 0.01]. Tukey’s
post-hoc test showed that stressed animals in EST increased the number of
avoidance behaviors compared to control in PRO. Moreover, females in PRO spent
less time performing social avoidance behavior when compared to control and
isolated in DIE (p <0.05). (Table 1).
55
Table 1. Effects of estrous cycle and social isolation in the social interaction test in rats
Values are expressed as mean ± SD. ap < 0.05 compared to control in DIE, bp < 0.05 compared to isolated in DIE, cp < 0.05 compared to control in PRO. Two-way ANOVA followed Tukey’s post-hoc test.
3.1.3 Sucrose preference test (SPT) Two-way ANOVA for sucrose consumption revealed condition [F (1,56) =
12.32; p = 0.01] and phase [F (91,57) = 13.58; p = 0.01] effects. Tukey’s post hoc
test showed a significant increase in the sucrose consumption by animals in PRO
and EST. Moreover, stressed female in DIE decreased sucrose consumption when
compared to control in DIE (Fig. 3).
Fig. 3. Effects of social isolation and estrous cycle on sucrose consumption in female rats. Percentage of total sucrose consumed into 24h. Data are expressed as mean ± SEM. #p < 0.05 compared to control group. *p < 0.05 compared to DIE **p < 0.05 compared to control in DIE (Two-way ANOVA followed Tukey’s post-hoc test).
CONTROL SOCIAL ISOLATION
Behavior DIE (n=19) PRO (n= 7) ES (n =7) DIE (n=13) PRO (n=7) EST(n = 7)
Sniffing
Number 25±2 41±16ab 32±7 26±2 26±9.1 33±9
Time 49±6b 123±41ab 111±46ab 15±2 81±17b 76±17b
Latency 37±5 5±4ab 7±4ab 58±9 17±12b 23±4b
Following
Number 4±1 12±7ab 4±2 4±1 5±3 3±1c
Time 16±3 25±8ab 24±16 2±1 13±12 22±17b
Latency 119±8 16±10ab 74±65 149±14 69±49b 35±8b
Avoidance
Number 10±1 4±1 12±4 9±1 10±5 17±9c
Time 17±2 2±1ab 5±1 16±1 4±3 11±9
Latency 13±4 40±62 30±17 10±2 10±6 11±9
56
3.2. Experiment II: Effects of the estradiol and tamoxifen on the depressive-like behavior induced by the social stress.
3.2.1 Sucrose splash test (SST) Two-way ANOVA revealed a significant effect of phase for time [F (5,44) =
9,171, p = 0.001] and latency [F (5,44) = 5.435; p = 0.01]. Tukey’s post hoc showed
increase grooming time by animals treated with 17β in DIE when compared to VEH
in DIE. Further, animals in PRO that received TAM showed less time spent of
grooming when compared to VEH and 17β in DIE. Considering the latency, the post
hoc showed that animals in PRO and EST treated with TAM increased the latency
to grooming behavior when compared to VEH and 17β in DIE (Fig. 4). No
differences were found to frequency of grooming behavior (data not shown).
Fig. 4. Effects of treatment 17β-estradiol (17β), tamoxifen (TAM) and vehicles (VEH) on grooming behavior of socially isolated female rats. (A) time spent of grooming behavior and (B) latency to start the grooming behavior in sucrose splash test. Data are expressed as mean ± SEM. *p< 0.05 compared to VEH in diestrus, **p < 0.05 compared to respective VEH, +p < 0.05 compared to 17β in diestrus (two-way ANOVA followed by Tukey’ post hoc test).
3.2.2 Social interaction test (SIT)
Sniffing behavior Two-way ANOVA revealed effect of treatment, but not phase, for frequency
[F (5,44) = 9.671; p = 0.01] and time [F (2,44) = 7.17; p = 0.01]. In addition, analysis
revealed phase x treatment interaction for latency [F (2,44) = 4.222; p = 0.01].
Tukey’s post hoc test showed that 17β-treated isolated females in DIE increased
57
frequency of sniffing when compared to VEH and both TAM-treated in PRO and
EST. Likewise, TAM-treated isolated animals in PRO reduced sniffing behavior
when compared to VEH. Regarding time, 17β-treated females in DIE spent more
time displaying sniffing behavior when compared to VEH and all TAM treatment. As
expected, VEH in PRO and EST spent more time displaying sniffing when compared
to VEH in DIE. Further, TAM-treated females in PRO and EST spent less time of
sniffing behavior when compared to VEH (Table 2). Considering the latency, 17β-
treated animals in DIE spent less time to start sniffing behavior when compared to
VEH-treated and all TAM treatment (p < 0.05). Besides, TAM-treated animals in
PRO increased the latency when compared to respective VEH (Table 2).
Following behavior Two-way ANOVA revealed phase x treatment interaction [F (2,44) = 4.616; p
= 0.03] for frequency of the following behavior. Also, analysis showed treatment
effect for time [F (2,44) = 7.486; p = 002] and latency [F (2,44) = 7.148; p = 002].
Tukey’s post hoc test showed that 17β-treated animals in DIE increased frequency
when compared to VEH in DIE and TAM-treated animals in PRO. Regards time,
17β-treated animals spent more time displaying the following behavior when
compared to VEH and all TAM-treated animals. Further, TAM-treated rats in PRO
demonstrated reduction in the time of the following behavior (p < 0.05). Considering
latency to start this behavior, 17β-treated animals reduced time to start following
behavior when compared to VEH in DIE and all TAM treatment (p < 0.05). Further,
TAM-treated animals increased latency for the following behavior in PRO and EST
(Table 2).
Avoidance behavior Two-way ANOVA revealed treatment effect for frequency [F (2,44) =
4.835; p = 0.13], time [F (2,44) = 6.615; p = 0.03] and latency [F (2,44) = 9.436; p =
0.01] of the avoidance behavior. Also, phase [F (2,44) = 7.228; p = 002] and
treatment F (2,44) = 4.304; p = 0.02 effect were observed for latency. Tukey’s post
hoc test showed that 17β-treated animals in DIE exhibited lowest frequency and
time for this behavior when compared to VEH and TAM-treated animals in PRO and
EST. In contrast, TAM-treated increased the time displaying avoidance behavior
when compared to VEH-treated in PRO (p < 0.05). Overall, animals in DIE phase
58
had lowest latency to start avoidance behavior. Unlike, VEH-treated animals in PRO
and EST showed higher latency when compared to VEH-treated animals in DIE that
was countered by TAM treatment.
Table 2. Effects of estrous cycle (DIE, PRO and EST) and pharmacological treatment of 17β-estradiol (17β), tamoxifen (TAM) or vehicle (VEH) on social behavior of isolated female rats. Total number, time and latency were registered for sniffing, following and avoidance behavior in social interaction test.
DIESTRUS PROESTRUS ESTRUS
Behavior VEH 17β VEH TAM VEH TAM
Sniffing Number 15±18 29±10abc 33±18 11±7d 29±10 15±9
Time 29±60 67±36abc 71±41a 13±8d 67±36a 20±7e
Latency 33±33 18±25abc 15±6 59±18d 12±5 50±28
Following Number 6±7 23±16ab 14±9 4±2 13±6 6±2
Time 11±7a 38±31abc 28±12 4±1d 21±15 7±2 Latency 64±7 16±13abc 20±16 75±23d 24±27 119±74e
Avoidance Number 8±4 6±9c 5±2 15±5 6±7 22±9
Time 13±14 9±3bc 6±3 35±21d 8±6 36±13
Latency 13±7 37±32 89±68a 8±2d 95±78a 14±5e Values represented in mean ± SD. ap < 0.05 compared to VEH in DIE phase, bp < 0.05 compared to TAM in PRO, cp < 0.05 compared TAM in EST, dp < 0.05 compared to VEH in PRO, ep < 0.05 compared to VEH in EST (two-way ANOVA followed by Tukey’s test).
3.2.3 Sucrose preference test (STP) For the consumption of sucrose test, two-way ANOVA revealed significant
difference for phase [F (5,44) = 3.194, p = 0.05] and treatment effect [F (5, 44) =
49.95; p = 0.001]. Tukey’s post hoc test showed VEH-treated animals in EST
increased consumption of sucrose when compared to VEH-treated rats in DIE.
Moreover, 17β-treated animals in DIE increased consumption when compared to
VEH in DIE. Besides, TAM-treated rats in PRO and EST reduced sucrose
consumption when compared to VEH, this same effect was observed when
compared to 17β-treated animals (Fig. 5).
59
Fig. 5. Percentage of sucrose consumption by socially isolated females treated with estradiol (17β), tamoxifen (TAM) or vehicle (VEH) in diestrus, proestrus and estrus phases. Data are expressed as mean ± SEM. *p < compared to VEH in DIE, **p < 0.05 compared to 17β, #p < 0.05 compared to VEH in DIE, +p < 0.05 TAM compared to matched VEH in PRO an EST. Two-way ANOVA followed Tukey’s post-hoc test.
3.3 Experiment III: Effects of the progesterone and mifepristone on the depressive-like behavior induced by the social stress.
3.3.1 Sucrose splash test (SST)
Two-way ANOVA revealed treatment effect [F (5, 41) = 2,431; p = 0.04] for
latency of the grooming behavior. Tukey’s post hoc showed that VEH and PROG-
treated animals in DIE increased latency to start the grooming behavior when
compared to VEH-treated in PRO and EST.
Fig. 6. Effects of estrous cycle and treatment with progesterone (PROG), mifepristone (MIF) and vehicles (VEH) in the latency to start the grooming behavior in sucrose splash test. Data are expressed as mean ± SEM. *p< 0.05 compared to VEH-treated in PRO and EST. (Two-way ANOVA followed by unequal Tukey’ post hoc test).
60
3.3.2 Social interaction test (SIT)
Sniffing behavior Two-way ANOVA revealed phase effect and treatment for frequency [F (2,42)
= 4.475; p = 0.01; F (2, 42) = 10.707; p = 0.001] and time [F (2,42) = 4.093; p = 0.02;
F (2,42) = 4.105; p = 0.02], respectively. Tukey’s post hoc showed that VEH-treated
in PRO increased frequency of sniffing behavior when compared to VEH-treated
animals in DIE. In contrast, MIF-treated rats in PRO reduced this behavior.
Moreover, VEH-treated animals in PRO increased time of sniffing when compared
to DIE phase. MIF treatment reduced time of sniffing in PRO phase (p < 0.05). No
effects of latency were detected for this evaluation.
Following behavior No differences were found to following behavior.
Avoidance behavior Two-way ANOVA revealed for time of avoidance behavior phase [F (2,42) =
3.756; p = 0.03] and treatment [F (2,42) = 3.472; p = 0.04]. Tukey’s post hoc
revealed that PROG treatment increased latency for avoidance behavior when
compared to all MIF-treated animals (Table 3).
Table 3. Effects of estrous cycle (DIE, PRO and EST) and pharmacological treatment of progesterone (PROG), mifepristone (MIF) or vehicle (VEH) in the social behavior of isolated female rats in social interaction test. Total number, time and latency were registered for sniffing, following and avoidance behavior.
DIE PRO EST
Behavior VEH (n=14) PROG (n=13) VEH (n=6) MIF (n=7) VEH (n=6) MIF (n= 7)
Sniffing
Number 18±6 30±8 34±3a 19±6d 29±10 19±8 Time 33±8 47±22 63±10a 37±19d 67±36 41±20 Latency 51±25 25±24 32±22 46±19 12±5 55±28
Following Number 11±2 13±6 16±8 10±1 13±6 8±4
Time 21±21 23±14 23±11 16±9 21±15 15±9 Latency 52±31 32±20 32±19 36±34 24±27 59±28
Avoidance Number 15±9 8±4 10±6 11±2 6±7 14±11
Time 29±24 10±8 11±9 23±9 8±6 47±23 Latency 53±58 79±52bc 67±32 49±13 95±78 50±11 Values represented in mean ± SD. ap < 0.05 compared to VEH in DIE, bp < 0.05 compared MIF in PRO, cp < 0.05 compared TAM in EST, dp < 0.05 compared to VEH in PRO, ep < 0.05 compared to VEH in EST (two-way ANOVA followed by Tukey’s post hoc test).
61
3.3.3 Sucrose preference test (SPT) Two-way ANOVA revealed phase effect [F (5,41) = 5.8; p = 0.002]. Tukey’s
post hoc showed that animals in PRO and EST had increased consumption of
sucrose when compared to rats in DIE (Fig. 7).
Fig. 7. Effects of estrous cycle and treatment of progesterone (PROG), mifepristone (MIF) and vehicles (VEH) on sucrose consumption of social isolated female rats. Data are expressed as mean ± SEM. #p < 0.05 PRO and EST compared to DIE. Two-way ANOVA followed unequal Tukey’s post-hoc test.
4. Discussion
In this present study, our results showed that there was oscillation of
emotional response through of the estrous cycle in stressed female rats. In addition,
we demonstrated the dissociative effects of the estradiol and progesterone on
depressive-like behaviors. Our main results showed that females in proestrus and
estrus phase: (1) increased social interaction behaviors in the SIT; (2) increased of
grooming behavior in the SST; (3) increased sucrose consumption in SPT.
Furthermore, the administration of 17β in females decreased the depressive-like
behaviors whereas the treatment with tamoxifen in proestrus phase increased these
behaviors. Unlike, progesterone treatment had no effect on depressive-like
behaviors.
In this present study, we used one social stress model in order to induce
depressive-like behaviors (Ieraci et al, 2016). It is known that chronic glucocorticoids
induce depressive-like behaviors and in the last decades a number of studies about
underlying molecular and cellular mechanisms altered in the chronic stress, which
62
may increase the vulnerability of individuals to develop depressive symptoms (Khan
& Khan, 2017; Ross et al, 2017). The causes of depression remain uncertain,
although most of studies have provided evidence about for neuron atrophy and loss
in limbic brain regions, such as hippocampus. Indeed, one of the most replicated
findings has been that hippocampus and pre-frontal cortex volume is decreased in
patients with depression by magnetic resonance imaging (Frodl et al 2007;
Videbech et al, 2002; Sheline et al, 1999; McQueen et al, 2003). As mentioned, in
our study female rats submitted to stress by social isolation showed depressive-like
behaviors (grooming reduction; poor social interaction and decreased sucrose
intake), and these behaviors have been correlated to social withdrawal, anhedonia,
self-neglect and motivational behavior in human (Toth & Neumann, 2013; Krishnan
& Nestler, 2011; Nollet et al, 2013). Interestingly, the depressive-like behaviors
induced by social isolation were estrous cycle phase-dependent, isolated females
in diestrus showed lowest social interaction and sucrose consumption when
compared to control in diestrus, indicating that this phase stress can aggravated
these behaviors. On the other hand, animals in proestrus and estrus phases appear
to have mitigated the depressive effect elicited by social isolation.
From this standpoint, sex differences in the stress response exist throughout
the lifespan and relate to both the organizational and functional effects of sex
steroids (Gillies & Mcarthur, 2010; Arthur, 2009; Silbergeld et al, 2002). In
adulthood, females experience higher rates of depression symptoms after puberty
and persist by adulthood (Studd & Nappi, 2002). This long-term effect on emotional
behavior can be associated with the cyclic release of ovarian hormones in the
beginning of sexual activity. Indeed, clinical findings reported that estrogen
oscillation related to emotional changes occur in the premenstrual syndrome, major
depression, anxiety disorders, bipolar disorders, borderline personality and post-
traumatic stress disorder (Backstrom et al, 2003; Berardies et al, 2007; Walf & Frye,
2006; Meinhard et al, 2014; Eisenlohr-moul et al, 2015; Glover et al, 2012). In
addition, the estrogen hormone has a well-known neuroprotective activity, which
could be related with the gender prevalence of selected neurodegenerative
diseases, such Alzheimer’s and Parkinson’s disease (Vegeto et al, 2008). In our
study, control and stressed animals in proestrus and estrus exhibited less
depressive-like behaviors when compared to control animals in diestrus, moreover,
63
this condition was aggravated by social isolation stress. These results corroborate
other studies that reported alterations in the emotional response throughout estrous
cycle of rodents, where social stress can be negative modulator (Palanza et al,
2001).
In this point, the negative effect of the stress and beneficial effect of estrogen
on depressive-like behaviors were also found when 17β-treated animals in diestrus
demonstrated better social interaction, increased of the grooming behavior and
sucrose intake. Notwithstanding, tamoxifen-treated animals in proestrus showed
decrease of the grooming, social interaction and sucrose intake. These results
suggest that the estradiol neuromodulation was blocked by administration of the
antagonist. All these findings are in accordance others studies that reported high or
low levels of estradiol can modulate positive or negatively emotional responses,
respectively.
We also observed that progesterone fluctuations through estrus cycle can
influence some depressive-like behaviors. In the estrus phase which progesterone
level is increased did not changes in the social interaction parameters, grooming
behavior and sucrose consumption. Nonetheless, mifepristone-treated animals did
not demonstrate impairment on beneficial effects observed in proestrus and estrus.
Moreover, animals in estrus showed decrease in depressive-like behavior, but this
effect did not reproduce in the treatments with progesterone and mifepristone. One
possible explanation could be because of the long-lasting effect of estradiol started
in proestrus phase with everlasting effects on estrus phase (Zhu et al, 2017; Barker
& Galea, 2008). Besides, our findings in relation to progesterone manipulation
suggest no accurated effect on depressive like behaviors.
From another standpoint, previous studies reported that ovariectomized rats
reduce the sucrose intake (Curtis et al, 2005) and estradiol treatment increase
resilience on learned helplessness test (Bredemann & McMahon, 2014). In addition,
Li et al. (2014) observed increase of nociceptive hypersensitivity response and
depressive-like phenotype in ovariectomized animals, however, this condition was
abolished by estradiol replacement. In this respect, parameters of social interaction
as sniffing behavior were enhanced intact female rats in proestrus phase (Frye et
al, 2000). In this sense, our data corroborate these findings, suggesting the potential
role of estrogen on mood alterations. This indicates that the amount of estrogen
64
circulation in proestrus phase could improve depressive-like behaviors, but not in
diestrus phase. We also observed that this condition can be reverting by
replacement with exogenous estradiol. As mentioned, evidence had associated
hormones fluctuations through estrous cycle with mood alterations in human and
animal studies (Romans et al, 2012; chen et al, 2012; Horst et al, 2012; Donner &
Lowry, 2013). Furthermore, the dissociative effect of sex steroids in women should
have great clinical relevance. For example, in premenstrual dysphoric disorder
(PMDD), one disabled condition marked by a cluster of affective, cognitive and
behavioral symptoms that occurs over luteal phase of menstrual cycle. The
manifestation of symptoms is marked by a cyclic pattern that begins after ovulation
and then continue through luteal phase. It is known that follicular phase is
characterized by the main controlling of estradiol that ends after ovulation, after, in
luteal phase, under the corpus luteum action the body receives a large amount of
progesterone (revised by Rapkin & Akopians, 2012). Controversially, the incidence
of depressive symptoms on PMDD is associated to role of low progesterone levels
early in the menstrual cycle (Toffoletto et al 2014). Although other studies report that
low estrogen levels after ovulation may correlate with depressed mood during the
late luteal phase (Cunningham et al, 2011; Freeman, 2002). Noteworthy, oral
treatment with progestin and estrogen combination showed efficient in reducing
PMDD symptoms (Berardis et al, 2007). Thus, while studies address the
neuromodulation of estrogens on female emotional responses, there are several
contradictions in the literature regarding progesterone effects. Nevertheless, in the
present study estradiol regulated affective responses, but not progesterone.
Most of replicated beneficial effects of estrogen found in literature are
explicated by the genomic effect of the estradiol on neurotrophic expression can be
related to neuroprotection, especially the brain derived neurotrophic factor - BDNF.
Indeed, the high levels of the estradiol in proestrus phase or 17β-estradiol
administration provoke alterations in hippocampus and pre-frontal cortex
morphology, these areas are associated with improvement of cognitive and
emotional processes, besides stress coping (Luine & Frankfurt, 2013; McEwen &
Morrison, 2013). The neurotrophic hypothesis of depression can be related to
estradiol and BDNF expression and maladaptive stress response (Zunszain et al,
2011; Duman & Nanxin li, 2012). Chronic stress lead to exacerbated glucocorticoids
65
release as consequence decreased BNDF levels and this compromise the
maintenance of neuroplasticity and neurogenesis in hippocampus. In the absence
of estradiol circulation, the vulnerability of hippocampus is amplified to deleterious
effects of glucocorticoids (Albert et al, 2015).
5. Conclusion The results suggest endogenous variations in estrogen levels across estrous
cycle modulate of depressive-like behaviors induced by social isolation. Indeed,
estradiol had a positive effect on depressive-like behaviors and this effect was
counteracted by previous exogenous administration of the antagonist. Moreover,
progesterone or mifepristone did not produce significant alterations.
Acknowledgements
This research was supported by fellowships from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Pró-reitoria de Pesquisa
da Universidade Federal do Rio Grande do Norte (PROPESQ/UFRN) and
Fundação de Apoio à Pesquisa do Estado do Rio Grande do Norte (FAPERN).
66
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PARTE II
Papel dos hormônios sexuais femininos sobre os processos de aprendizagem/memória, ansiedade
e atividade locomotora de ratas socialmente isoladas.
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Introdução
Além de papel clássico na regulação das funções reprodutivas, os hormônios
esteroides sexuais também influenciam aspectos da cognição do indivíduo. Os
hormônios sexuais estrógeno e progesterona são os mais abundantes nas fêmeas
de mamíferos, e podem ter vários efeitos em outros sistemas não reprodutivos, com
o sistema nervoso central. Os receptores desses hormônios são encontrados
extensivamente distribuídos no encéfalo, especialmente em estruturas límbicas,
como a amígdala, hipocampo, giro do cingulado, além de estruturas envolvidas nos
ajustes comportamentais como córtex pré-frontal, e em estruturais envolvidas na
regulação autonômica e controle homeostático, a saber, locus ceruleus, núcleos da
rafe, hipófise, hipotálamo e substância cinzenta periaquidutal (Shughrue et al 1997;
Hagihara et al 1992). O receptor de progesterona (PR) é uma proteína nuclear
encontrada em duas formas: PRA e PRB, e o receptor de estrogênio (ER) apresenta
dois subtipos o ERα e ERβ (Luine, 2014).
Tradicionalmente, a ativação do ER promove uma cascata de alterações
intracelulares que resultam em processos de transcrição e expressão gênica, sendo
este o mecanismo predominante pelo qual o estradiol medeia seus efeitos
biológicos no organismo, especialmente na modulação neural e consequentemente
em aspectos cognitivos. O estradiol, estrógeno mais abundante, também pode
ativar receptores de membrana (mERs), como receptores acoplados a proteína G
(GPER, conhecido como GPR30), um receptor não nuclear recentemente
descoberto, através do qual esse hormônio promove efeitos rápidos e não
genômicos (Prossnitz & Barton, 2014). A ativação do GPER inicia uma cascata
intracelular de segundos mensageiros, que incluem mobilização de cálcio, ativação
de quinases e produção de óxido nítrico. Esse efeito rápido do estradiol está
associado à excitabilidade dos neurônios e também pode regular o equilíbrio de
neurotransmissores como o GABA, o glutamato, acetilcolina, serotonina e a
noradrenalina (Revisado por Scharfman & MacLusky, 2006). Por exemplo, o 17β-
estradiol pode agir rapidamente alterando as respostas dos receptores
glutamatérgicos do tipo AMPA e do NMDA (Foy et al., 2008). Na realidade, a ligação
do hormônio com seu receptor ativa ambos os eventos de sinalização e respostas
transcriptacionais (DeWire et al. 2007).
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Os estrogênios exercem efeitos organizacionais e funcionais significativos
no hipocampo de roedores adultos. Prévio estudo demonstrou que o 17β-estradiol
que é o estrógeno mais abundante, é capaz de melhorar, prejudicar ou não ter
efeito nos processos de aprendizagem e memória hipocampo-dependente (Bean
et al, 2014). Além disso, está bem estabelecido que o 17β-estradiol é um potente
agente capaz de induzir aumento das concentrações de fatores de crescimento e
neurotróficos no encéfalo de mamíferos (Tuscher et al, 2016). Assim este hormônio
influencia aspectos de neurogênese, da diferenciação neuronal e a da
sobrevivência neuronal em todos os estágios da vida do indivíduo (Toran-Allerand,
2004), sendo capaz de melhorar o processo de potenciação de longa duração
(LTP), um mecanismo envolvido na melhoria duradoura na transmissão sináptica
do sinal entre dois neurônios envolvidos na codificação da memória (para revisão,
Luine, 2014).
A resposta genômica do estradiol tem efeito profundo na gênese e
manutenção celular. Brann e colaboradores (2007) revisaram vários estudos
envolvendo os efeitos neuroprotetores do estradiol, dentre eles sua ação protetora
contra os efeitos excitotóxicos do glutamato, diminuição da apoptose mediada por
ERα e aumento da síntese de transportadores de glicose, o que aumenta a
sobrevivência das células em eventos de isquemia. Além disso, o estradiol está
associado à redução de risco para demências tardias, além de ser um fator de
proteção contra a doença de Alzheimer (para revisão Lan et al, 2015). Portanto, os
efeitos de neuroproteção do estrogênio devem ocorrer por vias genômicas e de
efeito prolongado.
A aprendizagem é o processo no qual novas informações são adquiridas,
requerendo um conjunto de modificações celulares, por exemplo algumas
memórias do tipo declarativa, associativa e de valência emocional geralmente
requerem circuitarias que envolvem a formação hipocampal e o complexo
amigdaloide (para revisão Cameron & Glover, 2015).
O mesmo processo de formação da memória compreende os estágios de
aquisição, consolidação e evocação. É bem aceito que fatores internos e externos
podem influenciar esses diferentes estágios da formação da memória, facilitando
ou até mesmo podendo comprometendo a formação da memória. Sabe-se que
estados emocionais, ansiedade, atenção e picos de alerta/vigilância são
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necessários fatores psicofisiológicos inerente ao processo de aquisição (Eysenck
1976). Durante a consolidação há um conjunto de alterações fisiológicas que
participam do processo de estabilização da informação recém adquirida, essa fase
é descrita como sendo lábil e facilmente modulada por fatores diversos, como
estresse e hormônios sexuais (Roozendaal & McGaugh, 2011). A consolidação da
memória garantirá o acesso a informação previamente aprendida.
No que diz respeito aos efeitos da resposta de estresse e hormônios sexuais
sobre os aspectos de aprendizagem e memória, alguns estudos em nosso
laboratório vêm demonstrando efeitos em ratas testadas na tarefa de esquiva
discriminativa em labirinto em cruz elevado (EDLCE), uma versão modificada do
labirinto em cruz elevado que permite avaliar simultaneamente aspectos de
ansiedade, atividade exploratória, aprendizagem e memória aversiva (Silva &
Frussa-Filho, 2000). Por exemplo, De Macedo Medeiros (2013) observou prejuízos
na aquisição da memória aversiva em fêmeas que realizaram a tarefa de EDLCE
em diestro, mas não nas demais fases. Esse mesmo estudo demonstrou que
administração de estradiol reverte os prejuízos de memória induzidos por
escopolamina, sugerindo o papel dos hormônios sexuais na transmissão
colinérgica envolvida no processo mnemônico.
No estudo de Melo e colaboradores (2012) foi observado que ratas
aprenderam a tarefa de EDLCE e também evocaram a memória aversiva, contudo,
somente as fêmeas tratadas com fluoxetina foram capazes de extinguir a memória
aversiva. A memória aversiva formada na EDLCE parece ser robusta nas fêmeas.
De fato, Ribeiro et al (2010) observaram a ausência da extinção da memória
aversiva em ratas na mesma tarefa. Além disso, em um estudo anterior
observamos que prejuízos na memória aversiva de ratas estressadas por
isolamento social e estresse de contenção eram ausentes na fase proestro e estro
(Nascimento et al., em preparação, Anexo 1). Portanto, a circulação endógena de
hormônios sexuais, em especial o estradiol, parece modular aspectos envolvidos
na formação da memória aversiva, algo já relatado em outras diferentes tarefas,
tanto em modelos animais como em humanos (Revisado por Luine, 2014).
A progesterona, assim como estradiol, é um hormônio que também
desempenha um papel modulatório nos processos cognitivos (Lovick, 2012). No
entanto, os efeitos da progesterona sobre os processos de aprendizagem/memória
são frequentemente contraditórios. Esse hormônio pode se ligar a receptores
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nucleares ou também a receptores de membrana (mPRs), estes últimos podendo
ser ativados pela progesterona, mas também pelo seu metabolito
(alopregnanolona, ALLO). Os receptores de membrana da progesterona quando
ativado alteram rapidamente a sinalização celular via modulação de cascatas de
sinalização intracelular. Os mPRs parecem estar envolvidos nos efeitos
neuroprotetores e também antigonadotrópicos, reduzindo as concentrações de
outros hormônios como o luteinizante (LH), folículo estimulante (FSH) e estradiol
(Giatti et al, 2016). Além disso, a ALLO é um potente modulador alostérico positivo
do receptor GABAA em áreas do sistema límbico como a amigdala, ínsula, giro do
cingulado anterior e área ventral tegmentar. Essas estruturas estão relacionadas
com a resposta emocional, controle da ansiedade, estado de alerta e
responsividade ao estresse (Lovick, 2012). Nesse sentido, a progesterona também
é capaz de modular os processos de aprendizagem e memória emocional.
A crônica ativação da resposta de estresse por meio dos glicocorticoides
(cortisol em humanos e corticosterona em ratos e camundongos) promove
hiperativação da amígdala que por sua vez atua modulando a ativação do
hipocampo, o que comprometeria os aspectos da neuroplasticidade e
subsequentemente os processos mnemônicos (Kim and Diamond, 2002). De fato,
os núcleos basolateral (BLA) e basomedial (BM) da amígdala via transmissão
gabaérgica, inibem a atividade pós-sináptica de neurônios da região CA1 do
hipocampo ventral (vHPC, Bazelot et al, 2015). Agís-Balboa e colaboradores (2007)
observaram que a ALLO reduziu a atividade de diferentes núcleos da amígdala,
incluindo a BLA, e consequentemente promoveu alterações comportamentais
induzidas por isolamento social em roedores. Assim, a ativação da amígdala é
essencial para a aprendizagem/memória emocional, mas a sua ativação excessiva
ou combinada a ativação da resposta de estresse prejudica o funcionamento
hipocampal e consequentemente promove déficits, sendo que os esteroides
sexuais também modulam este circuito.
O hipocampo é alvo principal do efeito dos glicocorticoides durante a
resposta de estresse. Os efeitos produzidos por estes hormônios podem ser
bifásicos promovendo respostas que facilitem a atividade neuronal nessa estrutura,
mas que em uma ação crônica tem um efeito deletério, podendo reduzir as
concentrações de fatores neurotróficos, como o BDNF, que estão envolvidos no
processo de consolidação das memórias (para revisão Cameron & Glover, 2015).
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Essa hipótese é corroborada por estudos que mostram que o estradiol pode
aumentar as concentrações de BDNF, e produzir a reversão dos prejuízos
promovidos pelo estresse (Luine & Frankfurt, 2013).
Como mencionado anteriormente, embora vários estudos relatam os efeitos
do estradiol e progesterona sobre os déficits de aprendizagem/memória eliciados
pelo estresse, ainda não são totalmente esclarecidos os efeitos de cada hormônio
na aprendizagem e memória de uma tarefa com conteúdo aversivo em ratas
fêmeas estressadas.
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Artigo 2
Distinct roles of estradiol and progesterone in learning, anxiety and exploratory behavior of socially stressed female rats
Ezequiel Batista do Nascimento1,2, Aline Lima Dierschnabel1,2, Antônio Carlos
Queiroz2, Sara Sophia Guedes2, André de Macêdo Medeiros3,4, Ramon Hipólito
Lima1,2, Deborah Suchecki5, Regina Helena Silva1,3, Jeferson de Souza
Calvacante2, Alessandra Mussi Ribeiro1,6*
1Memory Studies Laboratory, Department of Physiology, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
2Laboratory of Neurochemical Studies, Department of Physiology, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
3Laboratory of Behavioral Neuroscience, Department of Pharmacology, Universidade Federal de São Paulo, SP, Brazil
4Center of Health and Biological Sciences, Universidade Federal Rural do Semiárido, Mossoró, Brazil
5Department of Psychobiology, Universidade Federal de São Paulo, SP, Brazil 6Laboratory of Neuroscience and Bioprospecting of Natural Products, Department of Biosciences,
Universidade Federal de São Paulo, Santos, SP, Brazil
*Corresponding author: Alessandra M. Ribeiro
Departamento de Biociências
Universidade Federal de São Paulo
Rua Silva Jardim 136 – CEP: 11015-020, Santos/SP
Tel: (55) 13 3229-0137
E-mail: [email protected]
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Abstract A growing body of evidence has shown the role of sex steroids and
corticosterone (CORT) in cognition and emotion. It is suggested that ovarian hormones may have beneficial actions on several neurophysiological processes, mainly on memory systems. On the other hand, chronic exposure to stressful conditions clearly exerts negative effects on neural structures related to learning and memory. In addition, ovarian and stress-related hormones may decrease or improve mammals’ performance in hippocampus-dependent tasks. In the present study, we used classical open field (OF) test and the plus-maze discriminative avoidance task (PMDAT) to evaluate the influence of endogenous variation of sex hormones and exposure to long-term of social isolation stress on learning, memory, anxiety-like behavior and exploratory behavior. Female Wistar rats were submitted to seven consecutive days of social isolation and tested in different estrous cycle phases (diestrus, proestrus and estrus), each phase received pharmacological treatment, e.g., females in diestrus (low steroids profile), received treatment of 17βestradiol or progesterone in order to antagonizes the estrous phase whereas proestrus and estrus, high estradiol and progesterone profile, received antagonist tamoxifen (TAM) and mifepristone (MIF), respectively. The main results showed that: (1) neither stress conditions nor hormonal treatment modified the acquisition of the task; (2) overall, social isolation and estrogen blockade induced memory impairments; (3) there was no impairment in stressed female treatment with estradiol; (4) Animal treated with progesterone reduced anxiety behavior; (5) There is a hyper-locomotor activity induced by social isolation. Taken together, our findings suggest that sex-steroids is an important modulatory factor of cognitive processing disrupted by stress in female rats. This effect was observed in proestrus treatment, moreover, progesterone treatment appears to influence in emotional response, both hormones is related to buffer the effects of stress on learning/memory processes. Keywords: Estradiol; progesterone; memory; stress; estrous cycle
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1. Introduction
Evidence points to high prevalence of anxiety disorder in women than in men
(Alexander et al,2007; Steel et al, 2014; Kessler, 1994; Caballo et al,2014). It is
widely documented that females are at increased risk for anxiety disorders and
reproductive factors are supposed to contribute to the majority of this vulnerability
(Soares & Zitek, 2007; Piccinelli & Wilkinson, 2000). Still, there are a clear sex
differences in prevalence and onset of stress-related mood disorders in humans.
These differences also need to be clarified in order to have a better understating in
the gender difference observed in emotional response, which are likely to be
contributed by the sex difference in stress reactivity.
Female sex hormones are widely described to be a potent modulator of
emotional response (Wald and Frye, 2010; Zeidan et al 2011; Lynch III et al, 2014).
Further, most of these effect on emotional response is followed by cyclic pattern of
estrous cycle (Mora et al 1996; Marcondes et al, 2001; D'Souza & Sadananda,
2017). As revised by Milad and colleagues (Cover et al, 2014) mechanisms of
estradiol may contributes to underlying sex difference on emotional response and
neural pathophysiology of fear conditioning and aversive memory. Intriguingly,
disorders such posttraumatic stress disorders (PTSD) or major depression occur
more frequently in woman then man. Also, these disorders are extensively related
to stressful situations. Regarding to stress response, evidences in rodents suggests
that female subject is more sensitive for stressful situations, have low adaptation to
stressors, highest corticosterone levels and high stress responsivity (Bangasser et
al, 2010; Viau, 2017; Babb et al 2014; Goel et al, 2014).
The hippocampus is a limbic structure important in learning and memory
process. This area is a target to action of glucocorticoids (GC) and are linked with
different modulation of GC over this structure (Kim et al 2015;). Studies are
described to show that chronic can compromise the hippocampus by reducing
neurogenesis, neuroplasticity and producing cell death. Such changes in
hippocampus make this structure vulnerable to abnormalities and altered
functionality (Moreira et al, 2016). In this sense, experimental animal models of
learning and memory are sensitive to detect changes in mnemonic processes
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suggesting predictive evidences about hippocampal functionality (Jarrard, 1993;
Gluck and Myers, 1997). Effects of chronic stress in hippocampus are reported to
memory impairment in different memory tasks (for review Conrad, 2010). Moreover,
studies in human have reported changes in hippocampus in patient with cognitive
deficits (Cornwell et al 2010; Sapolsky 2001; Sawyer et al, 2012). The relation
between stress and memory is already well investigated and it is known that an
optimum level of stress, arousal and anxiety are necessary for learning to occur,
causing distinct cognitive outcome under low or high stress conditions (Silva &
Frussa-Filho, 2000; Kameda et al., 2007; Joels, 2006; Forster, 2012), and these
variations on stress intensity are related to sex differences (Luine, 2014; Zorawski
et al., 2006). Acute or chronic stress protocols shows distinct fear response (Wood
& Shors, 1998; Shors, 2004; Zorawski et al., 2006), as well as exploratory activity
(Dubovicky et al., 1999), motivation (Kleen et al., 2006) and anxiety levels (Ieraci,
et al, 2016).
From another standpoint, sex steroids are hormones that are mainly
synthesized in the gonads under influence of neural axis initiated in hypothalamus.
The 17b-estradiol and progesterone are two steroid hormones that modulates the
in oscillatory expression across estrous cycle. Both hormones have been reported
to promotes effects on cognition and brain function. It seems several cognitive and
emotional processing appear to be dependent on the level of estradiol and
progesterone in pattern of inverted u-shaped dose curve (Revised by Foster,2012).
Studies in animal models indicated cognitive function can be modulated by the
estrous cycle or menstrual cycle in women (Scharfman et al, 2003; Poromaa &
Gingnell, 2014). Clinical evidences research linked women following surgical
removal of their ovaries or during menopause to memory impairment, dementia and
Alzheimer (Bove et al, 2014; Kurita et al, 2016; Rocca et al 2014). Hence, most of
deficits can be reversed by estrogen replacement therapy (Hogervorst et al, 1998;
Maki & Sundermann, 2009). Thus, the aim of the present study was to investigate
the dissociative effect of estradiol and progesterone over cognitive impairments,
anxiety and exploratory behavior elicited by social isolation. We used classical
models of anxiety investigation (open field), also, we used plus-maze discriminative
avoidance task (PMDAT: Silva & Frussa-Filho, 2000) in order to evaluate anxiety
levels and motor activity related to learning and memory performances.
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2. Materials & Methods
2.1. Animals 104 three-month-old Wistar female rats with regular estrous cycle from our
colony were used. Each animal was allocated in plastic cages (30 x 37 x 16 cm,
fiver per cage) under controlled temperature (22-24C) and light-dark cycle of 12/12
h (lights on ate 6:00 am). Conditions of food and water were available ad libitum
over the whole experiments. All procedures described in this study were approved
by the local ethical committee (CEUA/UFRN Nº 006/2014) and are in total
accordance to the Brazilian law for the use of animals in scientific research (Law
Arouca nº 11.794).
2.2. Drugs
The 17β-estradiol soluble water (17β, Sigma, USA) was diluted in saline
solution 0.9 % and given i.p. at 0.2 mg/kg. A selective estrogen-receptor modulator
(SERM) - Tamoxifen citrate (TAM, Sigma, USA) was dissolved in vehicle (saline
solution containing 10% of Tween 80) and given i.p. at 1 mg/kg. Progesterone
(PROG, Tocris, USA) was diluted in sesame oil and given subcutaneous at 0,5
mg/kg. A competitive progesterone receptor antagonist - Mifepristone (MIF, Tocris,
USA) was diluted in a solution with absolute ethanol, sesame oil and tween 80 and
given at 10 mg/kg. Saline solution or sesame oil in a volume of 10 ml/kg were
administered in vehicle animals of both groups. Doses of 17β-estradiol and
tamoxifen was chosen based in previous studies (Sandstrom & Williams, 2001;
Sharma, 2007; Packard & Theater, 1997) that showed the enhancement effect of
estradiol on cognition. For progesterone and mifepristone administration, doses
were chosen based on study of Aisa et al. (2006) that investigated the modulation
effect of progesterone on cognition and emotional response.
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2.3. Estrous cycle Samples of vaginal smears was collected daily during the whole experimental
protocol idetermine the estrous cycle phase. For this procedure, tips of a plastic
pipette filled with 100 µL of distilled water was gently introduced into the rat vagina,
then the bulb of the pipette was slightly pressured and the vaginal fluid entered the
interior of the pipette upon release. This material was placed on glass slides, dyed
with methylene blue and examined under a light microscope to examine the vaginal
cytology. The estrous cycle comprises four distinct phases: metaestrus (or early
diestrus), diestrus, proestrus and estrus, that are identified according to the
cytological features. The proestrus phase is characterized by the predominance of
epithelial nucleated cells, while the estrus phase is characterized by the presence
of cornified cells. Diestrus phase presents a predominance of leukocytes and the
metaestrus shows similar proportion of the features of the other phases (Byers et
al, 2012). To ensure that all female rats were cycling regularly, the estrous cycle
was determined for 10 days, at least, before the beginning of the experimental
procedures. We considered metestrus and diestrus phases together for analysis,
based on evidences of similar estradiol profile (Spornitz et al, 1999; Paccola et al,
2013) and lack of basal behavioral differences in the present study.
2.4. General procedures and stress condition Animals were randomly assigned to two groups: control (CT, n = 48) and
social isolation (SI, n = 56). Control rats remained in their home cage. Rats that
underwent stress conditions were transferred to a separate room. In social isolation
stress condition, the animals were removed from their home cages and separated
into individual cages (42 cm length, 34 cm width and 18 cm height) during 24h for
seven consecutive days. The individual cages were placed in isolated environments
to prevent vocalization among animals.
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2.5. Open Field
After the last day of stress exposure, the animals were submitted to the
open field test. The apparatus was a circular arena (84 cm in diameter) with high
walls, made of wood and painted in black. Animals were placed in the center of
arena for free exploration during 5 min. After the session, animals were retired and
returned to home cage. Parameters of locomotor activity were quantified by total
distance travelled on apparatus (in meters), percentage of time spent on central
area, total time spent on central and peripheral areas (in seconds).
2.6. Plus-maze discriminative avoidance task (PMDAT) The wood-made apparatus used is a modified version of the conventional
plus-maze. The maze contains two enclosed arms (50 length x 15 width x 40 height
cm): one aversive arm (AV) and one non-aversive arm (NAV) opposite to two open
arms (OA; 50 length x 15 width cm). The PMDAT is conducted in two sessions:
training and test, each session lasting 10 minutes. In both sessions, the animals
were individually placed in the center of apparatus with body orientation toward the
intersection between the open arms (Fig. 1). In the training session, the aversive
stimuli were triggered each time animals entered with the whole body in the aversive
enclosed arm and turned off when the animal left the arm. The aversive stimuli were
an 80-dB noise and a 100W light produced by speakers and lamp, respectively,
placed over the aversive enclosed arm. Memory acquisition was measured during
the training session by percentage of time in aversive arm (%TAV). In the test
session (24h later), the animals were placed again in the center of the apparatus
and allowed to explore the apparatus without presentation of the aversive
stimulation. In this session, we evaluated memory retrieval was evaluated. All
behavioral experiments were performed between 1:00 and 5:00 p.m. The sessions
were recorded by a digital camera placed above the apparatus and the behavioral
parameters were registered by a video-tracking software (Anymaze, Stoelting,
USA). Learning and memory were evaluated by the percentage of time spent in the
aversive arm [%TAV = time in AV / (time in NAV + AV) x100] across the training and
test sessions (in three blocks of 200 seconds each). Anxiety-like behavior was
evaluated by the percentage of time spent in the open arms [%TOA = time in OA /
(time in NAV + AV + OA) x 100. We only considered the training session for
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evaluation of anxiety because the anxious response to novelty is absent in a second
exposure to a plus-maze apparatus (Pereira et al, 1999). Locomotion was measured
by the distance travelled in the apparatus. At the end of each behavioral session,
the apparatus was cleaned with a 5 % alcohol solution.
Fig. 1. Schematic diagram of the behavioral plus maze discriminative avoidance
task - PMDAT.
2.7. Drug administration After stress protocol female rats were assigned to groups according to their
estrous cycle phase (metestrus/diestrus = DIE: proestrus = PRO; estrus = EST).
Animals in DIE of both conditions received VEH (CT = 6, SI = 6), 17βestradiol (CT
= 6, SI = 8) or progesterone (CT = 6, SI = 8). For animals in proestrus or estrus, we
injected VEH (CT = 6, SI = 6), tamoxifen (CT = 6, SI = 8) or mifepristone (CT = 6, SI
= 8). After 1h, the animals were submitted to the open field and 15 minutes after to
the training in the PMDAT. The PMDAT test was performed 24h later, without
pharmacological treatment.
2.8. Statistical Analysis Analysis was conducted considering: (1) effects of social isolation and (2)
effects of hormones and antagonists in both groups. All data were checked for
normality with Kolmogorov-Smirnov test. Three-way ANOVA with repeated
measures was performed for the %TAV in time blocks across training and test
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sessions for the different conditions and treatments. Also, Two-way ANOVA was
performed to analyze %TOA and distance travelled in PMDAT; For % in central and
peripheral as well as distance traveled in open field. Tukey post-hoc test was applied
to all parameters. All results were considered significant at p < 0.05.
3. Results
3.1. Anxiety and exploratory behavior on OF
3.1.1. Open field test: 17β-estradiol treatment The two-way ANOVA for distance travelled revealed condition effect [F (1,25)
= 4.484, p = 0.04]. No alterations were found in percent of time spent in central or
peripheral area. Overall, social isolation increased distance travelled in open field.
3.1.2. Open field test: Tamoxifen treatment No alterations were found in tamoxifen-treated animals.
3.1.3. Open field test: Progesterone treatment A significant treatment effect was revealed by two-way ANOVA for
distance travelled [F (3, 23) = 22.982, p = 0.001], percentage time spent in central
area [F (1,25) = 24.047, p = 0.001] and peripheral area [F (3,23) = 5.066, p = 0.03].
Tukey’s post hoc showed increased distance traveled and time central area of
PROG-treated rats, but there was decrease of the time spent in peripheral area
(Table1). Moreover, there were condition effect for percentage time spent in central
[F (1, 25) = 4.296, p = 0.05;] and peripheral area [F (1,25) = 14.147, p = 0.001],
indicating reduced time spent in periphery of treated PROG-females when
compared to vehicle group (Table 1).
3.1.4. Open field test: Mifepristone treatment Two-way ANOVA revealed condition x treatment interaction [F (1,25) =
14.792, p = 0.004] for distance traveled [F (1,25) = 14.792, p = 0.004]. Indeed,
Tukey’s post hoc test demonstrated that isolated females increase the distance
traveled when compared to non-isolated animals. Moreover, MIF-treated isolated
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animals showed higher increase of the locomotor activity when compared to non-
stressed and vehicle groups.
A significant treatment x condition interaction for percentage of time in central
area [F (1,25) = 5.361, p = 0.03] and peripheral area [F (1,25) = 5.244, p = 0.03]
was revealed by two-way ANOVA. Firstly, MIF treatment increased exploration in
central area and consequently reduced time spent in peripheral area for isolated
animals. In addition, MIF-treated animals spent more time in central area compared
to other groups (Table 1).
Table 1
Effects of social isolation and hormonal manipulation in open field.
Values are expressed as mean ± SD. ap < 0.05 compared to control vehicle, bp < 0.05 compared to isolated vehicle, cp < 0.05 compared to control MIF. Two-way ANOVA followed unequal Tukey’s post-hoc test.
Condition Treatment Distance traveled (m) % In central area % In peripheral
Control VEH 5.2 ± 2 12 ± 4 88 ± 4
17β 6.7 ± 2 18 ± 12 82 ± 12
Isolation VEH 6.3 ± 5 12 ± 9 88 ± 9
17β 10.1 ± 3 15 ± 8 85 ± 10
Control VEH 7.8 ± 2 16 ± 2 84 ± 2
TAM 6.6 ± 2 18 ± 4 82 ± 4
Isolation VEH 10.2 ± 5 16 ± 4 85 ± 4
TAM 6.7 ± 4 15 ± 7 85 ± 7
Control VEH 3.3 ± 1 27 ± 4 93 ± 4
PROG 8.7 ± 2a 67 ± 26a 48 ± 29
Isolation VEH 4.9 ± 4 17 ± 5 66 ± 50
PROG 9.1 ± 2ab 79 ± 14ab 21 ± 20ab
Control VEH 5.2 ± 2 6 ± 3 94 ± 4
MIF 2.4 ± 1 12 ± 6 88 ± 6
Isolation VEH 7.3 ± 3c 35 ± 43 66 ± 44
MIF 12.8 ± 3abc 85 ± 24abc 15 ± 29abc
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3.2. Learning and memory on PMDAT
3.2.1. Plus-maze discriminative avoidance task: 17β-estradiol treatment In the training session, three-way ANOVA in time blocks for %TAV revealed
time effect [F (2,44) = 31.96, p = 0.001], indicating reduction of the exploration in
aversive arm (Fig 2A), this result suggest that all animals learned the task. In the
test session, three-way ANOVA for % TAV revealed time x treatment interaction [F
(2,44) = 6.11, p = 0.004]. Tukey’s post hoc showed that isolated females spent more
time in aversive enclosed arm in the first block, indicating a deficit of retrieval of the
aversive memory (Fig. 2B). Further, 17β- and vehicle-treated animals reduced the
time exploration in aversive arm in the first block, showing memory retrieval (Fig.
2B)
3.2.2. Plus-maze discriminative avoidance task: Tamoxifen treatment In the training session, three-way ANOVA for %TAV revealed time effect [F
(2,44) = 3.24, p = 0.04] (Fig 2C). Regarding to test session, three-way ANOVA for
%TAV revealed time x condition interaction [F (2,44) = 12.55, p = 0.004] and time x
treatment interaction effect [F (2,44) = 2.91, p = 0.04]. Tukey’s post hoc for time x
treatment interaction revealed that TAM-treated animals and stressed animals had
increase of exploration of the aversive arm in the first block when compared to
control group treated vehicle (Fig. 2D). This result indicates that tamoxifen treatment
may induce memory impairment as well as isolation social.
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Fig. 2. Effects of social isolation (SI), Control condition (CT) in Estradiol (17β, A-B), Tamoxifen (TAM, C-D), Progesterone (PROG, E-F) and Mifepristone treatment (MIF, G-H) on learning of female rats submitted to the plus-maze discriminative avoidance task (PMDAT). Mean ± SE for the percentage of time spent in aversive enclosed arm (%TAV) in 200s time blocks during training session. Left side, graphics represents exploration in training session; right side, graphics represents test session. Time effect, p < 0.05 in training session showed all experimental groups reduce aversive arm exploration. *p < 0.004, SI-VEH compared to SI-EST, CT-VEH and CT-EST (B); **p < 0.04, SI-TAM, SI-VEH, CT-TAM compared to CT-VEH (D); #p < 0.04, SI-VEH and SI-PROG compared to CT-VEH; +p <0.04, SI-PROG compared to SI-VEH (F); +p <0.004, SI-MIF compared to SI-VEH; ++p < 0.004, SI-VEH compared to CT-VEH (H). All pairwise comparison was performed by three-way ANOVA with repeated measures followed by Tukey’s post hoc test.
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3.2.3. Plus-maze discriminative avoidance task: Progesterone treatment Considering training session, three-way ANOVA in time blocks for
%TAV revealed time effect [F (2,46) = 14.08, p = 0.00]. Post hoc analysis showed
that all groups reduced the exploration in the aversive arm (Fig. 2E). In the test
session, when %TAV was analyzed in time blocks, three-way ANOVA time x
treatment interaction [F (2,46) = 3.21, p = 0.04]. Tukey’s post hoc demonstrated that
animals treated with progesterone spent less time in the aversive arm when
compared to isolated females in the first block. This result showed that progesterone
treatment countered the isolation effect (Fig. 2F).
3.2.4. Plus-maze discriminative avoidance task: Mifepristone treatment In the training session, three-way ANOVA revealed effect [F (2,46) = 15.86,
p < 0.001], indicating that all experimental groups exhibit significant decrease for
%TAV (Fig 2G). In the test session, when %TAV was analyzed, three-way ANOVA
indicated time [F (2,46) = 5.99, p = 0.004] and time x treatment effect [F (2,46) =
6.05, p = 0.004]. Tukey’s post hoc showed that in the first block again isolated female
treated with vehicle spent more time in aversive arm when compared to non-
stressed females. However, the treatment with MIF protected the animals of the
memory deficit induced by stress (Fig. 2H).
3.3: Anxiety and exploratory behavior
3.3.1. Plus-maze discriminative avoidance task: 17β-estradiol treatment For distance travelled on PMDAT, two-way ANOVA revealed condition effect
[F (1,25) = 1.34, p = 0.002]. It is shown that isolated females increased the distance
travelled on apparatus in training session (Fig. 3A). For percentage of time spent in
open arms (%OA), again a significant effect of the condition [F (1,25) = 7.46, p = 0.03]
and treatment [F (3,25) = 8.46, p = 0.008] was found. Tukey’s post hoc indicated
isolated females treated with vehicle decreased the time spent in open arms when
compared to EST-treated females (Fig. 4A)
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3.3.2. Plus-maze discriminative avoidance task: Tamoxifen treatment No differences were found for distance travelled (Fig. 3B) and percentage
spent in open arms (Fig. 4B) in animals treated with tamoxifen.
Fig. 3. Influence of social isolation and Control condition (CT) in the treatment of Estradiol (17β, A), Tamoxifen (TAM, B), Progesterone (PROG, C), Mifepristone treatment (MIF, D) effects on females tested in the plus-maze discriminative avoidance task. Mean ± SE for the distance travelled in all apparatus during training session. *p < 0.004 for condition effect on 17β and PROG treatment. All statistical analyses were performed by two-way ANOVA followed by the Tukey's post hoc test.
3.3.3. Plus-maze discriminative avoidance task: Progesterone treatment Two-way ANOVA for distance traveled revealed condition effect [F (1,
25) = 13.23, p = 0.002]. Further, a significant condition [F (1,25) = 18.671, p = 0.002]
and treatment effect [F (1,25) = 4.466, p = 0.04] were found to time spent in open
arms (%OA). Tukey’s post hoc showed that both control and isolated females
treated with progesterone increased percentage of exploration in open arms when
compared to non-stressed female treated with vehicle (Fig. 4C).
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3.3.4. Plus-maze discriminative avoidance task: Mifepristone treatment For distance traveled, two-way ANOVA found condition [F (1,25) =
7.77, p = 0.01] and treatment effect [F (1,25) = 9.852, p = 0.04]. Regarding %OA,
the analyses revealed a significant effect of condition [F (1,25) = 33.211, p = 0.007]
and condition x treatment interaction [ F (3,25) = 8.624, p = 0.0007]. Tukey’s post
hoc demonstrated that isolated females treated with mifepristone increased the time
spent in open arms when compared to all experimental groups (Fig. 4D).
Fig. 4. Influence of social isolation in the of Estradiol (17β, A), Tamoxifen (TAM, B), Progesterone (PROG, C), Mifepristone (MIF, D) effects on female tested in rats submitted to the plus-maze discriminative avoidance task. All treatment received respective VEH. Time spent in open arms (%OA) during training session. *p < 0.002 for condition effect; **p < 0.008 for SI-VEH compared to CT-17β; #p < 0.04 for treatment, PROG-treated females increased %OA when compared to all VEH conditions; *p < 0.007 for condition effect in MIF treatment and +p <0.007 for condition x treatment interaction, SI-MIF compared to others experimental conditions. All statistical analyses were performed by two-way ANOA followed by the Tukey's post hoc test.
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4. Discussion
In this study our main results showed that (1) Neither social isolation nor
hormone manipulation impaired the acquisition of PMDAT; (2) social isolation and
hormone manipulation affected the retrieval of PMDAT (3) progesterone and
estradiol treatments showed dissociative effects on memory in the PMDAT; (4)
social isolation induced an increase in locomotor activity evaluated in the open field.
PMDAT allows the evaluation of anxiety-related behavior, locomotor activity
and learning/memory simultaneously (Silva & Frussa-Filho, 2000). Previous studies
performed with this task have shown that this paradigm is useful for the evaluation
of learning and retrieval process by the analysis of animal behavior during training
and test sessions, respectively (Ribeiro et al, 2010; Silva et al, 2016). Thus, since
PMDAT allows concomitantly evaluate anxiety and locomotor activity, we
investigated whether anxiety-like behavior or locomotor activity interfere on the
aversive memory acquisition in stressed females. Our results suggest that the stress
did not affect the acquisition of the PMDAT. Additionally, no hormonal treatment
effects were observed in relation to the learning of the task. These findings
corroborate previous studies reported by our group (Ribeiro et al. 2010; Melo et al.
2012; De Macedo Medeiros et al. 2014).
The effects of stress on learning processes are highly influenced by the type
of stress, duration or stage (prior or after training session) (Cadle & Zoladz, 2015).
In general, studies showed acquisition enhancements, impairments or no effects on
learning processes (For review; Roozendaal, 2002). Indeed, it is well-known that
stress prior training session can facilitate acquisition of aversive memory, for
example, restraint stress ameliorates the acquisition in rats submitted to eyeblink
conditioning (Servatius et al, 2001), contextual fear conditioning (Sandi et al, 2001).
Conversely, long-term exposure to unpredictable stress and social isolation impairs
acquisition of spatial memory in Morris water maze, working and short-term memory
on Y-maze (Jung et al, 2017; Famitafreshi et al, 2015) In addition, stress effects on
learning process are related to an inverted-U-shape response curve, suggesting that
is required an optimal level of glucocorticoid involved in emotionally arousing during
acquisition stage (revised by, Roozendaal et al, 2009). Most of arousal status is
modulated by noradrenaline released into the amygdala during stressful or
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emotional experiences (revised by, Roozendaal et al, 2009). In this sense,
emotional events play an important role in the learning processes (Pessoa, 2010).
Estrogens and progesterone are sexual hormones that exerts a broad range
of effects on the systems, including the central nervous system (CNS). These
hormones long considered for their roles as primary in reproductive behavior, are
now being studied as cognition modulator. Previous studies have shown effects of
estrous cycle on acquisition (Rubinow et al, 2004; Conrad et al, 2004; Silva et al,
2016; De Macedo Medeiros et al, 2014; Mora et al, 1996). Moreover, estradiol
replacement in ovariectomized females enhanced eyeblink conditioning (Leuner et
al, 2004) and improved performed of aged rats in learning inhibitory avoidance or
reduced time to reach platform of water maze task (Frye et al, 2005). As mentioned,
in our study no changes were observed in acquisition of PMDAT independently of
the pharmacological manipulations, this is evidenced by the learning curve
performed by all conditions (Fig. 2).
In this study, memory retrieval of the task was investigated by the analysis of
%TAV in time blocks throughout the session. Social isolation provoked deficits in
the task retrieval dependent of the hormonal manipulation. Indeed, the
administration of the estradiol blocked the amnestic effect of the stress.
The blockade of proestrus phase by TAM in rats impaired of associative
memory formation in the T-maze (chen et al 2002), conditioning of appetitive context
(Esmaelli et al 2008), novel recognition object memory and performance in the
inhibitory avoidance task (Valvassori et al, 2017). In humans, cognitive impairments
have been associated with endocrine clinical therapy for breast cancer treatment
(Reviewed by Agrawal et al, 2010). Indeed, chronic treatment of TAM in women
impaired the declarative memory and verbal fluency (Boele et al, 2015). Bender et
al. (2007) evaluated women in early-stage treatment of breast cancer with
anastrazole have reported impairment in the attention, memory, and mental
flexibility, besides depressive symptoms and fatigue. Besides, women that
tamoxifen showed reduction of the negative effects on cognition and emotional
response. Hence, these studies are in consonance with our results, the treatment
with TAM impaired the memory retrieval of females in the PMDAT independent of
the condition. Thus, these deleterious effects provoked by TAM may involve various
structures and difference mechanisms of the memory formation (Janelsins et al
2014). For example, Valvassori et al. (2017) demonstrated that memory deficits
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provoked by TAM in females, but not male, are correlated with neurotrophic factors
(NGF) in hippocampus, such brain derived neurotrophic factor (BNDF). It should be
noted that NGF and BNDF are highly requested for neuroplasticity involved in
mnemonic processes (Cunha et al 2010), moreover, the expression of this factors
are profoundly influenced by estrogen circulation (Luine & Frankfurt, 2013).
The data presented here indicated that progesterone treatment counteracted
the negative effects of social isolation on the memory retrieval (Fig. 2F). Hence,
progesterone appears to ameliorate the damages caused by stress. Interestingly,
MIF treatment caused appositive effect in test session (Fig. 2H). This effect can be
related to influence of other hormones, MIF is an antiprogestogen and exerts effect
as an antiglucocorticoid, it seems to antagonize the glucocorticoids action
competitively at the receptor level (revised by, Gallagher & Young, 2006). Acute
administration of mifepristone in rats reversed memory impairment elicited by
chronic mild unpredictable stress and maternal separation (Wu et al, 2007; Loi et al,
2017). Furthermore, MIF treatment improved cognition and emotion response in
patients with neuropsychiatric disorders related to Cushing's syndrome (For review,
Pivonello et al, 2015).
It is well established that hormonal oscillation across estrous cycle or
exogenous administration of estradiol and progesterone can alter anxiety behavior
in rodents (D’souza & Sadananda, 2017; Marcondes et al, 2001; Mora et al, 1996;
Walf & Frye, 2010). In our study, stressed females reduced exploration in central
area and open arms, in open field (Table 1) and PMDAT (Fig. 4ABC), respectively.
These anxiogenic effects of the stress were observed in animals that performed
behavioral tasks during diestrus phases (low profile of estradiol and progesterone).
Thus, it seems that higher levels of anxiety and responsiveness to stress are
increased during diestrus phase compared to proestrus and estrus phases (Lovick,
2012). Despite of isolated animals showed increased anxiety during PMDAT training
no alterations were observed in PMDAT acquisition, because all groups
demonstrated reduced time in aversive arm during training session. In this sense,
deficits memory retrieval did not be associated with anxiety changes. Interestingly,
we observed anxiolytic effect in MIF-treated stressed animals in estrus phase (high
progesterone profile). Studies in rodents and humans reported that stress induce
increase in progesterone level (Kalasz et al 2014; Herrera et al, 2015). Hence,
100
anxioylitc effect found in our study may be associated with progesterone modulatory
effect.
In support of dissociative effect of progesterone and estradiol on anxiety, our
data demonstrated no effect of estradiol or tamoxifen treatment on anxiety
parameters. On the other hand, we observed anxiolytic effect of the progesterone,
animals treated with this hormone increased percentage time spent in central area
instead of peripheral area in open field. This anxiolytic effect of progesterone also
was observed in PMDAT. Indeed, the time spent in open arms was increased in
animals treated with progesterone. Controversially, mipefristone in estrus phase did
not induce anxiogenic effect instead of it promoted anxiolytic effect, but it could be
explained by the antagonism effect of mifepristone on glucocorticoids receptors, this
evidence was corroborated by other studies (Jakovcevski et al, 2011; Lefevre et al,
2017). Reduced expression of progesterone in the brain could therefore be the key
factor that triggers increased responsiveness to stressful situation in late diestrus in
rodents and late lutheal phase in women. This evidence is supported by clinical
studies about the premenstrual dysphoric disorder (PMDD), one mood disorder
associated with sexual steroids characterized by depressed mood, irritability and
high anxiety experience (Cunningham et al 2009). Some studies have proposed
pharmacological models of PMDD, these studies consist of progesterone withdrawal
in order to induce anxiety and depressive-like behaviors similar in PMDD (Smith et
al 1998; Li et al 2012). Although, progesterone treatment in women with PMDD did
not promote improvement in depressed mood or anxiety levels (For review, Ford &
Roberts, 2012). Apparently falling progesterone in beginning or late luteal phases
(diestrus in rats) triggers increased sensitivity to stress responsiveness and
contributes to changes in emotional, and cognitive response by modulatory effect
their metabolites – allopregnanolone (ALLO) – over gamma-aminobutyric acid
(GABA) receptor activity (Devall et al 2015; Review, Hantsoo & Epperson, 2017:
Lovick, 2012). Our data support this hypothesis, considering that anxiogenic effect
elicited by stress was observed in diestrus phase and only treatment of
progesterone was able to revert it.
It seems that anxiety alterations may not be due progesterone per se, but by
the ALLO, progesterone’s metabolite that exerts modulatory effects over GABAergic
transmission (Schumacher et al 2014). Indeed, previous studies suggest ALLO is a
potent positive allosteric modulator of GABAA receptor, as results it appears to
101
promote anxiolytic, analgesic and sedative effects. Thus, lack of progesterone and
ALLO could be seen in late dietrus phase and early/late of luteal phase (Backstrom
et al 2014; Lovick, 2012). Further, reduced ALLO level are also observed in
prefrontal cortex and limbic structures like amygdala and hippocampus, all them are
involved in neurobiology of stress response (Devall et al 2015; Shirayama et al 2011;
Agis-Balboa et al, 2007).
Overall, our data demonstrated increased locomotor activity in PMDAT and
OF (Fig. 3A-C) (Table 1). These data are in line with other studies (Gentsch et al
1981; Butler et al, 2014). Social isolation increased motor activity when animals are
exposed to novelty contexts (Morato & Brandão, 1997). Interestingly, induced motor
activity elicited by social isolation are linked to motor behavior associated with
behavioral sensitization to drug-seeking behavior, considering social isolation risk
factor for addiction (Blanco-Gandía et al 2015; Butler et al, 2014). Further, social
isolation is an animal model used to investigate the relation between stress and
schizophrenia disorders (For review, Jones et al 2011). In point of fact, social
isolation most appropriately mimic human’s symptoms in schizophrenia, such
increased aggressiveness, hyper-psychomotor and sensorimotor gating (Revised
by, Braff et al 1988; Li et al, 2017; Powell et al, 2006).
5. Conclusion
The results suggest endogenous variations in estrogen levels across the
estrous cycle can modulate aspects of cognitive function associated by social
stress. In fact, social isolation induced retrieval impairment and this deleterious
effect was counteracted by previous exogenous administration of estradiol, but do
not progesterone. Moreover, there are dissociative effect of progesterone and
estradiol over anxiety response, pre-training stress induced high anxiety, however
there is an evidence of modulatory effect of progesterone in GABA transmission,
since progesterone-treated animals showed anxiolytic effect, this interaction
between progesterone and GABA is only speculated.
102
Acknowledgements
This research was supported by fellowships from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Pró-reitoria de Pesquisa
da Universidade Federal do Rio Grande do Norte (PROPESQ/UFRN) and
Fundação de Apoio à Pesquisa do Estado do Rio Grande do Norte (FAPERN).
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4. CONSIDERAÇÕES FINAIS
Há inegáveis evidências que relacionam o papel dos hormônios sexuais e
adrenais com de respostas cognitivas, emocionais e comportamentais vistas em
dimensões disfuncionais nos distúrbios neuropsiquiátricos. Os hormônios sexuais,
em especial estradiol e progesterona, modulam emoções, processos cognitivos e
comportamento, tanto em modelos animais quanto em achados clínicos. As
investigações acerca da relação dos hormônios sexuais e transtornos psiquiátricos
tem auxiliado no esclarecimento do fenômeno de maior incidência desses
transtornos em mulheres, no sentindo de elucidar os mecanismos neuroendócrinos
envolvidos e traçar novas propostas terapêuticas.
Segundo a organização mundial de saúde – OMS, situações de estresse ou
estilo de vida estressante estão associadas ao surgimento de transtornos afetivos,
como depressão maior e transtornos de ansiedade (Post, 1992; Kendler et al.,
1995). Nesse sentido, a exposição ao estresse crônico é utilizada como modelo
animal para investigar as relações entre estresse e alterações emocionais e
cognitivas na etiologia dos transtornos mentais associados ao estresse. Nesse
estudo utilizamos o estresse social, uma condição bastante atual e próxima de
realidade humana, capaz de promover ativação da resposta de estresse. Em
acordo com a literatura foram avaliados efeitos deletérios nos comportamentos dos
animais estressados pelo isolamento social. Ainda, foi explorada a relação desses
prejuízos ao longo do ciclo estral das ratas, no intuito de estabelecer quais as fases
poderiam proteger ou favorecer essa ação deletéria.
Em sequência, investigamos, por meio de manipulação hormonal, o efeito
do estradiol e progesterona em diferentes tarefas de comportamentais. Os dados
obtidos sugerem tanto um efeito benéfico para o estradiol e progesterona, no
sentido que o estradiol parece exercer uma função na regulação de estados de
humor, associados à motivação, resposta prazerosa e interesse social. Além disso,
observou-se um papel de facilitador na formação da memória aversiva. Já a
progesterona mostrou-se importante na regulação da resposta emocional e
comportamento exploratório, além de atuar na responsividade ao estresse. Então,
os resultados obtidos corroboram outros estudos que mostram o papel modulatório
112
desses hormônios ao longo do ciclo estral que parecem influenciar, em curto e
longo prazo, os glicocorticoides, gerando saídas comportamentais diferentes
dependentes da expressão desses hormônios, uma vez que se observa indução de
prejuízos cognitivos e emocionais em fase específica (diestrus), enquanto outras
são capazes de amenizar prejuízos (proestrus e estrus). Em nosso estudo, essas
evidencias foram corroborados com a manipulação hormonal, onde foi possível
identificar os efeitos dissociados dos hormônios sexuais.
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5. CONCLUSÃO
Nesse estudo foi demonstrado que o isolamento social é capaz de eliciar um
padrão de alterações cognitivas, emocionais e comportamentais em ratas. Além
disso, essas alterações são dependentes da circulação endógena de hormônios
sexuais, demonstrada pelo não aparecimento de prejuízos no desempenho dos
animais quando em fases onde o perfil hormonal é elevado, ao mesmo tempo é
visto que em fases com baixo perfil hormonal torna-se um fator de risco para efeito
agravado do estresse. Por fim, foi possível distinguir o feito do estradiol sobre à
formação da memória aversiva e manutenção de estados de humor, bem como o
da progesterona sobre à resposta emocional e modulação da ansiedade.
114
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7. ANEXO
127
Memory impairment induced by different types of long-term stress is
modulated by the estrous cycle
Ezequiel Batista do Nascimento1, Aline Lima Dierschnabel1, André de Macêdo Medeiros2,
Deborah Suchecki3, Regina Helena Silva2, Alessandra Mussi Ribeiro4*
1Memory Studies Laboratory, Department of Physiology, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil 2Laboratory of Behavioral Neuroscience, Department of Pharmacology, Universidade Federal de São Paulo, SP, Brazil
3Department of Psychobiology, Universidade Federal de São Paulo, SP, Brazil 4Laboratory of Neuroscience and Bioprospecting of Natural Products, Department of Biosciences, Universidade Federal de São Paulo,
Santos, SP, Brazil
Abbreviations:
AV, aversive enclosed arm;
OA, open arms;
NAV, non-aversive enclosed arm;
SAP, stretched-attend posture;
PHD, protected head dipping;
UPHD, unprotected head dipping;
%TOA, percentage time in open arms;
%TAV, percentage time in aversive enclosed arm;
PMDAT, plus maze discriminative avoidance task;
M/D, Metestrus/Diestrus
P, Proestrus
E, Estrus
*Corresponding author: Alessandra M. Ribeiro
Department of Biosciences
Universidade Federal de São Paulo
Rua Silva Jardim 136 – CEP: 11015-020, Santos/SP
Tel: (55) 13 3878-3862
E-mail: [email protected]
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Abstract
A growing body of evidence has shown the role of estrogen and corticosterone
(CORT) in cognition and emotion. Additionally, it is suggested that ovarian hormones may
have beneficial actions on several neurophysiological processes, mainly on memory
systems. On the other hand, chronic exposure to stressful conditions clearly exerts negative
effects in neural structures related to learning and memory. In addition, ovarian and stress-
related hormones may decrease or improve mammals’ performance in hippocampus-
dependent tasks. In the present study, we used the plus-maze discriminative avoidance task
(PMDAT) to evaluate the influence of the endogenous variations of sex hormones and
exposure to different types of chronic stress on learning, memory, anxiety-like behavior and
locomotion. Female Wistar rats were submitted to seven consecutive days of restraint stress
(4h/day), overcrowding (18h/day) or social isolation (18h/day), and tested in different
estrous cycle phases. The main results showed that: (1) neither stress conditions nor estrous
cycle modified the acquisition of the task; (2) overall, restraint stress and social isolation
induced memory impairments; (3) this impairment was observed particularly in stressed
female in metestrus/diestrus; (4) stressed-female in estrus phase presented decreased risk
assessment behavior; (5) only restraint stress and social isolation conditions increased the
corticosterone levels. Taken together, our findings suggest that estrous cycle is an important
modulatory factor of the cognitive processing disrupted by stress in female rats. This effect
was observed in metestrus/diestrus phase, indicating that the peak of sex hormones in other
phases could buffer the effects of stress on memory.
Keywords: Estradiol; corticosterone; memory; stress; estrous cycle
1. Introduction
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Previous studies have demonstrated that exposure to many types of stressors
provokes alterations in cognitive processes such as learning, memory and emotion
(Roozendal et al, 2009; Ulrich-Lai & Herman, 2009; Herman et al, 2003). The hippocampal
formation has an integrative role on these processes (Izquierdo & Mendina, 1997; O’Reilly
& McClelland, 1994). The high concentration of glucocorticoid receptors (GR) in this
structure suggests that adrenal hormones can exert effects in the hippocampal formation, and
hence this area is susceptible to stress effects (Luine, 2014).
Indeed, some studies have reported that long-term stress can alter the hippocampus
morphology and functionality by promoting the reduction in dendritic ramifications, and
thus decreasing synaptic plasticity in the hippocampus (Kim & Diamond, 2002). These
alterations are accompanied by deficits in different behavioral tasks (Sauro et al, 2003).
Conversely, it is also known that the effects of stressors on memory performance can vary,
so that enhancing or impairing effects can be observed depending on the nature of the
stressor (i.e. type, intensity, duration), the type of the memory, lifespan, gender and gonadal
hormones status (Pacak & Palkovics, 2001; Green & McCormick, 2013; Toufexis et al,
2014; Herrera & Mather, 2015).
Despite the role of gonadal hormones in reproductive function is well described,
these hormones can influence neuronal activity and cognitive functions depending on the
estrous cycle phase of female animals (Luine, 2014; Tabatadze et al, 2015). In addition,
neuromodulatory actions of estrogens have been observed in brain areas such as pre-frontal
cortex, hippocampus and amygdala (Zeidan et al, 2011; Mukai et al, 2010). For example,
previous studies have reported that both estradiol replacement in ovariectomized rats or its
endogenous variation during the estrous cycle in intact rats can modulate the growth of
dendritic spines, synaptogenesis and neurogenesis in the hippocampus (Scharfman et al,
2003; Li et al, 2011; Fester et al 2012; Horst et al, 2012; Tabatadze et al, 2015).
Furthermore, the activation of the hypothalamic-pituitary-adrenal axis (HPA) can be
modulated by the hypothalamic-pituitary-gonadal (HPG) axis (Toufexis et al, 2014), insofar
there is a close interaction between both axis. For instance, some endogenous neuropeptides
and sex hormones may act reducing the activity of HPA axis (Bowman et al, 2002; Zhao et
al, 2004; Suzuki et al, 2006; Arevalo et al, 2015). Oscillations in the sex hormones levels
across the estrous cycle may also buffer deficits triggered by chronic stress exposure in
female rats (Pisu et al, 2016). For example, Mohammadkhani et al. (2015) demonstrated that
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estrous phases (particularly proestrus and estrus) may lead to an unaltered learning and
memory processes after stressful conditions.
Aside from the influence on cognitive processes, the effects of glucocorticoids and
estrogens on anxiety-like behavior are widely reported (see Maeng & Milad, 2015 for
review). In this regard, the performance in memory tasks may be influenced by the emotional
state (Silva & Frussa-Filho, 2000). In view of the aforementioned, the present study aims to
investigate the effects of estrous cycle and different type of stressors (restraint, isolation
social and overcrowding) in female rats submitted to the plus-maze discriminative avoidance
task (PMDAT). We used the PMDAT (Silva & Frussa-Filho, 2000) in order to assess
anxiety-like behavior and locomotor activity simultaneously to the learning and memory
performances.
2. Material and Methods
2.1. Animals
Adult female Wistar rats (200-250 g) were kept in groups (4-5 per cage) under
conditions of acoustic isolation, controlled airflow and temperature (25 ± 1° C) and a 12h
light/12h dark cycle (lights on 06:30 a.m.). Food and water were provided ad libitum. The
animals were handled according to the Brazilian law for the use of animals in scientific
research (Law Number 11.794) and all procedures were approved by the local ethical
committee (CEUA/UFRN Protocol 055/2011). All efforts were made to minimize animal
pain, suffering or discomfort and to reduce the number of animals. Before any experimental
protocol starts, all the animals were gently handled for 5 min/day during 3 days.
2.2. Estrous cycle
The estrous cycle was monitored by collection of vaginal smears daily during the
whole experimental protocol. Briefly, the tip of a plastic pipette filled with 100 µL of
distilled water was gently introduced into the rat vagina, the bulb of the pipette was slightly
pressured and the vaginal fluid entered the interior of the pipette upon release. This material
was placed on glass slides, dyed with methylene blue and examined under a light
microscope. The estrous cycle comprises four distinct phases: metestrus (or early diestrus),
diestrus, proestrus and estrus, that are identified according to the cytological features. The
proestrus phase is characterized by the predominance of epithelial nucleated cells, while the
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estrus phase is characterized by the presence of cornified cells. Diestrus phase presents a
predominance of leukocytes and the metestrus shows similar proportion of the features of
the others phases (Byers et al, 2012). To insure that all female rats were cycling regularly,
the estrous cycle was determined for 10 days, at least, before the beginning of the
experimental procedures. We considered metestrus and diestrus phases together for analysis,
based on evidences of similar estradiol profile (Spornitz et al, 1999; Paccola et al, 2013;
Haim et al, 2003) and lack of basal behavioral differences in the present study.
2.3. General procedures and stress conditions
All animals were randomly assigned to one of four groups: control (n = 27) and
stressful conditions: restraint (n= 22), social isolation (n = 21) and overcrowding (n= 23).
Control rats remained in their home cage. Rats that underwent stress conditions were
transferred to a separate room. In the restraint stress condition, the animals were exposed to
a 4h period of restraint (2:00 to 6:00 p.m.) during seven consecutive days. Each animal was
placed in a transparent plastic cylinder (18 cm length and 9 cm width). There was a 0.3 cm
hole at one extremity of the tube where the head of the animal was positioned for breathing.
After each restraint session, rats returned to their home cages.
In social isolation stress condition, the animals were removed from their home cages
and separated into individual cages (42 cm length, 34 cm width and 18 cm height) during
18h (6:00 p.m. to 11:00 a.m.) for seven consecutive days. The individual cages were placed
at isolated environments to prevent vocalization among animals.
Animals in overcrowding were kept in groups of five animals in a small cage (31 cm
length, 20 cm width and 22 cm height) during 18h (6:00 p.m. to 11:00 a.m.) for seven
consecutive days. In this group, two animals were excluded from the analysis due to
technical problems in video recording. The accommodation of the unfamiliar subjects in a
small space directly affects the social organization of animals within the group and resembles
the stress condition due to a high population density (Bartolomucci et al, 2005; D Lin et al,
2015; Uarquin et al, 2016).
The experimental design is schematized in figure 1.
2.4. Plus-maze discriminative avoidance task (PMDAT)
Twenty-four hours after the last day of stress exposure the animals were submitted
to the training session of the PMDAT. The wood-made apparatus used is a modified version
of the conventional plus-maze. The maze contains two enclosed arms (50 length x 15 width
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x 40 height cm): one aversive arm (AV) and one non-aversive arm (NAV) opposite to two
open arms (OA; 50 length x 15 width cm). The PMDAT is conducted in two sessions:
training and test, each session lasting 10 minutes. In both sessions, the animals were
individually placed in the center of apparatus with body orientation toward the intersection
between the open arms. In the training session, the aversive stimuli were triggered each time
animals entered with the whole body in the aversive enclosed arm, and turned off when the
animal left the arm. The aversive stimuli were an 80 dB noise and a 100W light produced by
speakers and lamp, respectively, placed over the aversive enclosed arm. Memory acquisition
was measured during the training session. In the test session (24h later), the animals were
placed again in the center of the apparatus and allowed to explored the apparatus without
presentation of the aversive stimulation (see Fig. 1). In this session, we evaluated memory
retrieval. All behavioral experiments were performed between 1:00 and 5:00 p.m. The
sessions were recorded by a digital camera placed above the apparatus and the behavioral
parameters were registered by a video-tracking software (Anymaze, Stoelting, USA).
Learning and memory were evaluated by the percentage of time spent in the aversive arm
[%TAV = time in AV / (time in NAV + AV) x100] across the sessions (in three blocks of
200 seconds each). Anxiety-like behavior was evaluated by the percentage of time spent in
the open arms [%TOA = time in OA / (time in NAV + AV + OA) x 100] and by the time
spent in risk assessment behaviors (stretched-attend posture – SAP and head dipping). SAP
was registered when the animals stretched forward and then retracted to the original position
without doing any forward locomotion. Head dipping behavior was registered when the
animals orientated the head over the side out of the maze and was divided in two categories:
protected head dipping (PHD) – when the animals displayed this behavior being in the
central area (relative security place) and unprotected head dipping (UPHD) – when animals
displayed it in the open arms (relative unsafe place). We only considered the training session
for the evaluation of anxiety because the anxious response to novelty is absent in a second
exposure to a plus-maze apparatus (Pereira et al, 1999). Locomotion was measured by the
distance travelled in the apparatus. At the end of each behavioral session, the apparatus was
cleaned with a 5 % alcohol solution.
2.5. Determination of the corticosterone levels
Immediately after the test session of PMDAT, the animals were euthanized by
decapitation and blood samples were collected. Samples were centrifuged at 3000 rpm for
10 min. Plasma was collected and stored at -20 ºC until the assays. Plasmatic corticosterone
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concentrations were determined by radioimmunoassay with a commercial
kit (ImmuChem Double Antibody Corticosterone RIA, MP Biomedicals, Orangeburg, NY)
and run in standard duplicates. The detailed procedure was conducted according to the
manufacturer’s instructions.
2.6. Statistical Analysis
Analysis was conducted in two phases: (1) effects of the different stressors and (2)
effects of the different stressors considering the estrous cycle phases. All data were
checked for normality with Kolmogorov-Smirnov test. Two-way ANOVA with repeated
measures was performed for the %TAV in time blocks across training and test sessions
for the different stress conditions. One-way ANOVA (stress condition) was performed
to %TOA, corticosterone levels and risk assessment parameters, as well as distance
traveled in the apparatus. Afterwards, three-way ANOVA with repeated measures was
run for the %TAV in time blocks across training and test sessions for the different stress
conditions and estrous cycle phases. Two-way ANOVA (stress condition and cycle
phase) was performed to %TAV in the first time block, %TOA, corticosterone levels and
risk assessment parameters, as well as distance traveled in the apparatus. Tukey post-hoc
test was applied (if necessary) to all parameters. All results were considered significant
at p < 0.05.
3. Results
3.1. Effects of stress conditions
3.1.1. Learning and memory
In the training session, two-way ANOVA with repeated measures for %TAV in time
blocks showed effect of time [F(2,88) = 27.931; p = 0.01] (Fig. 2A). Tukey’s post hoc
revealed that the third time block is lower than the first one for all conditions, indicating all
groups learned the task. In the test session, two-way ANOVA with repeated measures for
%TAV in time blocks showed time x stress condition interaction [F(6,178) = 1.317; p =
0.01]. Tukey’s post hoc revealed that females exposed to restraint stress and social isolation
displayed increased exploration of the aversive arm during the first time block compared to
control (Fig. 2B). This result indicates a worse performance of animals submitted to restraint
stress or social isolation (but not overcrowding) during memory retrieval.
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3.1.2. Anxiety-like behavior and locomotor activity
There were no statistically significant differences among groups for %TOA, distance
travelled or any of the risk assessment measures. Results are summarized in table 1.
3.1.3 Corticosterone levels
One-way ANOVA showed significant effect of stress condition [F(3,89) = 10.974;
p = 0.01] for corticosterone levels. Tukey’s post hoc revealed that restraint and social
isolation stressed-females exhibited increased corticosterone levels compared to control
(Fig. 5A).
3.2. Effects of stress conditions on female rats in different cycle phases
3.2.1. Learning and memory
In the training session, three-way ANOVA with repeated measures for %TAV
showed effect of time [F(2,162) = 46.471; p = 0.001] (Fig. 3). Tukey’s post hoc revealed
that the third time block is lower than the first one for all conditions, indicating all groups
learned the task.
In the test session, three-way ANOVA with repeated measures showed time x stress
condition x cycle phase interaction [F(12,160) = 2.058; p = 0.02]. Tukey’s post hoc revealed
that restraint and social isolation stressed-females exhibited higher %TAV than control
females in the first time block. On the other hand, restraint (Fig. 4C) and social isolation
(Fig. 4D) stressed-females in proestrus phase showed lower %TAV in the first time block
compared to stress-matched females in metestrus/diestrus. This result indicates a protective
effect against stress-induced memory deficit during the proestrus phase. Indeed, two-way
ANOVA followed by Tukey’s post hoc revealed that only social isolation and restraint
stressed-females in metestrus/diestrus phase presented memory impairment represented by
higher %TAV in the first time block of the test session (Fig. 4E). Interestingly, control
females in the proestrus phase exhibited low exploration of the aversive arm in the beginning
of the session but it increased as observed in the last time block (p < 0.05), suggesting
extinction of the aversive memory (Fig. 4A).
3.2.2. Anxiety-like behavior and locomotor activity
135
During the training session, two-way ANOVA for %TOA did not show effect of
cycle or stress condition. On the other hand, two-way ANOVA showed stress condition x
cycle phase interaction for the time in SAP [(F(6,81) = 4.526; p = 0.01]. Tukey’s post hoc
revealed that restraint and social isolation stressed-females in estrus phase presented lower
SAP time when compared to stress-matched females in proestrus. In addition, restraint and
social isolation stressed-females in estrus phase decreased time in SAP compared with
control-females in estrus (Table 2). Furthermore, two-way ANOVA revealed stress
condition x estrous cycle phase interaction [F(6,81) = 3.665; p = 0.04] for UPHD. Tukey’s
post hoc showed an increased UPHD time for restraint and social isolation stressed-females
in estrus phase compared to control group (Table 2). No significant effects for PHD behavior
or distance travelled were found (Table 2).
3.2.3. Corticosterone levels
Two-way ANOVA showed only effect of stress condition [F (2,81) = 12.401, p =
0.01]. Tukey’s post hoc revelead increases in the corticosterone levels of the restraint and
social isolation groups compared to control irrespective of cycle phase (Fig. 5B).
4. Discussion
In summary, the main results showed that chronic stress conditions (except
overcrowding) provoked impairment on retrieval of the aversive memory, despite those
procedures did not influence the acquisition of the task. In addition, hormonal variations
across estrous cycle had a modulatory effect on stress-induced memory impairment.
Specifically, only stressed-female rats in metestrus/diestrus were susceptible to the memory
deficits induced by long-term stress. Interestingly, despite those stressful procedures
increased corticosterone levels irrespective of the estrous phase, only metestrus/diestrus
females showed susceptibility to the hormonal elevation induced by stress on the memory
alteration. Concerning anxiety-like behavior, stressful conditions induced an anxiolytic
profile specifically in the estrus group, while no locomotor changes were observed for any
136
group. Interestingly, there was a dissociation between the effects of stress on memory
performance and emotional response, suggesting that the modified memory processing
induced by long-term stress in the PMDAT does not rely on alterations of emotional states.
In the present study, the different stressors did not disrupt learning, i.e. all groups
showed a decrement in %TAV across training session (Figure 2A). Previous studies using
restraint stress condition (6h/21 days) have shown that the acquisition of spatial memory in
the radial arm maze was spared in female rats (Bowman et al, 2001). Similarly, social
isolation stress did not provoke changes during acquisition of spatial memory tasks in male
(Wongwitdech & Marsden, 1996) and female (Sandstrom, 2005) rats. From the literature,
the inverted-U hypothesis states that both low levels of arousal and high responsiveness
triggered by stressful conditions would result in a poor performance in behavioral tasks.
Thus, optimal levels of stress and arousal are necessary for suitable attention levels, working
memory and other cognitive mechanisms required to the learning process (Akirav et al,
2004). The discrepancy regarding the sparing effect of stress on the acquisition reported here
can be related to the type of task used here. Indeed, learning of emotional tasks is robust
(Bradley et al, 1992; Roozendal et al, 1996), and the emotional input could prevail over the
stress-induced learning impairment. Moreover, studies that address overcrowding stress and
learning are scarce and controversial. Progesterone-treated male rats submitted to
overcrowding stress showed a deficit in the acquisition of the Morris water maze (MWM,
Diaz-Burke et al, 2010). Male rats reared in an overcrowded environment were unable to
learn appetitive and aversive tasks (Goeckner et al, 1973), while overcrowding applied after
the adulthood facilitates acquisition of complex tasks (Wood & Greenough, 1974). The
authors proposed that the debilitating effects of the first study result from rearing in the
overcrowded environment rather than from the effects of overcrowding held prior to
behavioral testing (Wood & Greenough, 1974). To our knowledge, an unaltered learning
process after overcrowding stress prior to behavioral testing is the first report in the literature,
at least for female rats. Taken together, these evidences suggest that the influence of different
types of stressors on learning process depends on the type of the task, the period when stress
is applied and the sex.
Regarding estrous cycle, animals in all phases showed the same pattern of avoidance
of the aversive arm, exhibiting similar acquisition slopes regardless stress conditions in the
training session (Figure 3). In line with our results, Sava & Markus (2005) reported that the
estrous cycle did not affect the learning of MWM task. In contrast, restraint-stressed female
rats submitted to Y-maze task demonstrated differences in acquisition performance among
137
estrous cycle phases (Conrad et al, 2004). Moreover, when only estrous cycle is considered,
estrus females submitted to MWM showed longer latencies to reach the platform (Frye,
1994). Rubinow et al. (2004) found that proestrus females showed worst performance, and
suggested that this outcome was due to the stressful condition to swim at low temperature.
It is important to note that the results considering estrous cycle replicated the previous one
– i.e. estrous cycle did not alter the absence of stress effect on the acquisition of the PMDAT.
Moreover, a recent study from our group using the PMDAT showed that only diestrus female
rats were susceptible to learning deficits induced by scopolamine and this outcome was
prevented by supraphysiological levels of estradiol (de Macêdo Medeiros et al, 2014).
Corroborating this finding, high, but not low doses of estradiol facilitate eye blink-
conditioning acquisition (Leuner et al, 2004). Similarly, Lipatova et al. (2014) reported that
cyclic, but not continuous, estradiol administration facilitated the spatial memory
acquisition. However, most studies showing estrogen beneficial effects on acquisition are
conducted with estrogen replacement in ovariectomized animals. In this respect, the use of
supraphysiological levels may not be representative of those seen endogenously in rodents
(Beach et al, 1983). From this point of view, we suggest that any influence of the estrous
cycle would only be observed in the case of a learning deficit caused by stress, which did
not occur herein.
In the test session, females that were stressed by restraint or social isolation showed
increased %TAV during the first time block, indicating a deficit in the aversive memory
retrieval (Figure 2B). Despite the task evaluated in PMDAT is based on aversive association
and dependent on amygdala function (Ribeiro et al, 2011), we have recently show that
hippocampus inactivation causes memory impairment in rats submitted to this task (Leão et
al, 2016). . Indeed, the hippocampal formation is an important target of glucocorticoid
actions, and a direct interaction between the stress response and cognition has been
demonstrated (Conrad, 2008). From this standpoint, the literature on chronic stress and
memory has extensively considered the glucocorticoid receptors (GRs) located in
hippocampus areas to be a core feature of stress-induced memory impairment (Kim et al,
2015). The activation of these receptors reduce (1) transmission and release of glutamate
(Popoli et al, 2013); (2) long-term potentiation (LTP) at CA3 and CA1 (Pavlides et al, 1993;
Conrad et al, 2007; Chen et al, 2008; Kumar, 2011; Popoli et al, 2012) and (3) dendritic
spine and branching (McEwen et al, 2015). Furthermore, it is well established that exposure
to chronic stress decreases neurotrophins expression in the hippocampus (Duman & Thome,
1999; Becker et al, 2007; Pittenger & Duman, 2008; Calabrese et al, 2009). In light of this,
138
and considering the increase in the corticosterone levels of these experimental groups, the
stress-induced deficits found here may be related to functional abnormalities in the
hippocampus.
In line with memory retrieval impairment, there was a differential responsiveness of
the HPA axis to the different types of stressors applied. Intriguingly, only females submitted
to restraint and social isolation conditions showed higher corticosterone levels (Figure 5A).
The restraint stress is a well-established condition to induce HPA axis activation and elicits
acute and chronic stress responses in both male and female rodents (Darington et al, 1981;
Zavala et al, 2011). Social isolation was the only social condition leading to increased
corticosterone levels, corroborating the evidence of sex-specific neuronal excitability of
CRH neurons in female mice submitted to social isolation (Senst et al, 2016). On the other
hand, females submitted to overcrowding did not present alterations. Many species,
including nonhuman primates, cope with stress using social support – an essential
mechanism for maintaining physical and psychological health (Meyer & Hamel, 2014).
Female rodents demonstrate increased sensitiveness to social stressors compared to male,
suggesting that social isolation elicits HPA responsiveness (For review, see Beery & Kaufer,
2015) Thus, the absence of social support during social isolation could be a powerful stressor
for females, whereas the overcrowding situation has no detrimental effect on them.
Taylor and colleagues (2000) proposed that most of the female mammals evolved to
present a stress response pattern build on affiliative behavior with social groups in order to
reduce risk and increase survivor of themselves and offspring. In other words, female stress
response is believed to rely on attachment-caregiving processes, which could suppress
sympathetic and HPA responses to stress. Several neuroendocrine studies using different
species indicate that oxytocin affects affiliative, pro-social empathy and attachment
behaviors (Ross & Young, 2009; Shelley et al, 2006; Macdonald & Macdonald, 2010; Nave
et al, 2015; Galbally et al, 2011). Oxytocin (OT) decreases HPA responsiveness, possible by
acting in the inhibition or delaying of CREB transcriptional coactivator (CRTC3), this
mechanism of translocation is responsible for binding CRF promoter to trigger the CRF gene
expression during stress response. As consequence, the OT could reduce the triggers
response of corticotrophin releasing factor in the paraventricular nucleus of the
hypothalamus (Jurek et al, 2015). In this respect, the aversiveness of some social conditions
is sex-specific. The overcrowding condition may be a stressor for male subjects when there
is a hierarchic system of dominants and subordinates (Bartolomucci, 2002; Brown &
Grunberg, 1995). Thus, the social isolation could be not stressful due to the absence of
139
hierarchic conflicts (Palanza et al, 2001; Bartolomucci et al, 2003; Hoshaw et al, 2006;
Beery & Kaufer, 2015). Thus, one possible explanation comes from the evolutional social
ecology of the rodents. As discussed by Taylor and colleagues, most of the male mammals
evolved to present a stress response built on fight-or-flight mechanisms.
Despite restraint and social isolation conditions lead to a memory deficit in female
animals altogether (Figure 2B), when the estrous cycle is considered, only stressed-female
in metestrus/diestrus showed this impairment (Figure 4E). Because both proestrus and estrus
display high levels of endogenous sex hormones, as opposed to metestrus/diestrus (Becker
et al, 2005), we propose that the hormonal elevation during those phases overcame the
detrimental impact of chronic stressful conditions on memory. Accordingly, studies
demonstrate the protective role of estradiol in stressed-rats during the transition between
proestrus and estrus (Wei et al, 2013; Mohammadkhani et al, 2015) or when this hormone is
replaced in ovariectomized animals (McLaughlin et al 2010; Takuma et al, 2007). Estradiol
diffuses freely across the blood-brain barrier binding to ERα and ERβ receptors (Oren et al,
2004). There are two pathways through estradiol acting in the brain’s functionality: ER-
mediated rapid signaling and the long-lasting response by ER-mediated gene expression
(reviewed by Moriartry et al, 2006). Estradiol rapidly leads to changes in neuronal
excitability, suggesting that non-genomic actions have an impact on neurotransmission
effectiveness, stemmed from estrogen-mediated signaling on synaptic proteins, connectivity
and synaptic function of neuronal circuitry (Luine, 2014; Srivastava et al, 2011). For
example, estradiol has a role on the regulation of the cholinergic projections to the
hippocampus and cortex, and this modulation seems to be relevant to working memory and
visual-spatial attention (Revised by Dumas & Newhouse, 2015). Accordingly, exogenous
administration of estradiol counteracts the scopolamine-induced amnesia in the PMDAT (de
Macêdo Medeiros et al, 2014).
Thus, estradiol could act over cholinergic transmission to suppress the detrimental
effect of stress on memory found here, but this hypothesis remains to be investigated. .
Notwithstanding, there is evidence that the memory impairment induced by chronic stressors
is related to changes in cholinergic transmission. Restraint-stressed male Wistar rats
(2h/30days) decreased acetylcholine levels in hippocampus and pre-frontal cortex
accompained by memory deficits in radial arm maze (Srikumar et al, 2006). In contrast, a
study conducted by Mizoguchi and colleagues (2001) using male rats found that restraint
stress (6h/30days) did not decrease cholinergic transmission on hippocampus, and working
memory performance was not altered. Interestingly, a study has demonstrated the role of
140
gonadal hormones in acetylcholine levels during stress response, in this study, gonadectomy
in both male e female interrupts stress-induce ACh releases in hippocampus, but the
testosterone and estradiol replacement restores it, this evidence suggests the corresponding
sex hormone has a role to maintain the stress response of ACH liberation, this supports some
evidences of learning and memory modulated by cholinergic system ( Mitsushima et al,
2008; Ragozzino et al 1996; Gold, 2003;). In addition, some evidences have linked sex
differences in HPA activation to modulation of hippocampal cholinergic transmission is
more robust in female (Gentile et al, 2012; Rhodes et al, 2002).
Furthermore, memory enhancement could be explained by estradiol modulation on
the glutamatergic transmission (Bean et al, 2014; Frick, 2015; Herrera & Mather, 2015).
Recently, studies have demonstrated the participation of ERα and ERβ in signaling through
metabotropic glutamate receptor 1a (maGluR1a) triggering a sequential of intracellular
cascades pathway, suggesting that these receptors influence positively memory formation
by rapid activation of hippocampal cell signaling (Srivastava et al, 2011; Boulware et al,
2013; Oberlander & Woolley, 2016; Sárvari et al, 2016).
Another possible explanation is that estradiol would act like a natural buffer on the
negative actions of chronic stress. This reasoning would be related to the genomic and long-
lasting estradiol mechanism. For example, estradiol induces neuroplasticity mediated by
LTP, dendritic sprouting and neuronal growth in the hippocampal neurons (Scharfman et al,
2003; Woolley & McEwen, 1994; Finocchi & Ferrari, 2011; Smith & McMahon, 2006;
Gould et al, 1990; Smejkalova & Woolley, 2010; Frick et al, 2015; Mclaughlin et al, 2010),
and promotes the enhancement of the neurotrophic factors as BDNF (see Luine & Frankfurt,
2013). In turn, BDNF would exert neuroprotective effects against the harmful actions of
glucocorticoids, oxidative stress and neurodegenerative diseases (Fiocchetti et al, 2012;
Numakawa et al, 2011; Wei et al, 2014; Asimiadou et al, 2005). This alternative view is in
line with our results given that BDNF is reduced under lower levels of estradiol
(metestrus/diestrus) and increases concomitantly to the estradiol elevation (Gibbs, 1998;
Scharfman et al, 2003). It is worth stressing that glucocorticoids could compromise the
memory formation process by decreasing BDNF expression, which is important for memory
consolidation (for review, see Cunha et al, 2010). Other studies observed that
proestrus/estrus or exogenous administration of estradiol leads BDNF expression in
hippocampus CA1 and CA3 areas, resulting in unaltered memory formation (Mclaughlin et
al 2010; McEwen et al, 2012).
141
The memory extinction is also related to the estrous cycle. Higher %TAV in last time
block compared to the first (Figure 4A) indicate that only female control in proestrus
exhibited an extinction profile. Milad and colleagues (2009) reported that females in
proestrus showed extinction of a fear conditioned memory. Interestingly, in our study this
effect was absent in all other phases and stress conditions – i.e. stress interfered in extinction
process of the aversive memory in an estrous phase-specific manner.
Conversely, in our study, the hormonal status across the estrous cycle did not modify
the enhancement of corticosterone levels. The classical model of HPA and HPG cross-axis
communication proposes an inhibitory expression of estradiol triggered by stressful
conditions (Rivier & Rivest, 1991). Indeed, high levels of corticosterone suppress the
gonadotropin-releasing hormone (GnRH) by binding on GRs located in the hypothalamus
(Chandran et al, 1994) or increase the expression of gonadotropin-inhibitory hormones
(Kirby et al, 2009). For example, the hypothalamic inhibitory peptide, RFamide-related
peptide-3 (RFRP3) is expressed during stress response and inhibits reproductive functions
(Murakami et al, 2008; Ubuka et al, 2009). During the estrous cycle, the expression of
RFRP3 is elevated only during diestrus, suggesting a role of this peptide in maintaining of
the estradiol low levels during this phase (Salehi et al, 2013). Moreover, Geraghty and
colleagues (2015) demonstrated that RFRP3 high expression caused by stress (18 days of
immobilization) had an impact on GnRH levels, but did not affect the estrous cycle. Thus,
we suggest that the counterbalance to memory impairment during the hormonal changes
present in the estrous cycle is related to another point on the HPA-HPG cross-axis other than
that modulation by corticosterone.
Emotional state have a consequence on fear and motivation to explore the plus-maze
in the PMDAT task (Silva & Frussa-Filho, 2000). In this regard, no stress effect on
locomotion or anxiety-like behavior was detected when females in all cycle phases were
considered together for analysis – i.e. all animals showed similar distance traveled, %TOA
and risk assessment behaviors during the training session (Table 1). Nonetheless, anxiety
levels were influenced by estrous cycle phase. Restraint and social isolation stressed-females
in estrus had lower SAP behavior than control in the same phase as well as than stress-
matched females in proestrus (Table 2). Similarly, for the UPHD, restraint and social
isolation stressed-female in estrus increased the time exhibiting “unsafe” behavior in the
open arms. Taken together, these data suggest an anxiolytic effect of estrus phase in animals
that were stressed by restraint and social isolation. Furthermore, no changes in anxiety-like
behavior were observed in metestrus/diestrus phases, indicating a specific influence of the
142
estrous cycle on memory processing in stressed-female rats. These effects could be related
to progesterone levels, insofar as the expression of this hormone reaches the highest levels
in the first hours of estrus phase (Lovick, 2012). Previous studies reported a potent
neuromodulatory action on GABAergic neurotransmission by direct activation of
progesterone receptors (Auger & Forbes-Lorman, 2008; Maguire & Istavan Mody, 2007) or
by its metabolites, such as allopregnanolone (Bitran et al, 1995; Darbra & Pallarès, 2012) on
anxiety-like behavior. Allopregnanolone is a positive allosteric modulator of GABAA
receptors able to promote anxiety in animal models (Schule et al, 2014). Perhaps, the
anxiolytic effect observed in our study can be explained by an increase in allopregnanolone
during stress response in animals submitted to restraint and social isolation (Evans et al,
2012; Bruton et al, 2013). However, the effects of stress on anxiety-behavior were
dissociated from the effects on memory, because those actions occurred in rats that were in
different cycle phases. This outcome reinforce an specificity of stress effects on memory
processes.
In conclusion, the results suggest a modulatory effect of estrous cycle on stress-
induced memory impairment in PMDAT. Indeed, restraint and social isolation stressed-
female rats demonstrate memory impairment specifically when trained in metestrus/diestrus.
Despite corticosterone levels endorse the behavioral alterations induced by stress, the estrous
cycle did not modify such parameter. Moreover, the alterations promoted by stress were
memory-specific and dissociated from any interference of the emotional status or
locomotion. Our data support the hypothesis that the elevation of sex hormone levels in
proestrus and estrus phases could counteract cognitive deficits promoted by stressors.
Further investigations must clarify the mechanisms by which sex hormones could prevent
memory deficits.
Acknowledgments
We would like to thank Mr. Vinicius Bunscheit for technical support. This research
was supported by fellowships from Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPQ); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES); Pró-reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte
(PROPESQ/UFRN) and Fundação de Apoio à Pesquisa do Estado do Rio Grande do Norte
(FAPERN).
143
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Figure 1
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Figure 2
Figure 3
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Figure 4
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Figure 5
Figure 1. Experimental design. Figure 2. Effects of stress conditions (control, social isolation, restraint and overcrowding) on learning and memory of female rats submitted to the plus-maze discriminative avoidance task (PMDAT). Mean ± SE for the percentage of time spent in aversive enclosed arm (%TAV) in 200s time blocks during training (A) and test (B) sessions. *p < 0.05 compared to the first block; #p < 0.05 restraint and social isolation compared to control (two-way ANOVA with repeated measures followed by Tukey’s post hoc test). Figure 3. Effects of stress conditions (A-D: control, overcrowding, restraint and social isolation) on learning of female rats submitted to the plus-maze discriminative avoidance task (PMDAT) in different estrous cycle phases (metestrus/diestrus – M/D; proestrus – P and estrus – E). Mean ± SE for the percentage of time spent in aversive enclosed arm (%TAV) in 200s time blocks during the training session. *p < 0.05 compared to the first block (three-way ANOVA with repeated measures followed by Tukey’s post hoc test). Figure 4. Effects of stress conditions (A-D: control, social isolation, restraint and overcrowding) on the memory of female rats in different estrous cycle phases (metestrus/diestrus – M/D; proestrus – P and estrus – E) submitted to the plus-maze discriminative avoidance task (PMDAT). Mean ± SE for the percentage of time spent in aversive enclosed arm (%TAV) in time 200s time blocks and in the first block of the test session (E). #p < 0.05 compared to the first block. *p < 0.05 compared to proestrus (three-way ANOVA with repeated measures followed by Tukey’s post hoc test); +p < 0.05 compared to metestrus/diestrus control (two-way ANOVA followed by Tukey’s post hoc). Figure 5. Effects of stress conditions (control, social isolation, restraint and overcrowding) on the corticosterone levels of female rats in different estrous cycle phases (metestrus/diestrus – M/D; proestrus – P and estrus – E). Mean ± SE for the corticosterone levels [ng/ml] considering only stress condition (A) and considering stress condition and estrous cycle phases (B). *p < 0.05 compared with control group (one-way ANOVA – A; two-way ANOVA – B followed by Tukey’s post hoc test). Table 1. Effects of stress conditions on anxiety-like behavior and locomotion of female rats in the plus-maze discriminative avoidance task (PMDAT). Mean ± SD for the percentage of
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time spent in open arms (%TOA), stretched-attend posture (SAP), protected head dipping (PHD), unprotected head dipping (UPHD) and distance travelled (m) in the training session.
Table 2. Effects of the estrous cycle [metestrus/diestrus (M/D), proestrus (P) and estrus (E)] and stress conditions on anxiety-like behavior and locomotion in the plus-maze discriminative avoidance task (PMDAT). Mean ± SD for the percentage of time spent in open arms (% TOA), stretched-attend posture (SAP), protected head dipping (PHD), unprotected head dipping (UPHD) and distance travelled (m) in the training session
*p < 0.05 compared to estrus control, #p < 0.05 compared to stress-matched in proestrus phase; +p < 0.05 compared to stress-matched metestrus/diestrus (two-way ANOVA followed by Tukey’s post hoc test).
Anxiety-like behaviors Locomotion Stress condition %TOA
SAP PHD UPHD
Time (s) Time (s) Time (s) Distance (m) Control 23 ± 20.4 7.9 ± 3.6 8.9 ± 3.2 8.9 ± 3.2 4.6 ± 4.1 Overcrowding
23 ± 24.5
6.4 ± 3.8
6.7 ± 3.5
8.2 ± 3.5 7.4 ± 5.9
Restraint 28 ± 19.9 6.2 ± 5.3 8.8 ± 3.6 10.3 ± 3.6 6.5 ± 7.1 Social Isolation
29 ± 24.5
7.3 ± 5.0
9.4 ± 4.0
11.1 ± 4.0 8.3 ± 7.3
Anxiety-like behaviors Locomotion Stress condition Estrous Cycle % TOA SAP PHD UPHD Time (s) Time (s) Time (s) Time (s) Distance (m)
Control M/D 4.4±2.9 5.9±1.8 8.3±3.6 8.1±2.4 5.1±0.9 P 8.9±1.8 8.7±3.3 9.7±3.2 12.2±2.5 2.0±0.1 E 6.6±4.6 11.2±2.1 8.9±2.6 6.5±1.4 5.7±0.8
Overcrowding M/D 9.8±8.6 6.7±1.1 4.4±1.6 8.7±0.9 8.2±1.4 P 5.4±5.1 8.9±1.7 9.9±3.6 8.6±4.7 5.4±1.1 E 9.8±1.2 4.1±0.8 6.0±1.8 10.9±3.9 7.4±1.5
Restraint M/D 4.4±9.3 6.8±4.3 9.3±3.2 10.5±7.0 3.9±0.5
P 6.6±6.7 11.4±2.8 8.6±3.5 7.8±3.2 10.9±0.1 E 14.5±10.3 1.1±0.3*# 8.4±3.9 14.0±1.6* 3.1±0.3
Social Isolation M/D 12.4±11.3 9.5±3.4 11.7±0.7 11.5±2.9 9.1±1.9 P 2.5±1.2 12.0±0.8 10.5±4.6 9.0±4.0 5.9±1.4 E 15.5±16.1 1.5±0.9*# 6.9±3.2 14.3±3.3* 6.7±1.5