FLÁVIA SANTOS DA SILVA - repositorio.ufrn.br · Table 3 – Statistical values, GLM test and LSD post-hoc, and direction of acute alterations ... O chá de ayahuasca, tradicional

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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

PRÓ-REITORIA DE POS GRADUAÇÃO

CENTRO DE BIOCIÊNCIAS

PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA

FLÁVIA SANTOS DA SILVA

ESTUDO DO EFEITO AGUDO DOS COMPOSTOS ATIVOS DO CHÁ DE

AYAHUASCA (BANISTERIOPSIS CAAPI E PSYCHOTRIA VIRIDI), EM SAGUIS

(CALLITHRIX JACCHUS) COMO MODELO ANIMAL DE DEPRESSÃO JUVENIL.

NATAL

2017

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Defesa da dissertação apresentada ao Programa de

Pós-graduação em Psicobiologia da Universidade

Federal do Rio Grande do Norte, como requisito

para obtenção de título de mestra em Psicobiologia.

(Área: Psicologia Fisiológica).

Orientadora: Profa. Dra. Nicole Leite Galvão Coelho

NATAL

2017

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Natal, 05 de julho de 2017.

Banca Avaliadora

___________________________________________

Profa. Dra. Nicole Leite Galvão Coelho

Departamento de Fisiologia – UFRN

Orientadora

___________________________________________

Prof. Dr. Bruno Lobão Soares

Departamento de Biofísica e Farmacologia (DBF) – UFRN

Membro interno

___________________________________________

Profa. Dra. Marília Barros

Faculdade de Ciências da Saúde – UnB

Membro externo

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Universidade Federal do Rio Grande do Norte - UFRN

Sistema de Bibliotecas - SISBI

Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Leopoldo Nelson - Centro de Biociências - CB

Silva, Flávia Santos da.

Estudo do efeito agudo dos compostos ativos do chá de

Ayahuasca (Banisteriopsis Caapi e Psychotria Viridi), em saguis

(Callithrix jacchus) como modelo animal de depressão juvenil /

Flávia Santos da Silva. - Natal, 2017.

96 f.: il.

Dissertação (Mestrado) - Universidade Federal do Rio Grande do

Norte. Centro de Biociências. Programa de Pós-Graduação em

Psicobiologia.

Orientadora: Profa. Dra. Nicole Leite Galvão Coelho.

1. Depressão juvenil - Dissertação. 2. Cortisol - Dissertação.

3. Estresse crônico - Dissertação. 4. Comportamento -

Dissertação. 5. Primata não-humano - Dissertação. 6. Modelo animal

translacional - Dissertação. I. Coelho, Nicole Leite Galvão. II.

Universidade Federal do Rio Grande do Norte. III. Título.

RN/UF/BSE-CB CDU 159.9.019.4

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AGRADECIMENTOS

Agradeço primeiramente a Deus por ser meu fortalecimento todos os dias.

Aos meus queridos pais pela preocupação, zelo e presença constante na minha

vida.

Agradeço aos amigos queridos, que acompanharam de perto minha trajetória e

sempre apoiaram as minhas escolhas.

Agradeço a todos que contribuíram de alguma forma para a realização desse

estudo, ressaltando aqui, Ana Cecília Menezes Galvão, os funcionários do Núcleo de

Primatologia da UFRN, aos estudantes de Iniciação Científica que participaram das

coletas de dados e a Raissa Nóbrega de Almeida, técnica no laboratório de medidas

hormonais.

Deixo também um agradecimento especial para meus amiguinhos, os saguis, do

Núcleo de Primatologia, que tiveram papel fundamental para que alcançássemos os

resultados valorosos aqui obtidos.

Agradeço a duas importantes referências para mim, Maria Bernardete Cordeiro

de Sousa pelos ensinamentos e orientações valiosas que me prestou durante todo o

mestrado e a Nicole Leite Galvão Coelho, minha orientadora, quem contribuiu também

com o ensino, porém foi mais além na consolidação do meu crescimento acadêmico e

pessoal. Sou muito grata pelo o todo que pude aprender e vivenciar com você Nicole.

Mais grata ainda pela proporção da experiência vivida ao longo dessa jornada,

meu mestrado.

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GENERAL LIST OF TABLES, FIGURES AND ABBREVIATION

Tables

ARTIGO 1 – Common marmosets: A potential translational animal model of juvenile

depression.

Table 1. Description of behaviors. 33

Table 2. Statistical values of the protocol for induction of depression state (IDS).

37

Table 3. Significant Spearman correlation between behaviors, and behaviors versus cortisol.

38

Table 4 - Statistical values of the Protocol for depression state + pharmacological treatment

(DPT): Baseline (BL) + Isolated context (IC)

42

Table 5 – Protocol for depression state + pharmacological treatment (DPT): Vehicle (VE),

pharmacological (PH) + tardive Pharmacological Effects (tPE)

46

ARTIGO 2 – Acute antidepressant effect of Ayahuasca in juvenile non-human primate

model of depression.

Table 1. Statistical values, GLM test and LSD post-hoc, and direction of alterations of

physiologic and behavior parameters in response to social isolated context.

74

Table 2 – Statistical values, GLM test and LSD post-hoc, and direction of alterations of

physiologic and behavior parameters in response to pharmacological treatments,

comparing with IC.

75

Table 3 – Statistical values, GLM test and LSD post-hoc, and direction of acute alterations

of fecal cortisol in response to treatments.

76

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Figures

ARTIGO 1 – Common marmosets: A potential translational animal model of juvenile

depression.

Figure 1. Protocol for induction of depression state (IDS)

29

Figure 2. Protocol for depression state + pharmacological treatment (DPT)

31

Figure 3. Graphic 1: IDS protocol for family context group (FC) and social isolated context

group (IC) of male juveniles C. jacchus

39

Figure 4. Graphic 2: Protocol for depression state + pharmacological treatment (DPT):

predictive validity isolated context (IC)

43

Figure 5. Graphic 3: Vehicle, pharmacological, and post-pharmacological treatments (VE,

PH, and tPE)

47

ARTIGO 2 – Acute antidepressant effect of Ayahuasca in juvenile non-human primate

model of depression.

Figure 1. Experimental design. 70

Figure 2. Graphic 1 – Behavioural response after Vehicle, pharmacological, and post-

pharmacological treatments (VE, PH, and tPE) with ayahuasca tea.

73

Figure 3. Graphic 2 – Cortisol levels after Vehicle, pharmacological, and post-

pharmacological treatments (VE, PH, and tPE) with ayahuasca tea.

76

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Abbreviation

IDS – Induction of depression state

DPT – Protocol for depression state + pharmacological treatment

BL – Baseline

FC – Familiar context

IC – Isolated context

VE – Vehicle treatment

PH – Pharmacological treatment

tPEs – tardive Pharmacological Effects

W1; W2; W5; W7; W8; W9; W10; W11; W9; W12; W13 – weeks 1, 2, 5, 7, 8, 9, 10, 11,

12 e 13.

D, D1, D2 – Days 0, 1 e 2

PiloI – Individual piloerection

ScentM – Scent marking

Loc – Locomotion

Scrat – Scratching

GLM – General linear models

ACTH – Adrenocorticotropic hormone

CRH – Corticotropin-releasing hormone

SSRIs – Selective Serotonin Reuptake Inhibitors

HPA – Hypothalamic-Pituitary-Adrenal

N, N-DMT – N, N-dimethyltryptamine

MAOi – Monoamine Oxidase inhibitors

5-HT2A – Serotoninergic receptor

USP – University of São Paulo

THH – Tetrahydroharmine

GR – Glucocorticoid Receptor

MR – Mineralocorticoid Receptor

UFRN – Universidade Federal do Rio Grande do Norte

WHO – World Health Organization

IBAMA – Brazilian Institute of Environment and Renewable Natural Resources

CONCEA – National Council for Animal Experimentation Control

DSM-5 – Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition

https://en.wikipedia.org/wiki/Corticotropin-releasing_hormone

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SUMÁRIO

RESUMO GERAL E APRESENTAÇÃO 09

SUMMARY AND PRESENTATION 11

INTRODUÇÃO GERAL 13

OBJETIVOS 20

HIPÓTESES 22

ARTIGO 1 23

Common marmosets: A potential translational animal model of juvenile

depression 23

Resumo 24

Introdução 24

Materiais e Métodos 28

Resultados 34

Discussão 48

Referências 56

ARTIGO 2 65

Acute antidepressant effect of Ayahuasca in juvenile non-human primate

model of depression 65

Resumo 66

Introdução 67

Materiais e Métodos 68

Resultados 72

Discussão 77

Referências 81

CONCLUSÃO GERAL 88

REFERÊNCIAS GERAIS 90

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RESUMO GERAL E APRESENTAÇÃO

O Transtorno de Depressão Maior (TDM) é um distúrbio de humor de alcance global,

atingindo aproximadamente 350 milhões de pessoas em todo o mundo, capaz de induzir

prejuízos psicológicos, sociais e fisiológicos, que podem em alguns casos, levar à morte

por suicídio. 14 % dos jovens entre 15-18 anos apresentam TDM, o que desperta

atenção e preocupação, já que esta fase constitui um período ontogenético de grandes

modificações cerebrais que perduram por toda a vida. Atualmente, o tratamento

antidepressivo mais empregado em todas as faixas etárias é o farmacológico. Embora as

classes mais modernas de antidepressivos sejam mais específicas em sua ação, ainda

apresentam efeitos colaterais consideráveis, demoram até duas semanas para iniciar os

efeitos terapêuticos desejados e induzem baixa taxa de remissão. Sendo assim, há a

necessidade de se buscar novos tratamentos farmacológicos para essa doença, nesse

cenário algumas substâncias psicodélicas serotoninérgicas vêm sendo testadas. O chá de

ayahuasca, tradicional da Amazônia, tem particularmente chamado a atenção por seus

efeitos positivos na saúde, tanto na população geral de usuários quanto em pacientes

com transtornos de humor e viciados em drogas de abuso. Para testar a ação

antidepressiva aguda da ayahuasca utilizamos o Callithrix jacchus, um primata não-

humano que já vem sendo considerado um importante modelo em estudos biomédicos,

inclusive de desordens mentais, pois apresenta maior proximidade filogenética aos

humanos, possui um etograma bem definido, técnicas não invasivas para medição de

cortisol em fezes, boa adaptação em cativeiro e alta taxa de fecundidade. Entretanto,

inicialmente foi necessário proceder com a validação da espécie como modelo

translacional de depressão juvenil. Para isso foram utilizados dois procedimentos

experimentais de isolamento social crônico, em machos e fêmeas de C. jacchus juvenis,

que induziu um estado fisiológico e comportamental característico de depressão em

primatas não-humanos, o qual foi em grande parte revertido pelo tratamento com um

antidepressivo clássico, a nortriptilina, inoculada por 7 dias, e não por um veículo

(salina; 7 dias). Esse estudo e seus respectivos resultados estão descritos no artigo I

intitulado “Common marmosets: A potential translational animal model of juvenile

depression”, publicado no periódico Frontiers in Psychiatry. Em seguida, testamos o

potencial antidepressivo agudo do chá de ayahuasca, no modelo translacional

previamente validad. Assim, originou-se o artigo II intitulado “Acute antidepressant

effect of ayahuasca in juvenile non-human primate model of depression”, submetido no

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periódico Frontiers in Pharmacology. Observamos que uma dose única de ayahuasca, e

não do veículo (salina), induziu melhoras em parte dos sintomas depressivos por até 14

dias e reestabeleceu, de forma rápida, 24 horas após a ingestão, os níveis de cortisol aos

valores basais, encontrados quando os animais habitavam a gaiola familiar. Sendo

assim, observa-se que o chá de ayahuasca apresentou resultados antidepressivos mais

interessantes que a nortriptilina, uma vez que a ayahuasca induziu a reversão dos

sintomas mais rapidamente, de maneira mais ajustada e duradoura que a nortriptilina.

Ambos os estudos aqui apresentados são de grande relevância para área, uma vez que de

maneira inédita um modelo animal translacional de depressão com primatas não-

humanos atendeu a todos os critérios de validação, tanto os tradicionais, como; o

etiológico, de face, funcional e preditivo, quanto os critérios mais recentes: o inter-

relacional, evolutivo e populacional, possibilitando assim a sua utilização em áreas

complementares de investigações. Adicionalmente, foi apresentado não apenas ações

antidepressivas aguda do chá da ayahuasca, mais também ações mais eficazes quando

comparado com um antidepressivo clássico, nortriptilina, corroborando assim no

processo de validação desta substância como antidepressivo, estimulando novas

investigações farmacologicas, inclusive em adolescentes, que possibilitem a

consolidação do chá como antidepressivo, uma vez que ela não tem demonstrado

tolerancia à doses, nem efeitos colaterias de longo prazo em usuários recreacionais.

Palavras-chaves: Modelo animal translacional, Primata não-humano,

Neuroendocrinologia, Depressão juvenil, Substâncias psicodélicas.

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SUMMARY AND PRESENTATION

The Major Depression Disorder (MDD) is a mood disorder of global reach,

reaching approximately 350 million people, capable to induce psychological, social and

physiological impair, which in some cases can lead to death by suicide. 14% of young

people aged 15-18 have MDD that provoke attention and apprehension, since this phase

consist an ontogenetic period of major brain modifications that last for the all lives.

Currently, the most commonly used antidepressant treatment in all age groups is the

pharmacological treatment. Although the newer classes of antidepressants are more

specific in their action, they still have considerable side effects, take up to two weeks to

initiate the desired therapeutic effects and induce low rate of remission. Thereby, there

is a necessity to seek new pharmacological treatments for this disease. In this scenario,

have been tested some psychedelic serotonergic substances. The ayahuasca tea, a

traditional Amazonian tea, has particularly drawn attention by its positive effects on

health, both in the general population of users and in patients with mood disorders and

drug addicts. To test the acute antidepressant action of ayahuasca was used Callithrix

jacchus, a non-human primate that has already been considered an important model in

biomedical studies, including mental disorders, because it´s more phylogenetic

proximity to humans, has a well-defined etogram, invasive methods for measuring

cortisol in feces, good adaptation in captivity and high fecundity rate. However, it was

initially necessary to validate the species as a translational model of juvenile depression.

For this purpose, two experimental procedures of chronic social isolation were used in

males and females of C. jacchus juveniles, which induced a physiological and

behavioral state characteristic of depression in nonhuman primates, which was largely

reversed by treatment with an antidepressant nortriptyline, inoculated for 7 days, rather

than vehicle (vehicle, 7 days). This study and its results are described in article I entitled

" Common marmosets: A potential translational animal model of juvenile depression",

published in the Journal Frontiers in Psychiatry. We then tested the acute

antidepressant potential of ayahuasca tea in the previously validated translational model.

Thus, was originated the article II entitled "Acute antidepressant effect of Ayahuasca in

juvenile non-human primate model of depression", which was submitted in the journal

Frontiers in Pharmacology . We observed that a single dose of ayahuasca, not vehicle

(saline), induced improvements in part of depressive-like symptoms for up to 14 days

and quickly restored 24 hours after ingestion to cortisol levels at baseline, when the

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animals inhabited the family cage. Thus, it is observed that ayahuasca tea presented

more interesting antidepressant results than nortriptyline, since ayahuasca induced the

reversal of symptoms more quickly, in a more adjusted and lasting way that

nortriptyline. Both studies presented here are of great relevance to the area, since in an

unpublished way a translational animal model of depression with nonhuman primates

met all validation criteria, both traditional, the etiological, face, functional and

predictive, as well as the most recent criteria: the inter-relational, evolutionary and

population, thus enabling its use in complementary areas of investigation. In addition,

not only acute antidepressant actions of ayahuasca tea were presented, but also more

affective actions when compared with a classic antidepressant, nortriptyline, thus

corroborating in the validation process of this drug as antidepressant, stimulating new

pharmacological investigations, including in adolescents, that make possible the

consolidation of tea as an antidepressant, since it has not shown tolerance to doses, nor

long-term side effects in recreational users.

Keywords: Translational animal model, Non-human primate, Neuroendocrinology,

Juvenile depression, Psychedelic substances.

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INTRODUÇÃO GERAL

A depressão é uma psicopatologia reconhecida atualmente como uma das mais

prejudiciais a população mundial, acomete aproximadamente 350 milhões de pessoas no

mundo. A Organização Mundial da Saúde (OMS) aponta que a depressão será o maior

problema de saúde pública em 2030 [World Federation for Mental Health (WFMH)

2012] e a primeira causa de mortes (WHO 2017). O Transtorno Depressivo Maior

(TDM) é a forma mais comum entre os transtornos depressivos, sendo as mulheres as

mais afetadas, em episódio único ou na forma recorrente [Boletim Brasileiro de

Avaliação de Tecnologias em Saúde (BRATS) 2012, American Psychiatric Association

(APA) 2013]. Estima-se que 13% a 16% dos adultos experimentem sintomas de

depressão durante a vida, ocorrendo uma taxa de 4% a 8% em um determinado ano.

Porém, é preciso alertar que tais taxas podem ser maiores, uma vez que os índices de

diagnóstico e tratamento são particularmente baixas entre certos grupos, como; idosos,

homens adultos e afro-americanos (APA 2013).

Segundo o DSM-5, o diagnóstico do TDM considera a ocorrência de episódios

depressivos quando, por um período mínimo de duas semanas (em adultos), emergem

pelo menos cinco dos sintomas a seguir, e esses devem representar uma mudança em

relação ao funcionamento anterior e induzir sofrimento clinicamente significativo ou

prejuízo no funcionamento social, profissional ou em outras áreas importantes da vida

do indivíduo. Obrigatoriamente deve ocorrer o humor deprimido e/ou anedonia

(caracterizada pela perda de interesse ou prazer), entre os demais sintomas encontram-

se, por exemplo, alterações de apetite, de sono, de concentração, sentimentos de

desvalia e pensamentos com conteúdo mórbido. A apresentação desse transtorno dá-se

em episódios recorrentes na maioria das pessoas, com cerca de 20-25% expressando-os

cronicamente (Fava e Kendler 2000). Além da necessidade de se considerar tanto a

frequência quanto a intensidade dos episódios, características adicionais, tais como

ocorrência de sintomas ansiosos, melancólicos ou psicóticos devem ser observados no

ato do diagnóstico (Kavan e Barone 2014).

Poucas décadas atrás a TDM ainda era reconhecida como um distúrbio

específico de adultos, pois se acreditava que na infância o desenvolvimento era muito

imaturo para experienciar esse tipo de doença, de forma que comportamentos de mau

humor eram sempre compreendidos como sendo comuns às modificações vivenciadas

na adolescência (Maughan et al. 2013). Atualmente, o TDM acomete 15% dos jovens e

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observa-se uma maior incidência entre as meninas após a puberdade que pode chegar

até os 30% das jovens com idade próxima aos 17 anos (Sims et al. 2006). Essa crescente

expressão da TDM na juventude envolve um conjunto de fatores que vão desde os

genéticos até os psicossociais, como problemas acadêmicos, abuso físico e sexual e

divórcio dos pais (Lima 2004).

A adolescência é considerada um período crítico do desenvolvimento, com

grandes modificações sociais e biológicas, importantes para o desenvolvimento do

indivíduo, ocorre uma alta taxa de plasticidade cerebral, onde o sistema nervoso

encontra-se mais suscetível às influencias ambientais. Os hormônios, por exemplo,

atuam modulando a plasticidade e o funcionamento dos circuitos cerebrais envolvidos

em respostas sexuais, de recompensa e de perigo (Thapar et al. 2012, Young 2012).

Alguns estudos mostram que perturbações vivenciadas nessa fase podem induzir

adaptações negativas permanentes, podendo prejudicar a nível social, educacional e

mental, facilitando a incidência de desordens mentais que podem perdurar por toda a

vida (Blakemore 2008, Fletcher 2010).

No que se refere ao diagnóstico do TDM, observa-se uma relativa dificuldade

em se realizar o diagnóstico de maneira rápida e precisa, o que se relaciona

principalmente como a sobreposição de sintomas com outras desordens mentais

(Friborg et al. 2014; Butelman e Kreek 2017), mas também alta incidência de

comorbidades a esse quadro, sobretudo com transtornos ansiosos (Clark e Watson 1991;

Kessler et al. 2003). A identificação do TDM realiza-se prioritariamente por meio de

entrevistas clínicas, podendo haver auxílio de escalas, questionários e inventários para

esse fim (Cunha 2009). Sendo assim, a melhor compreensão desta patologia e

identificação de biomarcadores, principalmente nessa fase ontogenética, é muito

relevante porque favorecerá pesquisas que visam acelerar os diagnósticos.

Algumas alterações em mecanismos biológicos são hoje reconhecidamente

envolvidas na etiologia da TDM. Dentre as teorias que explicam tais alterações, a

“Teoria das monoaminas” é a mais consolidada e se centra principalmente na

desregulação do sistema de neurotransmissores monoaminérgicos, especificamente, o

esgotamento da serotonina, norepinefrina e dopamina na fenda sináptica (Anacker et al.

2011a, Palhano-Fontes et al. 2014). Apesar de essa ser a teoria mais consolidada, essa

não esclarece a causa dos distúrbios monoaminérgicos e a refratariedade ao tratamento

antidepressivo com fármacos que disponibilizam as monoaminas na fenda sináptica.

16

Com isso, outras teorias têm sido apresentadas, e que em conjunto têm norteado os

estudos quanto ao desencadeamento dessa doença.

A teoria neurodegenerativa se fundamenta na observação da redução do volume

hipocampal em pacientes com depressão, área do diencéfalo cuja função mais

importante é a formação de memórias declarativas de longo prazo e participação nos

processos de aprendizagem, além disto, o hipocampo compõe o sistema límbico,

participando também da regulação das emoções (Anacker et al. 2011b). A morte de

células neuronais, associada à redução da neurogênese e da expressão de proteínas

neurotróficas e de plasticidade neuronal, como BDNF (brain-derived neurotrophic fator

– fator neurotrófico derivado do encéfalo), induzem a atrofia hipocampal, prejuízos

cognitivos e as alterações no humor (Salposky 2004, Schmidt e Duman 2007, Palhano-

Fonte et al. 2014). Ainda não se tem certeza se é a depressão que gera a

neurodegeneração hipocampal, ou o inverso.

Outra importante teoria sugere que o eixo Hipotálamo-Pituitária-Adrenal (HPA)

esteja totalmente envolvido como mecanismo gerador desse distúrbio, já que pacientes

com depressão apresentam de forma consistente alterações na atividade do eixo HPA,

resultando na elevação ou redução crônica dos níveis de cortisol (Joca et al. 2003,

Romero 2011, Willard e Shively, 2012). O sistema de retroalimentação negativa do eixo

HPA parece ser o principal responsável por tal desregulação na atividade do eixo, uma

vez que são observadas alterações na sensibilidade de resposta do receptor de

glicocorticoides ao cortisol (Campbell 2004, Belzung 2014).

O cortisol é um hormônio pleiotrópico e tem papeis importantes na manutenção

do organismo, em situações de rotina e de desafios. Sua principal função é ser

hiperglicemiante, porém outras importantes funções também são atribuídas a esse

hormônio, por exemplo, regulação do sistema imunológico e da neurogênese, na

sobrevivência neuronal, na excitabilidade dos neurônios e aquisição da memória (Payne

e Nadel 2004, Moica et al. 2016). O aumento ou redução excessiva do cortisol pode

induzir desregulações significativas na fisiologia e cognição, além de contribuir para o

aparecimento de sintomas depressivos em função dos danos impostos às funções

cerebrais (Sen at al. 2008, Anacker et al. 2011). A teoria do U invertido sugere uma

modulação do cortisol sobre parâmetros fisiológicos em uma curva de U invertido, onde

altos e baixos níveis de cortisol são prejudiciais para o desempenho do indivíduo.

(Lupien et al. 2009, Zverová 2013).

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Evidências que demonstram excesso de citocinas pró-inflamatórias em pacientes

com depressão, tem favorecido a consolidação da teoria inflamatória da depressão

(Zunszain et al. 2011, Marques et al. 2007). Este ambiente inflamatório seria decorrente

da resistência dos receptores de cortisol presente em células do sistema imunológico, o

que levaria ao desequilíbrio do sistema imune, deslocando a produção de linfócitos do

tipo T-CD4 Th2 para o tipo Th1, diminuindo a concentração de citocinas anti-

inflamatórias (IL-4, IL-10) e aumentando citocinas pró-inflamatórias (IL-1, IL-6, TNF-

a) (Marques et al. 2007, Vismari et al. 2008).

Esse quadro geral de teorias que tentam explicar a etiologia do TDM favorece o

pensamento de que, provavelmente, não é apenas um sistema, ou alteração, o

responsável pelo desencadeamento dessa patologia, mas o conjunto de todas essas

alterações e sistemas. Apesar de a literatura científica apontar essas varias teorias

etiológicas para a depressão, na maioria das abordagens clínicas essas mudanças

fisiológicas não são investigadas, nem tratadas, considerando que, em geral, o

diagnóstico psiquiátrico não é etiológico, apenas a sintomatologia é considerada

(Schuder 2005). Porém é preciso novamente enfatizar que o entendimento das bases

biológicas desse transtorno e a elucidação de biomarcadores eficientes são de extrema

importância tanto para aprimorar os diagnósticos quanto os tratamentos

No que se refere aos tratamentos antidepressivos, o tratamento farmacológico é

o mais utilizado e eficaz em reduzir a morbidade e melhorar os sintomas clínicos de

milhares de casos de depressão em todo o mundo, mas não são completamente

satisfatórios (Lima et al. 2004, Belzung 2014). Os antidepressivos podem ser

classificados em dois grupos, os de primeira e segunda geração, a depender da ação

ativa que promovem no organismo. Os de primeira geração compreendem os tricíclicos

(ADT) e os Inibidores de Monoaminas Oxidase (IMAO). Os primeiros atuam

bloqueando a recaptação, pelo neurônio pré-sináptico, de monoaminas presentes na

fenda sináptica, aumentando assim a concentração destes neurotransmissores na fenda.

Entre os ADT mais utilizados estão a nortriptilina (PamelorTM

) e imipramina

(TofranilTM

). Já os IMAOs bloqueiam a enzima monoamina-oxidase, que metaboliza as

monoaminas, (Bolland and Keller, 2004) aumentando também a concentração dos

neurotransmissores dopamina, serotonina e norepinefrina na fenda sináptica, os

fármacos deste grupo são; fenelzina (NardilTM

) e tranilcipromina (ParnateTM

).

Por possuírem ação sobre vários sistemas de neurotransmissores, os

antidepressivos de primeira geração provocam efeitos colaterais impactantes ao

18

indivíduo, com alterações sobre os batimentos cardíacos e pressão arterial, induzem

efeitos hipotensores e cognitivos indesejados, dentre outros (Guimaraes and Graeff

2000, Belzung 2014). Por este motivo, houve a necessidade de desenvolver fármacos

mais seletivos e com ação mais específicas que os de primeira geração, surgindo os

antidepressivos de segunda geração. Os antidepressivos de segunda geração abrangem,

por exemplo, os inibidores da recaptação de serotonina (ISRS). Os ISRS apresentam um

menor número de efeitos colaterais e são mais toleráveis pelos pacientes, por isso são os

antidepressivos mais utilizados atualmente, alguns destes incluem o escitalopram

(LexaproTM

) e fluoxetina (ProzacTM

). (Kupfer et al. 2012, BRATS 2012, Belzung 2014).

Apesar dos avanços no desenvolvimento de novas classes de antidepressivos,

estes ainda são insatisfatórios, quanto aos efeitos colaterais induzidos e, principalmente,

por não apresentar uma resposta eficiente em todos os pacientes. Aproximadamente

30% a 40% dos pacientes tratados não apresentar remissão mesmo após quatro

tratamentos sistêmicos (Warden et al. 2007). Sabe-se que os antidepressivos

normalmente demoram em torno de duas semanas para iniciar os efeitos terapêuticos

desejados (Doris et al. 1999, Lima et al. 2004, Trivedi et al 2006). Discute-se que essa

ineficácia dos antidepressivos observada em grande parte dos tratamentos pode ser

ocasionada por fatores com diferenças individuais no processo de metabolização da

droga pelo fígado, em função de variações no citrocromo P540, e em função da

complexidade com que a TDM se apresenta (Belzung 2014, Hodgson 2015), uma vez

que é associada à alterações nos sistemas de neurotransmissores monoaminérgicos

(Canale e Furlan 2006, Palhano-Fontes et al. 2014), desregulações no eixo HPA

(Campbell 2004, Belzung 2014), alterações na neurogênese (Frodl 2008) e desregulação

no sistema imunológico (Vismari et al. 2008).

A partir do exposto acima, compreende-se a importância da investigação de

novos fármacos antidepressivos que contribuam de maneira mais eficaz para remissão

dos sintomas depressivos, com menor latência de ação e ausência de efeitos colaterais

graves. A fitoterapia data de décadas muito antigas e possibilita o ser humano

normalizar funções fisiológicas e cognitivas alteradas e restaurar a imunidade

enfraquecida (França et al. 2008). O chá da ayahuasca é originário de tribos indígenas

da região amazônica, utilizado tanto em rituais religiosos, quanto na medicina popular,

aplicada por curandeiros, com base nos conhecimentos do vegetalismo. Seu uso vem se

expandindo nas últimas décadas em função de sua utilização em rituais religiosos do

19

Santo Daime nas principais capitais brasileiras, nos Estados Unidos e Europa. (Santos

2007).

O chá é produzido a partir da união do tronco do cipó da Banisteriopsis caapi

Morton e das folhas da Psychotria viridis (Mckenna et al. 1998, Santos 2007). A B.

caapi apresenta compostos alcalóides do tipo β-carbolina: a harmina, a

tetrahidroharmina (THH) e a harmalina, que inibem a enzima monoaminoxidase

(MAO), aumentando os níveis de serotonina, dopamina e norepinefrina na fenda

sináptica (Costa et al. 2005). Enquanto que as folhas da P. viridis possuem a N,N-

dimetriltriptamina (DMT), que é um alucinógeno com ação agonista para os receptores

serotoninérgicos 5-HT1a, 1b, 1d e do 5-HT2a e 2c (Callaway 2005, Dos Santos 2011,

De Souza 2011). Normalmente o DMT é inativado quando consumido por via oral,

como é o caso do chá, pois sofre desaminação pela enzima MAO presente no intestino e

fígado (Mckenna et al. 1998, Santos 2007). Entretanto, as β-carbolinas presentes na B.

caapi inibem a desaminação do DMT e permitem seu acesso ao cérebro (Callaway et al.

1999).

Por ter ampla atuação em sistemas biológicos envolvidos na etiologia da

depressão, acredita-se que este chá apresente potencial antidepressivo (Palhano-Fonte et

al. 2014). Taylor et al. (2015) observaram aumento no transporte de serotonina em

usuários do chá em rituais religiosos e usuários do chá, em contextos religiosos,

também apontam que a substancia apresenta efeitos ansiolíticos (Meneguetti e

Meneguetti, 2014). Os usuários regulares da ayahuasca, nos contextos religiosos,

apresentaram baixo nível de psicopatologias (Barbosa et al 2009; Bouso et al. 2012),

baixos escores nas escalas estatais relacionadas ao pânico e à desesperança (Santos et al

2007), bem como bons desempenhos em testes neuropsicológicos cognitivos (Bouso et

al 2013, 2015).

Considerando a toxicidade da substância, a ayahuasca não produz dependência

fisiológica, não induz desenvolvimento de tolerância, ou evidências de qualquer

distúrbio psiquiátrico que caracterize dependência, do tipo, de abuso, perda social ou

abstinência (Santos 2007). Além disto, estudos apontam que a administração de doses

repetidas de DMT em humanos não induz tolerância aos efeitos alucinógenos da

substancia ou toxicidade, além de qualquer dependência fisiológica ou comportamentos

associados com dependência. (Strassman et al. 1996, Callaway et al. 1999, Jacob e

Presti 2005). Dos efeitos colaterais, os usuários do chá relatam sensações de vigilância,

excitação, hipertensão, taquicardia, tremores, midríase, náuseas, vômitos e, em alguns

20

casos, diarreia após ingestão do chá (Cazenave 2000, Costa et al. 2005, Martinez e Silva

2010). A ayahuasca ainda induz “estados alterados de consciência” que podem ser

caracterizados como alterações da percepção (visuais, auditivas e olfativas), cognição,

volição e afetividade, que varia de acordo com as experiências individuais (Labigaline

1998). Os efeitos visuais, subjetivos, são os mais relatados e se apresentam na forma de

visões, com olhos fechados, e delírios em forma de sonhos, contudo o indivíduo

permanece consciente de que está sob o efeito do chá e tem controle de suas ações

(Meneguetti e Meneguetti 2014).

Sendo assim, estudos científicos que visem buscar cada vez mais informações

quanto aos benefícios e prejuízos fisiológicos, cognitivos e comportamentais induzidos

pela ayahuasca no organismo precisam ser realizados, a fim de contribuir para o

tratamento de pacientes com depressão. No Brasil, o uso da ayahuasca é regulamentada

pela resolução datada de 4 de novembro de 2004 do Conselho Nacional Antidrogas

(National Anti-Drug Council 2004, Coutinho 2017). Em 2010, o Conselho Nacional de

Políticas de Drogas divulgou uma resolução que estabeleceu regras e princípios éticos

para o usuário ayahuasca em rituais religiosos e proibiu sua comercialização, consumo

ilícito ou terapêutico de chá. No entanto, esta resolução incentiva a pesquisa científica

sobre o uso terapêutico da ayahuasca. Dessa forma, a realização de estudos com

modelos animais translacionais podem auxiliar na elucidação dos mecanismos de ação

desse chá e apontar sua real eficácia no tratamento de desordens de humor, como a

depressão.

O modelo animal de depressão mais tradicional e amplamente utilizado tem sido

os roedores, a ampla utilização destas espécies deve-se a existência de padrões

comportamentais para depressão já bem estabelecidos, resposta eficaz aos

antidepressivos tradicionais, com reversão do quadro patológico, somando-se a isto o

curto tempo de vida e ótima reprodução em cativeiro destas espécies, características

importantes para o desenvolvimento de novas terapias farmacológicas (Willard e

Shively 2012).

Apesar das importantes contribuições obtidas com os roedores em estudos de

depressão, estes apresentam grandes distinções estruturais e funcionais com relação ao

sistema nervoso central humano, além das distintas histórias evolutivas e organizações

sociais, caracterizando um modelo animal filogeneticamente distante do modelo ideal

de representação da depressão (Pryce et al. 2005). Novas espécies que expressem

melhor os diferentes aspectos da complexa etiologia da depressão humana precisam ser

21

estudadas para ampliar o conhecimento a cerca dos mecanismos envolvidos no

transtorno depressivo, aumentando e gerando novas possibilidades para o tratamento

dessa doença. (Willard e Shively 2012). Alguns primatas não humanos vêm sendo

utilizados com sucesso como modelo animal de depressão e tem contribuído fortemente

no melhor entendimento da doença (Magness et al. 2005).

O Callithrix jacchus, conhecido popularmente como Sagui, é um pequeno

primata neotropical, originário da Mata Atlântica brasileira (Stevenson e Rylands 1988)

que vêm sendo considerado um importante modelo em estudos biomédicos em função

de apresentarem uma boa adaptação ao cativeiro, alta taxa de fecundidade e tamanho

relativamente pequeno, quando comparado com primatas do velho mundo (Dixson e

Lunn 1987, Lacreuse et al 2014). Esta espécie possui ainda um etograma bem definido

(Stevenson e Poole 1976) e técnicas não invasivas para a medição de hormônios

esteroides (cortisol, progesterona e andrógenos), a partir das fezes (Sousa e Ziegler

1998), por exemplo, favorecendo sua utilização como modelo biomédico. Há algum

tempo esta espécie vem sendo utilizada em estudos de estresse e apresentam diversas

alterações de estados fisiológicos e comportamentais que se assemelham aos observados

em humanos, de forma que podem contribui para investigações de patologias associadas

ao estresse, como é o caso da depressão (Saltzman et al. 2006, Galvão-Coelho et al.

2012, De Sousa 2015). Contudo, a maior parte dos estudos sobre estresse com esta

espécie, utilizam animais adultos (Leuner et al. 2007, Galvão 2015) ou infantes (Pryce

et al. 2002), apenas mais recentemente, após o entendimento que a fase juvenil é um

período crítico de plasticidade neuronal, estudos com juvenis começaram a surgir

(Sousa et al. 2015, Taylor et al. 2015).

Diante do exposto, espera-se contribuir para a validação desta espécie como

modelo animal translacional de depressão juvenil, para o desenvolvimento de novas

terapias farmacológicas antidepressivas, e para o entendimento da etiologia e

sintomatologia da depressão.

OBJETIVOS

Objetivo geral

22

Validar o Callithrix jacchus como modelo translacional de depressão juvenil e

avaliar os efeitos antidepressivos agudo do chá de ayahuasca no modelo previamente

validado.

Objetivo específico

Artigo 1 – Investigar os efeitos do isolamento social crônico e do antidepressivo

Nortriptilina sobre os parâmetros fisiológicos (cortisol e peso) e comportamentais em

machos e fêmeas juvenis de Callithrix jacchus, para validação dessa espécie de primata

não-humano como modelo translacional de depressão juvenil.

Artigo 2 – Avaliar o potencial antidepressivo do chá de Ayahuasca na reversão do

estado depressivo previamente induzido por isolamento social crônico (60 dias) em

machos e fêmeas juvenis de Callithrix jacchus.

23

HIPOTESES

24

ARTIGO 1

Front Psychiatry. 2017 Sep 21.8,175. doi: 10.3389/fpsyt.2017.00175.eCollection 2017.

Common Marmosets: A potential translational animal model of juvenile

depression

Nicole Leite Galvão-Coelho1,2,3

, Ana Cecília de Menezes Galvão1,2

, Flávia Santos da

Silva1,2

, Maria Bernardete Cordeiro de Sousa 1,2,4

. 1

Laboratory of Hormone Measurement, Departament of Physiology, Federal University

of Rio Grande do Norte, Campus Universitário Lagoa Nova, 59078-970 Natal, RN,

Brazil; 2

Postgraduate Program in Psychobiology, Federal University of Rio Grande do

Norte, Natal, Brazil; 3

National Institute of Science and Technology in Translational

Medicine Natal, Brazil; 4 Brain Institute, FederalUniversity of Rio Grande do Norte,

Natal, Brazil

Running head: Callithrix jacchus as a depression animal model

Address correspondence to:

Nicole L. Galvão-Coelho

Universidade Federal do Rio Grande do Norte

Departamento de Fisiologia

Caixa Postal, 1511

59078-970 NATAL, RN, BRAZIL

Tel 55 84 3215-3410

Fax 55 84 3211-9206

E-mail: [email protected]

Highlights

1. Several species have been tested to develop translational models of animal

depression.

2. C. jacchus were chronic social isolated and treated with an antidepressant drug.

3. The alterations found were characteristic of the depressive-like state.

4. The depressive-like state was in large part reversed by treatment.

5. This model meet all validation criteria of one translational animal model.

https://www.ncbi.nlm.nih.gov/pubmed/28983260

25

ABSTRACT

Major depression is a psychiatric disorder with high prevalence in the general

population, with increasing expression in adolescence, about 14% in young people.

Frequently, it presents as a chronic condition, showing no remission even after several

pharmacological treatments and persisting in adult life. Therefore, distinct protocols and

animal models have been developed to increase the understanding of this disease or

search for new therapies. To this end, this study investigated the effects of chronic

social isolation and the potential antidepressant action of nortriptyline in juvenile

Callithrix jacchus males and females by monitoring fecal cortisol, body weight, and

behavioral parmeters and searching for biomarkers and a protocol for inducing

depression. The purpose was to validate this species and protocol as a translational

model of juvenile depression, addressing all domain criteria of validation: etiologic,

face, functional, predictive, interrelational, evolutionary, and population. In both sexes

and both protocols (IDS and DPT), we observed a significant reduction in cortisol levels

in the last phase of social isolation, concomitant with increases in autogrooming,

stereotyped and anxiety behaviors, and the presence of anhedonia. The alterations

induced by chronic social isolation are characteristic of the depressive state in non-

human primates and/or in humans, and were reversed in large part by treatment with an

antidepressant drug (nortriptyline). Therefore, these results indicate C. jacchus as a

potential translational model of juvenile depression by addressing all criteria of

validation.

Keywords: behaviors, cortisol, chronic stress, early-age depression, non-human

primate, translational animal model.

1 Introduction

Major depression is a mood disorder, which is ranked as the most prevalent

disease in the population. According to the World Health Organization (1), the

estimated number of people with depression globally is over 300 million. Moreover,

depression is ranked as the largest contributor to disability in the world (1, 2).

26

The symptoms of depression are expressed in varied and sometimes opposing

ways: sleep, feeding and body weight alterations, fatigue, irritability, depressed mood,

loss of interest or pleasure in almost all daily activities, accompanied by feelings of

guilt. In addition, psychomotor disorders can occur, although they are less common, and

they are an indicator of severity in the individual's situation (DSM-V). These symptoms

are associated with neuroendocrine modifications and in severe cases, can lead to death

by suicide (1–3).

Despite substantial progress in the understanding of depression and in the

development of new antidepressants drugs, fewer than half of cases achieve clinical

recognition, that is, are diagnosed. In diagnosed people, approximately half are treated,

the same proportion receives adequate treatment, and 65% of this group achieves

remission after satisfactory treatment (4, 5). Even after many systematic treatments,

approximately one-third of patients do not achieve remission (6).

Some studies suggest that pharmacological resistance to the treatment, observed

in a substantial number of patients, could be in part due to the use of inadequate

protocols and/or animal models to investigate depression and to test antidepressants,

demonstrating the need of different approaches (7). Rodents are commonly used to

study depression and pharmacological antidepressant drugs due to the easy handling,

possibility of using transgenic animals, and the presence of well-established protocols

(2, 8, 9). However, several species and numerous stress protocols have been tested to

develop other translational models that mimic the physiological and behavioral states

observed in human depression (2, 10, 11).

Nevertheless, the validity of the model, that is, its consistency and predictive

value, needs to be taken into account (12). In principle, in psychiatric studies, four

criteria need to be considered when validating an animal model. First, regarding

etiologic criteria, the model should develop the disease by the same cause or agent that

induces it in humans. Second, the model should address the face criteria, which

correspond to the ability of the proposed model to present behavioral and other

symptoms observed in humans. Third, the functional or content criteria involve the

capacity of the model to show similar physiological alterations to those observed in

human patients. The last criteria include the predictive value, which is the reversion of

these symptoms by effective pharmacological treatments used in humans (7, 13, 14).

Therefore, in practice, only face and predictive criteria are considered in the

studies. The induction of a state of depression in animal models is accomplished by

27

protocols applying acute physical stressors such as restraint, food or water restriction,

continuous light exposure, physical or chemical lesions or genetic knockout (14–16) or

social stressor paradigms such as social defeat (17) and early social separation (18),

whereas in humans, chronic psychosocial stress is typically the agent of onset of this

disease (19). In addition, the protocols used in rodents to investigate depressive state

sometimes do not have etiological validity, i.e., the forced swimming (20) test normally

used in studies of depression is not a relevant stressful situation for all species of

rodents in nature, and exhibits low ecologic validity for some species (15, 21).

Therefore, the etiologic criteria are not addressed in various studies, and this is a cause

for concern, because the face aspects observed are not likely an evolutionary trace.

Moreover, investigations with molecular biomarkers such as hormones,

neurotransmitters, or cytokines associated with behaviors are scarce in animal models of

depression (22); therefore, the content criteria are also not addressed in most studies

(15, 22–24).

More recently, to improve studies conducted in animal models, other criteria have

been considered to validate translational models in psychiatry: inter-relational,

evolutionary, and population (25). Inter-relational criteria propose to investigate a

model in various and interacting disordered domains such as behaviors, molecular

biomarkers, and cognition (25). Evolutionary criteria reflect the ability of the proposed

model to investigate determinate disordered domains in a similar manner across various

species. In depression disorders, feeding, somnolence, motor alteration, and anhedonic

behaviors, as well as cortisol levels and body weight, are altered in humans, non-human

primates, rodents, and other species and can be used to investigate evolutionary criteria.

Therefore, these indicate conserved phenomena in this pathology along with evolution

with great importance placed on its understanding, mainly because it can be associated

with subjective states that cannot be measured directly in animals (12, 25, 26). The

population validity criterion is the capacity of the proposed model to reflect the natural

variance in phenotypes observed in the general population. The use of inbred or

knockout strains to reduce random noise in studies also reduces the variability and does

not reflect the heterogeneity of the human population. In this context, it is better to use

outbred or wild-type populations (25).

The brain morphology, neural functional organization, social organization, and

evolutionary history of non-human primates are more closely related to those of humans

than are those of other species commonly used as animal models of depression, such as

28

rodents and fish (27, 28). Consequently, the use of non-human primates as animal

models in studies of depression addresses both traditional and newly proposed

validation criteria.

Moreover, it was recently observed that macaques exhibit naturally occurring

depression attributed to chronic psychosocial stress, similar to that observed in humans.

Consequently, in non-human primates, depending on their social organization, the stress

protocols involving changes in social rank or chronic social isolation can induce similar

physiological and behavioral symptoms to those observed in depressive disorders in

humans (11, 28–30). The majority of studies of mood disorders are developed in adult

or infant animals and only a small proportion are conducted in juveniles. It is well

known that juvenile age is an important ontogenetic phase because it is a biological

window of plasticity of the nervous system, showing considerable susceptibility to

environmental influences that might induce permanent changes in cognition and in the

stress response system. If these changes are maladaptive, they can induce serious and

permanent damage in cognitive, behavioral, and physiological parameters in adulthood,

increasing the probability of the emergence of mood disorders such as depression (31–

33). The incidence of depressive episodes in adolescents has increased (34), reaching

14% of the youth population aged between 15 and 18 years, with a recurrence rate of

approximately 40% in 3–5 years following the first episode (31).

In this context, the present study investigated the effects of chronic social

isolation and the potential antidepressant effect of nortriptyline on physiological

parameters (fecal cortisol and body weight), as well as on the behavioral repertoire, in

juvenile male and female common marmosets (Callithrix jacchus) to characterize

biomarkers that respond to an etiological protocol for the study of depression in non-

human primates. Thus, our intention is to validate this animal model and protocol with

respect to distinct criteria including etiologic, face, functional, predictive, inter-

relational, evolutionary, and population, in order to provide evidence for the utility of

this species as a translational juvenile animal model of depression.

The common marmoset, C. jacchus, is a small and social non-human primate that

adapts well to captivity and has a high fertility rate, when compared with Old World

primates (35–37). This species exhibits a range of changes at the physiological and

behavioral levels that resemble those observed in humans when facing stress (11, 38–

40). C. jacchus also display complex social organization and a number of similar social

behaviors to humans and alloparental care (41), making this species relevant for the use

29

as a model in several areas of research, including affective disorders such as anxiety and

depression (42, 43). Moreover, C. jacchus has a well-defined ethogram (44) and a non-

invasive technique to measure steroid hormones (cortisol, progesterone, and androgens)

in feces (45, 46), which facilitates its use as an experimental model in disorders

associated with the hypothalamic–pituitary–adrenal axis (HPA), such as depression

(38).

2 Materials and Methods

2.1 Study design

This study includes two experimental procedures to validate the use of the

common marmoset as an animal model of juvenile depression. The first protocol was

performed to validate chronic social isolation as a protocol for induction of depression

state (IDS) and to provide evidence validating the etiologic, face, and functional criteria

of this depression animal model. To address the predictive criteria of validation, a

second protocol was developed, namely, depression state + pharmacological treatment

(DPT), to attempt to reverse the symptoms and physiologic alterations observed in

isolated context (IC) by using antidepressants.

2.2 Protocol for IDS

This experimental procedure included two phases, and the objective of this

protocol was to induce a state of depression using the paradigm of chronic social

isolation in common marmoset juveniles Figure 1.

(1) Baseline (BL): ten juvenile males (according to the classification of age by Leão et

al. (47)) of common marmosets, aged approximately 7 months, were monitored for 4

weeks living in cages with their families. Fecal samples for cortisol measurements and

behavioral data were collected on alternate days to establish the hormonal and

behavioral profiles of the animals of the animals.

(2) Social context [familiar context (FC)/ isolated context (IC)]: after BL, the animals

were divided randomly in two groups with distinct social context and were monitored

for 13 weeks: (a) FC—five juvenile males remained in their home cages with their

30

respective families; (b) IC—five juvenile males were socially isolated, separated from

their families, and placed alone in new cages.

During 13 weeks of social context, data collection (fecal sampling and behaviors)

was performed daily in the 1st, 5th, 9th, and 13th weeks (W1, W5, W9, and W13,

respectively) for both groups (FC and IC). The duration of social isolation was

determined according to similar studies of depression in non-human primates (48, 49).

Figure 1 – Protocol for induction of depression state (IDS).

2.3 Protocol for DPT

The second experimental procedure included five phases Figure 2. The objective

was to evaluate whether the depressive state could be reversed by nortriptyline, an

antidepressant drug commonly used in humans, to demonstrate whether this animal

model adhered to the predictive validity criterion. Moreover, this protocol was

developed to investigate whether sexual dimorphism occurred in the depressive state in

this species and at this age. To that end, eight juvenile males and seven juvenile females

of common marmosets were observed consecutively in four situations:

(1) Baseline: similarly to the IDS protocol, in the BL phase 15 juvenile common

marmosets, 8 males and 7 females, aged approximately 7 months, were observed for 4

31

weeks living with their families. Fecal sampling for cortisol measurement and

behavioral data collection were performed on alternate days to establish the hormonal

and behavioral BL profiles of the animals. After the BL period, all animals were

sequentially monitored across four conditions:

(2) Isolated context: the animals were separated from their families and were socially

isolated for 8 weeks in new cages. Under this condition, data collection (feces and

behaviors) was performed daily in the first 2 weeks of the first month (W1 and W2) and

in the last 2 weeks of the second month (W7 and W8, respectively).

(3) Vehicle (VE) treatment: after the eighth week of isolation, three males and three

females were randomly selected to be treated with saline solution as a VE, prepared

using 9.8 mL of saline mixture to 0.2 mL of Tween 80. All animals received one daily

intraperitoneal administration of saline (0.2 mL/100 g animal), for 7 days in week 9

(W9). Behavioral and fecal data collections were performed daily.

(4) Pharmacological treatment (PH): following the VE, the same animals received one

daily intraperitoneal administration of nortriptyline hydrochloride (Pamelon Novartis)

(12.5 mg/mL) for 7 days in week 10 (W10) at the same volume used for saline solution

(0.2 mL/100 g of nortriptyline). The dose of nortriptyline was determined based on a

study with rats, whereby the effective antidepressant dose was found to be

approximately 30 mg/kg (50). The ip administration method was chosen after a number

of unsuccessful attempts to induce the animal to ingest the drug together with several

different food items. Similarly, for the previous condition, behavioral and fecal data

collections were performed daily (29).

(5) Tardive pharmacological effects (tPEs): after PH, the same animals were monitored

for a further 21 days to observe postpharmacological symptoms. This period was

divided into 3 weeks (week 11 = W11; week 12 = W12; week 13 = W13) to facilitate

statistical analysis of the data. Fecal data collections were performed daily.

32

Figure 2 – Protocol for depression state + pharmacological treatment (DPT).

2.4 Animal maintenance

All 25 animals used in this study (IDS = 10 males and DPT = 7 males and 8

females) lived in captivity in the Laboratory of Advanced Studies in Primates of the

Universidade Federal do Rio Grande do Norte (UFRN), Natal, Brazil. In order to

address the population validity criteria and mimic the individual variability observed in

the human population, the animals were randomly selected from 19 different families.

In experiment 1, only juvenile males were available for experimental use in the primate

colony. However, after validation of the stressor protocol in experiment 1 and

considering the high prevalence of depression in females since the adolescence phase,

and the fact that the colony was able to provide animals of both sexes, we developed the

other experimental phases of the study using males and females.

In the BL periods, the animals lived with their families, in outdoor cages, under

conditions of natural lighting, humidity, and temperature. The cages measured 2.0 m ×

2.0 m × 1.0 m and were built of masonry. The front consisted of a glass wall with a

unidirectional visor and on the back wall, a wire mesh door, on which were placed a

water bottle and a plate of food. Inside the cage were placed a nest box in which to rest,

planks of wood, and branches of plants for environmental enrichment and to allow the

animals to move around the cage.

During the social isolation phases of both procedures (IDS and DPT), the animals

were placed in new masonry cages with different dimensions (1.0 m × 2.0 m × 1.0 m)

from those in which they were living in family groups, but without space restrictions.

33

These new cages were also located outdoors. In this condition, the animals did not have

any visual contact with conspecifics but had auditory and olfactory contact with other

conspecifics that were not members of their own families.

None of the animals had been separated from their respective family groups for

prolonged periods and were habituated to the presence of the researchers prior to the

study. Veterinary care was provided throughout the experiment. Water was available

without restriction during the entire study and all animals were fed twice a day with the

same diet, which included seasonal fruits such as banana, papaya, melon, and mango, as

well as potato and a protein potage containing milk, oats, egg, and bread. A

multivitamin supplement (Glicocan) was administered twice a week. In order to address

the inter-relational validity criteria for validation of this translational animal model, the

animals were weighed approximately every 15 days.

The animals were housed according to IBAMA (Brazilian Institute of

Environment and Renewable Natural Resources) guidelines (Normative Instruction no.

169 of February 20, 2008), and the care standards for animals established by

CONCEA—National Council for Animal Experimentation Control, Law No. 11.794

(October 8, 2008). In addition, the laboratory complies with international standards for

ex situ maintenance of animals as defined by the Animal Behavior Society and the

International Primatological Society. The study and experimental procedures were

approved by the Animal Research Ethics Committee (UFRN protocol No. 019/2013 and

protocol No. 034/2014).

2.5 Behavioral records

A continuous fecal sampling method was used to evaluate the frequency and/or

duration of the selected behaviors. Recording was performed continuously for each

animal over a 30-min period (51). Behavioral data were always collected between 06:30

and 07:30 a.m. to avoid the influence of circadian variation (52). Descriptions of

behaviors were according to the ethogram compiled by Stevenson and Poole (42)

(Table 1).

34

Table 1. Description of behaviors.

Typical behaviors for common marmosets, which included scent marking

(frequency), individual piloerection (frequency), scratching (duration), and

autogrooming (duration) were recorded. Indeed, behaviors that can be compared across

species to address evolutionary validity, such as locomotion (frequency), somnolence

(duration, investigated only in DPT), feeding (frequency for IDS/frequency and

duration for DPT), and anhedonia were also recorded. The frequency (for IDS and DPT)

and duration (DPT) of ingestion of an aqueous solution of sucrose (4.16%) was

measured to verify a possible state of anhedonia [adapted from Paul et al. (58)].

2.6 Fecal collection and cortisol assay

BEHAVIOR DESCRIPTION CONTEXT OF STRESS

Scent marking

Act of scrubbing the

anogenital and suprapubic

region on a substrate.

Expression of anxiety (62; 63).

Individual

piloerection

Act of erection of the pelage

and walking with an arched

back.

Expression of activation of the

sympathetic nervous system (63).

Autogrooming Act of self-grooming. Acts as a stress/tension reducer (69;

70).

Locomotion

Act of moving randomly

between four different

quadrants of the same size,

delimited in a cage.

Expression of anxiety (62; 63).

Scratching Act of using hands to scrub

some body region.

It is considered a stereotyped

behavior (49).

Somnolence Characterized by a slow

blink, sleepiness, and stare.

Feeding Act of taking a piece of food

to the mouth and ingesting it.

Ingestion of an

aqueous

solution of

sucrose (4.16%)

Act of drinking a palatable

substance (sucrose solution).

The reduction of this pleasurable

behavior can express a possible

state of anhedonia

(49; 71).

35

In order to address the functional and inter-relational criteria for validation of this

translational animal model, feces were collected for the measurement of cortisol. Fecal

collection was performed in the morning between 6:30 and 8:30 a.m. to avoid circadian

variation in cortisol measurement (59). Fecal cortisol reflects plasma cortisol with a

delay of approximately 8–10 h (45).

Prior to fecal collection, the cages were cleaned to facilitate sample identification.

The observer entered the cage shortly after animal defecation and collected the sample

with a clean wooden stick. Samples were identified and stored at approximately −4°C

until the day when they were processed for cortisol extraction and quantification at the

Hormonal Measurements Laboratory of Department of Physiology—UFRN, according

to the protocol of Sousa and Ziegler (45). Intra- and inter-assay coefficients of variation

for fecal cortisol obtained in this study were 1.78 and 15.47%, respectively, for samples

collect during IDS and 2.74 and 16.61%, respectively, for samples collected during the

DPT procedure.

2.7 Statistical analysis

Hormonal data were normalized by logarithmic transformation, and for both

hormonal and behavioral data, the statistical technique of bootstrap resampling was

applied to the multivariable analysis. For cortisol, one outlier four standard deviations

above the mean was excluded from the analysis in W5 of IDS.

General linear models (GLM) Fisher’s post hoc test were used to investigate the

variations of behaviors, cortisol, and body weight between groups (FC/IC) or sex (in

DPT) throughout the study phases. Moreover, the parametric Student’s t-test and the

nonparametric Mann–Whitney U and Wilcoxon’s sum-rank tests were used in some

investigations to analyze hormonal and behavioral data. The correlations between

behaviors and hormones and between behaviors were investigated in combined phases

by the Spearman correlation test. Results were considered statistically significant at p ≤

0.05 and 0.05 < p < 0.07 was considered to indicate a trend in all the tests.

3 Results

3.1 Protocol for IDS: Etiologic, face, and functional validity

3.1.1 Hormonal profile

36

No significant variation in cortisol levels was observed throughout the experiment

in the FC group (n = 5). On the other hand, in IC (n = 5) cortisol levels were statistically

higher in W1 than in BL, W5, W9, and W13. After W1, cortisol profiles showed a

gradual reduction along the weeks, with lower values in W13 as illustrated in Figure

3A. All statistical values are in Table 2.

3.1.2 Behavioral profile

No significant differences between groups nor variations across phases were

observed in scent marking, individual piloerection and feeding [GLM test,

Phases*Groups; scent marking: F = 0.77, p = 0.54, df = 4; individual piloerection: F =

2.03, p = 0.09, df = 4; feeding (frequency): F = 0.44, p = 0.77, df = 4]. However, during

the IDS protocol, significantly higher scores for individual piloerection for the IC

compared with the FC group were detected (GLM test, Group*; individual piloerection:

F = 5.22, p = 0.02, df = 1).

Regarding locomotion, a significant increased occurred in W1 relative to BL, W9,

and W13, but not to W5. In W13, the frequency of locomotion was similar to that of BL

(Figure 3B). All statistical values are in Table 2. In addition to IC, consecutive

significant increases in the frequency of scratching were observed in W5, W9, and W13

(Figure 3C). During the IDS protocol, analyzing all weeks together, significantly

higher scores for scratching were found for the IC group than in the FC group (GLM

test, Group*; scratching: F = 25.55, p = 0.01, df = 1).

For autogrooming (duration in seconds), significant increases were observed for

both groups, but those of the FC group occurred only between W13 vs. W1 and W5. For

IC, significant increases in autogrooming behavior were detected at W9 and W13

relative to BL, W1 and W5. All statistical values are in Table 2. Although both

conditions (familiar and socially isolated), showed increases in W13, the duration of

autogrooming was higher in IC than in FC at W13 and W9, but was higher in FC than in

IC at BL (Figure 3D). All statistical values are in Table 2.

We observed a significant reduction in the frequency of ingestion of sucrose

solution in FC at W13 relative to BL and W5. For IC were observed reduction in initials

and finals phases of isolation with respect to BL. All statistical values are in Table 2.

Moreover, the percentage of reduction in the frequency of ingestion of sucrose solution,

comparing BL and W13, was higher in IC than in FC (Mann–Whitney U = 1.00, p =

37

0.01), while IC reduced the intake by approximately 65.99%, and FC reduced it by

26.95% (Figure 3E).

For IC, correlation analysis along the experimental phases (W1, W5, W9, and

W13) showed significant negative correlations between cortisol and autogrooming and

scratching and positive correlations between locomotion and scent marking, individual

piloerection, and scratching (Table 3). No significant statistical correlations were found

in FC among the same comparisons.

3.1.3 Body weight

The body weight records over 13 weeks showed a parallel profile of weight gain

in both groups, except for the IC group in W9, when the values decreased. Statistically

significant differences between groups occurred at this point, where FC showed higher

weight than IC. It is interesting to note that at W13, IC animals showed a recovery of

weight and presented higher values than those observed in the initial phases, which was

similar to that observed in the FC group (Figure 3F). All statistical values are in Table

2.

38

Table 2. Statistical values of the protocol for induction of depression state (IDS).

General linear models (GLM) and Fisher’s post hoc tests were used to investigate the variations of behaviors, fecal cortisol, and body weight between groups (FC/IC) or sex (in DPT) throughout the

study phases. Results were considered statistically significant at p ≤ 0.05 and 0.05 < p < 0.07 as statistically trend. All p values in black correspond to statistical significance and values in gray to

non-significant ones.

BL, baseline; FC, familiar context; IC, isolated context; W1, Week 1; W5, Week 5; W9, Week 9; W13, Week 13.

Fecal Cortisol Autogrooming

Frequency of ingestion of sucrose solution

Body weight Locomotion Scratching

F = 3.92, p = 0.01, df = 4 F = 5.49, p = 0.01, df =4 F = 8.16, p = 0.00, df = 4 F = 3.44, p = 0.02, df = 4 F = 2.35 p = 0.05, df = 4 F = 13.98, p = 0.01, df =4

FC

W1 W5 W9 W13 W1 W5 W9 W13 W1 W5 W9 W13 W1 W5 W9 W13 W1 W5 W9 W13 W1 W5 W9 W13

BL p = 0.77 p = 0.12 p = 0.74 p = 0.98 p = 0.47 p = 0.31 p = 0.71 p = 0.20 p = 0.08 p = 0.91 p = 0.14 p = 0.01 p = 0.32 p = 0.05 p = 0.07 p = 0.00 p = 0.87 p = 0.86 p = 0.25 p = 0.44

p = 0.86 p = 0.27 p = 0.59 p = 0.34

W1 p = 0.20 p = 0.96 p = 0.76 p = 0.76 p = 0.28 p = 0.04 p = 0.06 p = 0.79 p = 0.27 p = 0.31 p = 0.38 p = 0.01 p = 0.99 p = 0.33 p = 0.55

p = 0.21 p = 0.71 p = 0.27

W5 p = 0.22 p = 0.12 p = 0.17 p = 0.02 p = 0.11 p = 0.01 p = 0.88 p = 0.09 p = 0.33 p = 0.55

p = 0.10 p = 0.88

W9 p = 0.73 p = 0.36 p = 0.17 p = 0.07 p = 0.71

p = 0.14

IC

BL p = 0.01 p = 0.49 p = 0.51 p = 0.01 p = 0.77 p = 0.40 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.61 p = 0,23 p = 0.02 p = 0.00 p = 0.01 p = 0.10 p = 0.68 p = 0.54 p = 0.58 p = 0.01 p = 0.01 p = 0.01

W1 p = 0.01 p = 0.01 p = 0.01 p = 0.26 p = 0.00 p = 0.00 p = 0.58 p = 0.43 p = 0.04 p = 0.48 p = 0.00 p = 0.00 p = 0.38 p = 0.03 p = 0.00 p = 0.01 p = 0.01 p = 0.01

W5 p = 0.18 p = 0.01 p = 0.00 p = 0.00 p = 0.81 p = 0.13 p = 0.00 p = 0.03 p = 0.22 p = 0.02 p = 0.24 p = 0.02

W9 p = 0.01 p = 1.00 p = 0.20 p = 0.00 p = 0.31 p = 0.25

F = 3.92, p = 0.01, df = 4 F = 5.49, p = 0.01, df =4 F = 8.16, p = 0.00, df = 4 F = 3.44, p = 0.02, df = 4 F = 2.35 p = 0.05, df = 4 F = 13.98, p = 0.01, df =4

BL p = 0.31 p = 0.05 p = 0.01 p = 0.68 p = 0.55 p = 0.90

IC W1 p = 0.15 p = 0.12 p = 0.37 p = 0.52 p = 0.06 p = 0.56

X W5 p = 0.04 p = 0.86 p = 0.15 p = 0.46 p = 0.99 p = 0.01

FC W9 p = 0.09 p = 0.01 p = 0.74 p = 0.01 p = 0.56 p = 0.01

W13 p = 0.01 p = 0.02 p = 0.01 p = 0.78 p = 0.01 p = 0.01

39

Table 3. Significant Spearman correlation (p < 0.05), between behaviors, and behaviors

versus cortisol, in both experimental protocols of the study, IDS (protocol for induction

of depression state; IC, isolated group, males = 5) and DPT (protocol for depression

state + pharmacological treatment; males = 8, females = 7).

IC group (IDS) Males + Females (DPT)

Cortisol*AutoG (rs = - 0.16) AutoG*Loc (rs = - 0.15)

Cortisol*Scrat (rs = - 0.19) Scrat*Cortisol (rs = - 0.21)

Loc*ScentM (rs = 0.46) Scrat*AutoG (rs = 0.53)

Loc*PiloI (rs = 0.15)

Loc*Scrat (rs = 0.22)

AutoG: autogrooming; PiloI: individual piloerection; ScentM: scent marking; Loc: locomotion; Scrat: scratching.

40

Figure 3: Mean of the week + SEM for: (A) fecal cortisol, (B) locomotion, (C)

scratching, (D) autogrooming, (E) ingestion of sucrose solution, and (F) body weight in

the IDS (protocol for induction of depression state) for family context group (FC) and

social isolated context group (IC) of male juveniles Callithrix jacchus in the baseline

(BL) and weeks of the experimental protocol: 1st week (W1), 5th week (W5), 9th week

(W9), and 13th week (W13). *Statistically significant (p ≤ 0.05) differences between

the phase in which the symbol is and the (s) phase (s) indicated next to the symbol;

#statistical difference (p ≤ 0.05) between the groups in the respective phase;

°statistically trend (0.05 < p < 0.07) of difference between the phase in which the

symbol is and the phase indicated next to the symbol or between the groups in the

respective phase. General linear models and post hoc Fisher.

41

2.2 Protocol for DPT: predictive validity IC


In last 2 weeks of social isolation (W7 and W8), the cortisol levels of males (n =

8) and females (n = 7) decreased significantly below BL levels and W1 (Figure 4A).

All statistical values are in Table 4.


No significant changes were observed in individual piloerection and somnolence

across phases or by sex and between interactions of them (GLM test; individual

piloerection, Phases*: F = 0.83, p = 0.50; Sex*Phase: F = 0.60, p = 0.65; somnolence,

Phases*: F = 1.52, p = 0.19; Sex*Phase: F = 1.44, p = 0.21).

Males and females showed similar profiles of changes in autogrooming,

scratching, and locomotion behaviors during social isolation phases. In the first 2 weeks

(W1 and W2) of isolation, significant increases were observed in locomotion relative to

the BL phase. Locomotion in W1 and W2 were also statistically higher when compared

with the last 2 weeks (W7 and W8) when the frequency of displacements returned to BL

values (Figure 4B). All statistical values are in Table 4.

Moreover, in both sexes, in W7 and W8, significant increases in autogrooming

were observed with respect to all anterior phases (BL, W1, and W2) (Figure 4C). All

statistical values are in Table 4. A weak negative correlation between autogrooming

and locomotion was detected (Spearman correlation: rs = −0.15, p < 0.05) (Table 2).

Scratching behavior also presented a significant increasing pattern in W2, W7, and

W8, as well as W2 with respect to W1, and W7 and W8 relative to BL, W1, and W2

(Figure 4D). All statistical values are in Table 4. A weak negative correlation between

scratching and cortisol (Spearman correlation: rs = −0.12, p < 0.05) and a positive

correlation between scratching autogrooming in the IC group were observed (Spearman

correlation: rs = 0.53, p < 0.05) (Table 3).

Scent marking and feeding behaviors showed a sexual dimorphic profile of

variation during the social isolation period. Males increased scent marking behavior

significantly in W7 and W8 with respect to anterior weeks. In contrast, females reduced

this behavior in both W2 and W7 relative to the BL phase and showed a strong

42

decreasing tendency in W8 relative to BL (Figure 4E). All statistical values are in

Table 4.

Males showed a significant reduction in feeding (duration) at W1, W7, and W8

relative to the BL phase. In females, reduced feeding occurred during all phases of

isolation in comparison with the BL phase (Figure 4F). All statistical values are in

Table 4.

For males and females grouped together, the frequency and duration of ingestion

of sucrose solution was significantly reduced between BL and W8 by 41.33 and

50.55%, respectively (Wilcoxon frequency: z = 3.14, p = 0.00; duration: z = 2.70, p =

0.00). Despite a large number of cases with zero frequencies, it was not possible to run a

GLM test due to a high frequency of zeros.

3.2.3 Body weight

Males and females showed a significant statistically dimorphic profile of

alterations in body weight (GLM; Phases*Sex: F = 10.68, p = 0.00). Males experienced

reduced body weight in the first week (W1) of isolation and maintained the reduction in

all subsequent weeks of isolation (W2, W7, and W8). In contrast, females showed

increased body weight at W1 and W2 with respect to BL. However, weight loss was

observed at W7 and W8 with respect to W1 and W2 (Figure 4G). All statistical values

are in Table 4.

43

Table 4 - Statistical values of the Protocol for depression state + pharmacological treatment (DPT): Baseline (BL) + Isolated context (IC)

General linear models (GLM) and Fisher’s post hoc tests were used to investigate the variations of behaviors, fecal cortisol, and body weight between groups (FC/IC) or sex (in DPT) throughout the study phases. Results were considered statistically significant at p ≤ 0.05 and 0.05 < p < 0.07 as statistically trend. All p values in black correspond to statistical significance and values in gray to non-significant ones. M, males; F, females; BL, baseline; W1, Week 1; W2, Week 2; W7, Week 7; W8, Week 8.

Fecal Cortisol Locomotion Autogrooming Scratching F = 3.88, p = 0.00, df = 4 F = 14.05, p = 0.00, df = 4 F = 9.47, p = 0.00, df = 4 F = 22.77, p = 0.00, df = 4

M + F

W1 W2 W7 W8 W1 W2 W7 W8 W1 W2 W7 W8 W1 W2 W7 W8

BL p = 0.44 p = 0.09 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.54 p = 0.82 p = 0.80 p = 0.60 p = 0.00 p = 0.00 p = 0.08 p = 0.23 p = 0.00 p = 0.00

W1 p = 0.36 p = 0.01 p = 0.01 p = 0.20 p = 0.00 p = 0.00 p = 0.78 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00

W2 p = 0.14 p = 0.14 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00

W7 p = 0.99 p = 0.70 p = 0.55 p = 0.41

Scent marking Feeding Body weight

F = 10.88, p = 0.00, df = 4 F = 3.05, p = 0.01, df = 4 F = 10.68, p = 0.00, df = 4

M

W1 W2 W7 W8 W1 W2 W7 W8 W1 W2 W7 W8

BL p = 0.24 p = 0.79 p = 0.02 p = 0.00 p = 0.00 p = 0.08 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.00

W1 p = 0.36 p = 0.00 p = 0.00 p = 0.03 p = 0.26 p = 0.63 p = 1.00 p = 0.41 p = 0.41

W2 p = 0.01 p = 0.00 p = 0.00 p = 0.11 p = 0.41 p = 0.41

W7 p = 0.00 p = 0.11 p = 1.00

F

BL p = 0.14 p = 0.04 p = 0.05 p = 0.06 p = 0.00 p = 0.00 p = 0.00 p = 0.00 p = 0.01 p = 0.01 p = 0.75 p = 0.75

W1 p = 0.55 p = 0.64 p = 0.70 p = 0.55 p = 0.04 p = 0.30 p = 1.00 p = 0.03 p = 0.03

W2 p = 0.90 p = 0.83 p = 0.14 p = 0.65 p = 0.03 p = 0.03

W7 p = 0.93 p = 0.30 p = 1.00

44

Figure 4: Mean of the week + SEM for (A) fecal cortisol, (B) locomotion, (C)

autogrooming, (D) scratching, (E) scent marking, (F) feeding, and (G) body weight in

male and females juveniles Callithrix jacchus along DPT (protocol for depression state

+pharmacological treatment) in baseline (BL) and Isolated context (IC; W1, W2, W7,

and W8). *Statistically significant (p ≤ 0.05) differences between the phase in which the

symbol is and the (s) phase (s) indicated next to the symbol; °statistically trend (0.05 < p

45

< 0.07) of difference between the phase in which the symbol is and the phase indicated

next to the symbol. General linear models and post hoc Fisher.

3.3 Vehicle effect, PH, and tPE


As described in the Methods section, from the 15 animals that were socially

isolated (IC) (M = 8; F = 7), 3 males and 3 females were randomly selected to

participate in a protocol of VE and pharmacological treatment (DPT) and were followed

during 5 additional successive weeks after isolation (Week 8). In the first week (Week 9

= W9 = VE), common marmosets were subjected to a daily injection of VE (saline). In

the 10th week (W10), a daily injection of antidepressant drug (nortriptyline chloride)

was injected into the same animal (PH). Then, in the 11th, 12th, and 13th weeks (W11,

W12, and W13) without any manipulation, all six animals were monitored for the tPEs.

The data collected under these three conditions (VE, PH, and tPE) were compared with

those obtained for the same animals in W8.

Statistical increases in cortisol levels were detected in W10 and W11 relative to

W8 (Figure 5A). All statistical values are in Table 5. The cortisol levels observed in

the pharmacological treatment week (W10) was higher than BL levels (t-test = −2.80, p

= 0.00) (Figure 5B).


Males and females reduced significantly the scratching and feeding in response to

pharmacological treatment (W10). This same reduction was not observed in the VE

(W9) with respect to W8 (Figures 5C, D, respectively). All statistical values are in

Table 5.

Females increased significantly the locomotion in the VE (W9) and

pharmacological weeks (W10) with respect to W8, whereas males reduced locomotion

in W10 with respect to W8 and W9 (Figure 5E). All statistical values are in Table 5.

Median somnolence in W8 and W9 were similar, μ = 0.00 •+ 0.00. However, an

increase in somnolence was observed in W10 with respect to W8 (μ = 3.77 •+ 2.88)

(Wilcoxon z = 2.36, p = 0.01).

46

Despite the low variability and large number of case with zero frequencies of

sucrose solution ingestion, in W8, W9, and W10, once again, the GLM test and

Wilcoxon test could not be conducted (median: W8, p = 0.00, W9, p = 0.00 and W10, p

= 0.00).

3.3.3 Body weight

Males and females did not present significant variations in body weight (GLM

test; Phases*Sex: F = 0.17, p = 0.83, df = 2) during the VE and pharmacological

treatments (Figure 5F).

47

Table 5 – Protocol for depression state + pharmacological treatment (DPT): Vehicle (VE), pharmacological (PH) + tardive Pharmacological

Effects (tPE)

General linear models (GLM) and Fisher’s post hoc test were used to investigate the variations of behaviors, fecal cortisol, and body weight between groups

(FC/IC) or sex (in DPT) throughout the study phases. Results were considered statistically significant at p ≤ 0.05 and 0.05 < p < 0.07 as statistically trend. All

p values in black correspond to statistical significance and values in gray to non-significant ones.

M, males; F, females; W8, Week 1; W9, Week 9; W10, Week 10; W11, Week 11; W12, Week 12; W13, Week 13.

Fecal Cortisol Scratching Feeding

F = 3.16, p = 0.00, df = 5 F = 5.78, p = 0.00, df = 2 F = 6.63, p = 0.00, df = 2

W9 W10 W11 W12 W13 W9 W10 W9 W10

M + F

W8 p = 0.14 p = 0.00 p = 0.00 p = 0.07 p = 0.91 p = 0.94 p = 0.00 p = 0.52 p = 0.00

W9 p = 0.23 p = 0.10 p = 0.72 p = 0.17 p = 0.00 p = 0.00

W10 p = 0.66 p = 0.40 p = 0.01

W11 p = 0.20 p = 0.00

W12 p = 0.09

Locomotion

F = 8.87, p = 0.00, df = 2

M

W9 W10

W8 p = 0.48 p = 0.01

W9 p = 0.00

F W8 p = 0.02 p = 0.00

W9 p = 0.34

48

Figure 5 - Mean of the week •+ SEM for: (A,B) fecal cortisol, (C) scratching, (D)

feeding, (E) locomotion, and (F) body weight in male and females juveniles Callithrix

jacchus along DPT (protocol for depression state + pharmacological treatment) in

baseline (BL) and isolated context (IC; W8), VE (W9), PH (W10), and tPE (W11, W12,

and W13). *statistically significant (p ≤ 0.05) differences between the phase in which

the symbol is and the (s) phase (s) indicated next to the symbol; °statistically trend (0.05

< p < 0.07) of difference between the phase in which the symbol is and the phase

indicated next to the symbol. General linear models and post hoc Fisher.

49

4 Discussion

The aim of this study was to validate C. jacchus as a translational juvenile animal

model using chronic social isolation as a protocol inductor of depression, meeting the

distinct criteria of validation: etiologic, face, functional, predictive, inter-relational,

evolutionary, and population.

To meet the population criteria and mimic the variability observed in human

populations, 25 common marmosets, males and females, were randomly selected from

20 different families of the Laboratory of Advanced Studies in Primates of the

Universidade Federal do Rio Grande do Norte, Natal, Brazil. This laboratory had

approximately 150 animals with fair genetic variability, living under captive conditions,

and their pedigree comes from animals captured from nature and those born in captivity.

To address the etiologic criteria of validation, we elected to use a chronic social

stressor protocol to induce the depression state in non-human primates, as social

stressors seem to be the most prevalent inductors of depression in humans. Social

species exhibit behavioral interactions with their conspecifics, who express adapted

neural, hormonal, cellular, and genetic mechanisms that support survival, reproduction,

and care of offspring. Consequently, social isolation causes disruptions in the social

relationship and dysfunctions in physiological mechanisms reducing the adaptability

and inducing the onset of physical and mental disorders (28, 60, 61).

To meet the face and functional domains of validation criteria, initially, 10

juvenile males were chronically socially isolated for 13 weeks, and modifications in

their behaviors were investigated, including hormonal profile (fecal cortisol) and body

like state. The alterations observed in socially isolated animals were not observed in

body weight. We observed that this procedure triggered significant physiological and

behavioral changes, and some of those showed intervariable correlations, which

supported the investigated inter-relational criteria. Several of these alterations were

similar to those observed in humans and/or in non-human primates in the depressive-

like state. The alterations observed in socially isolated animals were not observed in

common marmosets of the same sex and age that remained in their family

environments.

Moreover, to address inter-relational and evolutionary criteria of validation, we

analyzed species-specific behaviors such as individual piloerection, scent marking,

scratching, and autogrooming, as well as behaviors that can be compared among

50

species, such as locomotion, anhedonia, feeding, and somnolence. Indeed, body weight

and cortisol levels were analyzed, constituting parameters that can also be used for

comparison across species in other studies.

Our results showed that animals who remained in the family group (FC), in

protocol 1 (IDS) exhibited no significant alterations in cortisol levels throughout the 13

weeks of the study. This is expected, since the social and environmental conditions were

unchangeable and supportive. In accordance with the “theory of main effect,” social

support is a buffer against the challenges posed by stressors of the daily routine,

reducing stress responses and the onset of associated pathologies (62). In marmosets,

social and affiliative behaviors such as allogrooming and body contact, induce

reductions in cortisol levels (63). However, the benefits of social support are not a

general phenomenon and depend on many factors such as species, sex, age,

temperament, genetic relatedness, and familiarity (39, 64).

The changes in hyper and hypocortisolism are entirely linked to a variety of other

alterations in common marmosets, showing their regulatory influence to face isolation

from the family group. Hypercortisolemia was observed as an acute response, at the

beginning of isolation in the animals and might promote changes similar to those

observed in humans, such as hyperglycemia, increased cardiovascular and respiratory

system, inhibition of the reproductive system and cellular growth, imbalance of the

immune system, and a greater susceptibility to viral infections. While hypocortisolism

was recorded during the chronic phase of isolation and can cause damage to the animal

since it is associated with the expression of hypoglycemia and dysregulation of the

immune system and greater propensity for bacterial infections. Moreover, it can trigger

severe inflammatory processes, characterized by reduced energy in the animal and a

state of apathy, characteristic of depression associated with chronicity (49).

By contrast, animals of the IC group displayed increased cortisol levels and an

increased frequency of locomotion in the initial week of isolation (W1), characterizing a

typical endocrine and behavioral anxious stress response to an acute stressor. Afterward,

they displayed a reduction in both of these variables, indicating a recovery of the initial

stress response. It is important to note that the random displacement of the animals after

isolation observed in this study differs from the approximation and withdrawal directed

to another animal or object being considered as an anxious behavior, because it occurs

without a defined interest (65). These changes in locomotion were positively correlated

with scent marking and individual piloerection behaviors, which are also considered

51

anxious behaviors in a stress context (53, 54, 66) IC animals also exhibited higher levels

of individual piloerection than those in FC during W1 to W13 of the IDS protocol.

Likewise, individual piloerection also is considered an indicator of activation of the

sympathetic nervous system (54).

The continued exposure to stressors induced other alterations in IC animals,

showing that the ability to self-adjust allostatic systems fails. In subsequent phases, IC

animals showed an increase in scratching from W5 to W13, which remained higher than

that of FC in the IDS protocol. Additionally, a negative correlation between cortisol and

scratching in IDS (W1 to W13) was observed. Scratching also occurs as dislocate

behavior, without the cleaning function, and is also included as typical depression like

behavior in non-human primates (29, 65, 67). The occurrence of ethologically abnormal

patterns where the animals express stereotypic behaviors and/or self-mutilation might

indicate the low quality of life of animals and the presence of behavioral disorders (68,

69).

During the last two phases of IDS (W9 and W13), increases were registered in the

duration of autogrooming, which is a reducer of tension behavior, for socially isolated

animals. For the IC group, this increase in autogrooming correlated positively with a

reduction of cortisol levels. Autogrooming reduces tension by induction of oxytocin

release, an inhibitor of activation of the HPA axis (55, 56, 70). A similar result was

recorded for Callithrix geoffroyi when an inverse association between the frequency of

social grooming and urinary cortisol was demonstrated (63).

Anhedonia corresponds to an inability to feel pleasure, which is an important

symptom in human patients with depression. In non-human primates and in rodents, it

has been investigated by measuring the intake of an aqueous solution of sucrose (29, 57,

65, 71, 72). In this study, both groups decreased their consumption of sucrose solution

during the 13 weeks of IDS, as expected, since consumption declines when a sweet taste

is no longer a novelty.

However, the profile of the reduction was different between the two groups,

showing that the reduction in IC was faster than in FC, taking place in W1 in IC and

only starting in W13 in FC. Furthermore, the proportion of reduction of consumption of

sucrose solution between basal and final ingestion was higher in IC than in FC,

suggesting that IC animals developed an anhedonic state, similar to that of human

patients with depression.

52

The IC animals reached the end of the study showing lower cortisol levels than

those observed at BL and lower than those in FC. Studies in depressed human patients

or in animal models of depression found conflicting results regarding cortisol levels (49,

73, 74). The majority of animal studies used rodents as models, for which protocols

differ in nature and the duration of the stressor with respect to those used in this study

(8, 29). Most protocols available in the literature involve acute situations and frequently

use physical stressors known as “earned helplessness” (23, 75). Moreover, in protocols

in which social stressors are used, such as early maternal separation and social defeat,

the results should be evaluated with care because these stressors can have low

ecological validity depending on the rodent species used (76, 77). It is important to

consider that some rodents have complex social organization, such as free-living

populations of house mice (Mus musculus domesticus), in which females display social

cooperation such as regular nursing of non-offspring (76). However, some rodent

species display solitary habits and lower parental care in their natural environment, as

observed in montane (Microtus montanus) and meadow (Microtus pennsylvanicus)

voles and African mole-rats (78–83). In these distinct taxa the neurobiology, behaviors,

as well as reproductive and parental adaptations are at the opposite sides of the

spectrum. Therefore, social stressors may have low ecological validity to taxa that adopt

a strictly solitary lifestyle and lack the ability to form long-term social bonds.

However, recent studies that induced depression in non-human primates using

paradigms of chronic social stressors such as rank dispute or social isolation, similar to

that used in our study, found hypocortisolemia in response to these protocols. An

association between low levels of cortisol and depressive like behaviors in adult females

of Macaca fascicularis exposed to chronic changes in social position (22–26 months)

was demonstrated by Willard and Shively (49). For squirrel monkey (Saimiri sciureus)

juveniles, subjected to repeated separation from their mothers as infants revealed

cortisol hyporesponsiveness in adulthood when compared with a control group (84).

Juvenile common marmosets that were exposed to repeated separation of their families

in infancy present lower basal cortisol levels than animals of the same age that were not

exposed to this stressor (54).

In humans while major depression is traditionally characterized by

hypercortisolemia (85), specific groups have been showing hypocortisolemia, like

patients with atypical subtype major depression, patients with maladaptive coping styles

and major depression associated with severe, refractory and chronic conditions (86–90).

53

Currently, two theories attempt to explain the etiology of hypocortisolemia. The first

associates hypocortisolemia with increased sensitivity of negative feedback on the HPA

(91), and the second is linked to an adrenal insufficiency (92). Generally, the literature

associates depression with a hyperactivation of the HPA axis and hypercortisolemia in

depressed human patients or in animal models of depression (28, 73, 74).

Some studies have suggested that hypocortisolemia initially might be a protective

adaptation of the individual after abrupt rises in cortisol in response to chronic stressful

events (93). However, if this condition remains for an extended period, the ability to

self-adjust allostatic systems fails, and pathologies arise (94). Therefore, in this study,

one allostatic system that could likely have induced a severe reduction of cortisol in IC

animals to pathological levels after W9 and fail to return cortisol levels might be

autogrooming, as suggested by the negative correlation between cortisol and

autogrooming recorded for IC.

The failure of allostatic systems to self-adjust might induce permanent

physiological vulnerability, especially if it occurs during biological windows of

plasticity, such as adolescence, therefore facilitating the subsequent emergence of

psychiatric disorders such as depression (95, 96). Some studies show that when the first

signs of depression arise during adolescence, those symptoms frequently will also be

manifested in adulthood (97, 98).

In addition to alterations in behaviors and cortisol levels, during W9, a severe

reduction in body weight of the IC group, but not in FC, was found in this study. The

weight loss seems not to be related to a reduction in feeding, because the isolated

animals did not alter the amount of food ingested during the IDS protocol. After a

reduction of body weight in W9, the isolated animals recovered in W13, showing higher

values relative to the BL phase. Loss of weight is a typical response of non-human

primate adults subjected to depression protocols (29, 65). Nevertheless, the evaluation

of this parameter during ontogenetic phases of rapid growth, such as the juvenile stage,

might mask any results and generate false conclusions (99). Moreover, the relation

between chronic stress and feeding appears to be quite individualized and studies with

animal models suggest that stressful situations can lead to increases in some situations,

but in others, it leads to decreases in feeding (100). In humans, similarly to animal

models, stressful situations and depressive disorders can also act in both directions on

feeding (101).

54

In summary, the animals of IC showed correlated behaviors and physiological

(cortisol fecal and body weight) alterations, which included hypocortisolemia, high

levels of scratching, and anhedonia, composing a typical depression-like state that is

similar to alterations observed in other species of non-human primates or/and humans.

Therefore, the results exhibited for common marmosets corroborate the validation of

face, functional, inter-relational, and evolutionary criteria for this animal model.

After the above validation, to address predictive criteria of validation and to

investigate sexual dimorphism in this model, eight more males and seven female

juvenile common marmosets were randomly selected from different families to be

socially isolated by 8 weeks and to subsequently be treated sequentially with a VE and

nortriptyline (M = 3 and F = 3). The same parameters of the IDS protocol were

investigated namely, behaviors, fecal cortisol, and body weight.

In large part, the alterations observed in protocol for DPT were similar between

the sexes, with the exception of scent-marking and body weight. In common marmosets,

puberty starts around 6–7 months, whereas sexual maturity is achieved at approximately

at 16 months (35, 102). As a result, sexual maturity transitions of the animals used in

this study may have produced variability in terms of sexual dimorphism of stress

response, with some and not others being sexually dimorphic.

In the initial isolation phases of IC (W1 and W2), as expected, increases in

locomotion and anxious behavior were observed in both sexes, similarly to the results

observed in IDS protocols for inducing a depression-like state. Afterward, males and

females reduced their cortisol production in the two final phases of isolation (W7 and

W8), again consistent with the results of IDS. For common marmosets at this age, de

Sousa et al. (40) found similar cortisol responses between male and female juveniles (12

months) after social isolation (21 days), in which they observed a drop in the cortisol

response. Once again, probably, in DPT, autogrooming behavior acted as a

decompensated allostatic mechanism responsible for the pathological reduction of

cortisol, as both males and females showed an increase in the duration of autogrooming

concomitant with decreased cortisol.

Also in W7 and W8, both sexes showed similar increases in the frequency of

scratching, a stereotypical behavior. Moreover, the presence of anhedonia was inferred

in W8; the animals reduced the frequency of ingestion of sucrose solution by 41%, both

findings also analogous to those of the IDS protocol. By contrast, males and females

showed dimorphic alterations in scent marking. Whereas males increased this behavior

55

in the last two weeks of social isolation, females reduced scent marking between W2

and W8.

Moreover, males and females showed a reduction in feeding during IC and

consequently, reductions in body weight, but distinct gradations between the sexes were

observed. For males, weight loss was observed immediately (W1) and was sustained

until W8. On the other hand, females initially gained weight and only lost weight in the

last two phases of isolation (W7 and W8). These results in females corroborate the

discussion presented for animals of the IDS protocol, which indicate difficulties in the

analysis of body weight in this phase of ontogenetic development, because of intense

growth and large individual variability.

Similarly and more consistent than the results of IC (IDS protocol), physiological

and behavioral alterations observed in IC (DPT protocol) such as hypocortisolemia,

high levels of scratching, and anhedonia associated with a reduction in feeding and

body weight were typical of a depression-like state in non-human primates, allowing us

to investigate the action of a traditional antidepressant drug in these animals,

nortriptyline.

To address this, after the isolation phase (IC) of DPT protocol, three males and

three females were randomly selected to be treated for 7 days (W9) with a VE followed

by nortriptyline treatment for an equal period (W10). During W10, an increase was

observed in cortisol levels above the values observed during BL, which was not induced

by VE. This cortisol increase induced by drug was sustained for 7 additional days in

W11 after suspension of the treatment. Subsequently, in W12 and W13, cortisol

returned to similar levels to those observed in the final week of social isolation (W8).

The high cortisol levels detected in W10 might be related to the dose of

nortriptyline and the duration of the pharmacological protocol used for marmosets. The

dose used was the most effective(30 mg/kg ip) for rats in Moura (50), but our protocol

was more extensive than that used in their study. Based on these findings, future

protocols are needed that include different treatment dosages and/or durations for more

adjusted changes.

Furthermore, after nortriptyline, but not with a VE, males and females exhibited

reduced frequencies of scratching, reversing the high levels induced by isolation. This

observation is very important, as scratching is a stereotypical behavior permanently

observed in non-human primates and associated with a depression-like state (29, 65,

67).

56

Different profiles were observed for locomotive behavior in the sexes. Whereas

females showed an increase in locomotion after both treatments using VE and

nortriptyline, males reduced locomotion only with nortriptyline use. This evidence of a

sexual dimorphic response shows the importance of using both males and females in

protocols involving antidepressants drugs. For instance, in humans, different responses

are observed between the sexes after the use of antidepressants (103).

An increase in somnolence and a reduction in feeding during the pharmacological

phase, but not the VE phase, were observed in marmosets. These alterations can be

considered as side effects of drugs that frequently induce sedation and loss of appetite in

humans (104). Nevertheless, these results corroborate the validation of predictive

criteria in our model, since nortriptyline reversed the hormonal and behavioral

expressions of depression-like changes exemplified by cortisol and scratching levels,

respectively.

In summary, both physiological and behavioral alterations found for male and

female juvenile common marmosets in response to chronic social isolation can be

characterized as a depressive-like condition, which was, in large part, reversed by

nortriptyline. Thus, the present study supports the validation of C. jacchus as one

potential translational model of juvenile depression. Additionally, the study

demonstrated that social isolation is an efficient paradigm that works as an inducer of

depression in this model. We have provided important evidence that this model and

protocol meet all validation criteria such as etiologic, face, functional, predictive, inter-

relational, evolutionary, and population, which allow its use in complementary areas of

investigations, including neurochemistry studies to disclose new biomarkers as well as

the development of new antidepressant drugs, such as those derived from natural

compounds, which could be more effective in alleviating depression symptoms in

humans.

Ethics statement

The animals were housed according to IBAMA (Brazilian Institute of Environment and

Renewable Natural Resources) guidelines (Normative Instruction no. 169 of February

20, 2008), and the care standards for animals established by CONCEA—National

Council for Animal Experimentation Control, Law No. 11.794 (October 8, 2008). In

addition, the laboratory complies with international standards for ex situ maintenance of

animals as defined by the Animal Behavior Society and the International Primatological

57

Society. The study and experimental procedures were approved by the Animal Research

Ethics Committee (UFRN protocol No. 019/2013 and protocol No. 034/2014).

Author contributions

NG-C and MS designed the experiments; FS and AG collected the experimental data,

carried out statistical analysis, and prepared figures. NG-C, MS, FS, and AG prepared

the manuscript.

Acknowledgments

We would like to thank Edinolia Câmera, Antonio B. da Silva, Geniberto C. dos Santos,

and Janaína Nitta for animal and veterinary care and Raíssa Nóbrega de Almeida for

hormonal measurements.

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66

ARTIGO 2

Acute antidepressant effect of ayahuasca in juvenile non-human primate model of

depression

Flávia Santos da Silva1, 2

, Erick Allan dos Santos Silva2, Geovan Menezes de Sousa

Junior2, João Paulo Maia-de-Oliveira

3,4, Vanessa de Paula Soares Rachetti

5, Draulio

Barros de Araujo6, Maria Bernardete Cordeiro de Sousa

1,2,6, Bruno Lobão Soares

1,4,5 &

Nicole Leite Galvão-Coelho1,2,4,7

1

Postgraduate Program in Psychobiology, Federal University of Rio Grande do Norte,

Natal, RN, Brazil; 2

Laboratory of Hormone Measurement, Department of Physiology,

Federal University of Rio Grande do Norte, Natal, RN, Brazil; 3 Department of Clinical

Medicine, Federal University of Rio Grande do Norte, Natal, RN, Brazil; 4

National

Institute of Science and Technology in Translational Medicine Natal, RN, Brazil; 5Department of Biophysics and Pharmacology, Federal University of Rio Grande do

Norte, Natal, RN, Brazil; 6Brain Institute, Federal University of Rio Grande do Norte,

Natal, RN, Brazil; 7Department of Physiology, Federal University of Rio Grande do

Norte, Natal, RN, Brazil.

Running title: Antidepressant effect of Ayahuasca in a non-human primate model of

depression.

Corresponding Author:

Nicole L. Galvão-Coelho

Universidade Federal do Rio Grande do Norte

Departamento de Fisiologia

Caixa Postal, 1511

59078-970 NATAL, RN, BRAZIL

Tel 55 84 3215-3410

Fax 55 84 3211-9206

[email protected]

67

ABSTRACT

The incidence of major depression in adolescents, aged between 15 to 18 years,

reaches approximately 14%. Usually, this disorder presents a recurrent way, without

remission of symptoms even after several pharmacological treatments, persisting

through adult life. Due to the relatively low efficacy of commercially available

antidepressant, new pharmacological therapies are under continuous exploration. Recent

evidence suggests that classic psychedelics, such as ayahuasca, produce rapid and

robust antidepressant effects in treatment-resistant depression patients. In this study, we

evaluated the potential of antidepressant effects of ayahuasca in a juvenile model of

depression in a non-human primate, common marmoset (Callithrix jacchus). The model

induces depressive-like symptoms by chronic social isolation (60 days) and

antidepressant effects monitoring included fecal cortisol, body weight, and behavioral

parameters. The animals presented hypocortisolemia and the recovery of cortisol to

baseline levels started already at 24h after the ingestion of ayahuasca, but not the

vehicle. Moreover, in males, ayahuasca, and not the vehicle, reduced the scratching, a

stereotypic behavior, and increased the feeding. Ayahuasca also improving body weight

to baseline levels in male and female common marmosets. The behavioral response

induced by ayahuasca shows long effect, lasting 14 days. Therefore, for this

translational animal model of juvenile depression, it could be proposed that ayahuasca

treatment presented more notable antidepressant effects than tricyclic antidepressant

nortriptyline, investigated by our group, using this same protocol in an anterior study.

Ayahuasca produced faster and more durable effect on reversion of physiological

changes and depressive-like symptoms. Therefore, the results found for ayahuasca

treatment corroborates in the validation of this substance as an effective antidepressant

drug and encourages the return of studies with psychedelic drugs in the treatment of

humor disorders, including adolescents with early-age depression.

Keywords: translational animal model, non-human primate, common marmoset,

behaviors, cortisol, early-age depression, psychedelic drugs.

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INTRODUCTION

Major depressive disorder (MDD) is characterized by depressed mood, anhedonia,

weight alterations (loss or weight gain), sleep disorders (insomnia or hypersomnia) and

psychomotor alterations (motor retardation or agitation) [Diagnostic and Statistical

Manual of Mental Disorders 5, 1, 2]. Depression is currently ranked by World Health

Organization (WHO) as the major contributor to global disability and suicidal deaths

[3].

MDD has been associated with physiological dysregulation and a consistent

finding is related to the monoaminergic unbalance, where decreased levels in

dopaminergic, noradrenergic and serotoninergic neurotransmission pathway have been

frequently observed [4]. However, antidepressants drugs that target these systems and

increase the levels of monoamines in synaptic cleft have failed to revert depressive

symptoms in totality [5] and only around 50% of patients achieve remission of

symptoms after several treatments [6]. Moreover, commercially available

antidepressants usually take around two weeks to achieve the desired therapeutic effects

[8, 9]. Therefore, enormous efforts have been devoted to the search for alternative

pharmacological treatments that improve efficacy, with a faster onset of therapeutic

effects [10].

Ayahuasca is a decoction made from a combination of two plants from Amazon

rainforest: Psychotria viridis and Banisteriopsis caapi [11]. Recent studies suggest that

ayahuasca does not exhibit tolerance and is not addictive [12, 13, 14, 15]. Different

ayahuasca compounds act on biological systems involved in the etiology of depression.

For instance, N,N-dimethyltryptamine (N,N-DMT) besides acting as a serotonergic (5-

HT2A) agonist [16, 17, 18] also modulates sigma-1 receptors (σ1R) that, in turn, has

recently been implicated in depression [19, 20, 21]. When consumed orally DMT is

inactivated by monoamine oxidase (MAO) present in the intestine and liver [22].

Ayahuasca also contains β-carbolines (Tetrahydroharmine, Harmine and Harmaline)

that act as reversible monoamine oxidase inhibitors (MAOi), allowing monoamines to

remain more time within the synaptic cleft space, and protecting DMT against

degradation when ingested [23].

Besides MAOi properties, THH is also a serotonin reuptake inhibitor [24] and

recent studies suggest significant antidepressant effects of Harmine, in a rodent model

of depression [25]. Furthermore, it has been observed that ayahuasca modulates plasma

cortisol, which is also involved in etiology of depression disorders, and patients with

69

major depression show consistent altered levels in plasma and saliva cortisol [26, 27,

28] whereas healthy volunteers show increased cortisol levels 2 hours after ayahuasca

intake [29, 23].

In fact, positive health benefits have been found in regular ayahuasca users in

religious contexts [30, 15, 31]. Furthermore, the antidepressant effects of ayahuasca

have been explored and a recent randomized controlled trial suggested a fast onset of

antidepressant effect in patients with treatment-resistant depression [32, 9, 33].

Adolescents, aged between 15 to 18 years, have shown incidence rates of

depression that reach to 14%, with approximately 40% of recurrence in the next 3 to 5

years following the first episode [34]. The influence of sexual steroids at this age opens

an important biological window of plasticity in the nervous system, which turns the

brain largely susceptible to environmental influences. If the stimulus induces

maladaptive changes in brain morphology and functions, these can lead to permanent

impairment of cognitive, behavioral and physiological mechanisms, increasing the

probability of the emergence of mood disorders that can persist into adulthood [35, 36,

37]. Therefore, the rise of sexual hormones at puberty turns adolescents into a higher

risk group of presenting depressive episodes. According to recent findings, a fast

antidepressant action of ayahuasca in adult patients with treatment-resistant depression

has been reported [32, 9, 33]. Thus, a question that arises from such findings is whether

ayahuasca might be effective to other age ranges also susceptible to depression such as

adolescents.

Therefore, this study evaluated the acute antidepressant effects of ayahuasca on

physiological (cortisol fecal), body weight and behavioral parameters in a juvenile

model of depression, common marmoset (Callithrix jacchus), after induction of a

depressive-like state by chronic social isolation.

METHOD

Animal maintenance

All animals were housed according to the guidelines of the Brazilian Institute of

Environment and Renewable Natural Resources (IBAMA) (Normative Instruction no.

169 of February 20, 2008), and care standards for animals established by the National

Council for Animal Experimentation Control (CONCEA), Law No. 11.794, (October 8,

70

2008). In addition, the laboratory complies with international standards for ex situ

maintenance of animals as defined by the Animal Behavior Society and the

International Primatological Society. The study and experimental procedures were

approved by the Animal Research Ethics Committee, (UFRN protocol No. 034/2014).

To allow genetic variability, all juvenile animals (7 to 9 months) used in this study

(n = 15; 8 males and 7 females) were randomly selected from 10 different families from

approximately 150 marmosets living in captive conditions in the Laboratory of

Advanced Studies in Primates, at the Federal University of Rio Grande do Norte

(UFRN), Natal, Brazil. The marmoset colony is formed by animals that were born in

captivity as well as those captured from nature and introduced in the colony.

At baseline conditions, animals were living with their families in masonry cages

(2.0 × 2.0 × 1.0 m3), located outdoors, under natural conditions of lighting, humidity,

and temperature. The cage has on the front a glass wall with a unidirectional visor and

on the back wall, a wire mesh door where water bottle and food plate are available.

Inside the cage there is a nest box for resting, planks of wood, and branches of plants for

environmental enrichment, allowing the animal´s displacement within the cage.

The model of depression was induced by social isolation [38]. During this

procedure, animals were moved from their family groups and placed alone in a new

smaller masonry outdoor cage (1.0 × 2.0 × 1.0 m3), but without space restrictions.

During this condition, animals did not have any visual contact with related conspecifics

but had auditory and olfactory contact with other conspecifics living at the colony.

None of the animals had been used in previous scientific study neither separated

from their respective family groups for prolonged periods. All animals were habituated

to the presence of the researchers prior to the study. Veterinary care was provided

throughout the experiment. Water was available without restriction during the entire

study and all animals were fed the same diet, twice per day, which included seasonal

fruits such as banana, papaya, melon, and mango, as well as potato and a protein potage

containing milk, oats, egg, and bread. Twice a week, a multivitamin supplement

(Glicocan) was diluted in the food. The animals were weighed every 15 days, in order to

monitor animal health.

Study design

A previous study has validated the induction of depression-like state for juvenile

common marmoset [38], and the same design was used herein.

71

During baseline phase (BL), juvenile marmosets (8 males and 7 females) were

observed for 4 weeks while living within their families, which were followed by an

eight weeks period of social isolated context (IC). Subsequently, 5 males and 4 females

were randomly selected to be treated, first with one administration of vehicle containing

a pure saline, vehicle treatment phase (VE), which lasts one week and comprised

behavioral and physiological sampling. In the following phase, pharmacological

treatments (PH), that also lasts one week, animals received one dose of the ayahuasca

and were sampled daily. PH was followed by one more week of sampling that

corresponded to tardive-pharmacological effects (tPE) phase. Figure 1 shows the

experimental design. Besides behaviors and fecal cortisol were monitoring, body weight

was also recorded during all five phases of the study. For more detailed description of

the protocols, see Galvão-Coelho et al. [38].

Differently of traditional antidepressants that are administered usually daily,

ayahuasca treatment consisted in a single dose, considering the observation that in

previous studies ayahuasca provoked acute increases in cortisol levels in plasma [32],

we decided to investigate behavioral and physiological parameters 24 and 48 hours after

administration, day 1 (D1) and day 2 (D2) respectively, for both treatments, vehicle

(VE) and ayahuasca (PH).

Figure 1 – Experimental design, comprising baseline (BL, 4 weeks), social isolated context (IC,

8 weeks), vehicle treatment (VE, 1 week), pharmacological treatments (PH, 1 week) and tardive-

pharmacological effects (tPE, 1 week) phases, where marmosets were sampled for behaviors,

fecal cortisol and body weight.

Treatments

All animals were treated with one administration of the vehicle, and seven days

after, the same animals received a single dose of ayahuasca. In both cases, we used a

72

dose of 1.67 ml/ 300g of animal weight, via gavage. The dose of ayahuasca established

for marmosets was transposed from the dose used in humans, using a recommended and

effective allometry procedure [39].

The ayahuasca used was produced and provided free of charge by the religious

organization called "Barquinha" (Brazil). A unique ayahuasca batch was used

throughout the whole study, for all animals. The preparation of the tea followed a

traditional recipe, by infusing 50% of leaves of Psicotria viridis with 50% of the stem

of Bansteriopsis caapi. The batch was stored in bottles in a refrigerator. The

quantification of alkaloids was determined by mass spectroscopy by the Laboratory of

Toxicological Analysis at the University of São Paulo. Results showed 0.36 ± 0.01mg

of DMT/mL, 1.86 ± 0.11mg of Harmine/mL, 0.24 ± 0.03mg of Harmaline/ml of and

0.20 ± 0.05mg of tetrahydroharmine (THH)/mL [40].

Behavioral recordings

The recorded behaviors were the same validated by Galvão-Coelho et al. [38] for

juvenile marmosets, and included: species-specific behaviors, as scent marking

(frequency), individual piloerection (frequency), scratching (duration), autogrooming

(duration), and behaviors associated with depressive-like state that can be compared

across species such as locomotion (frequency), somnolence (duration), feeding

(duration) and anhedonia. Anhedonia was measured by frequency and duration of

ingestion of an aqueous solution of sucrose (4.16%). For more detailed description of

behavioral data and its implications in stress context, see Galvão-Coelho et al. [38].

The selected behaviors were recorded between 6:30 and 7:30 a.m. to avoid the

influence of circadian variation, over a 30-min period by the focal continuous method

[41].

Fecal collection and cortisol assay

Fecal samples were collected between 6:30 and 8:30 a.m. to avoid circadian

variation in cortisol profile in feces [42]. Cages were cleaned prior to fecal collection, to

avoid collecting samples expelled prior to 6:30 a.m. Samples were stored at −4 °C until

cortisol extraction and quantification, which was measured at the Hormonal

Measurements Laboratory of the Department of Physiology (UFRN), according to the

protocol of Sousa and Ziegler [43]. Fecal cortisol reflects plasma cortisol with a delay

73

of approximately 8–10 hours. Intra- and inter-assay coefficients of variation were 2.74%

and 16.61%, respectively.

Statistical analysis

Hormonal data were normalized by logarithmic transformation and for both

hormonal and behavioral data, the statistical technique of bootstrap resampling was

applied to the multivariable analysis. General linear model (GLM) and Fisher's post hoc

tests were used to investigate the variations of behaviors, cortisol, and body weight,

between sex, throughout the study phases. Additionally, the parametric Student’s t-test

was used to analyze hormonal and behavioral data between specific phases. Statistically

significant results were considered for p < 0.05.

RESULTS

The analysis of studied variables showed at the end of IC, a significant decrease in

cortisol levels, increase in autogrooming, scratching and somnolence, as well as

reductions in feeding and sucrose ingestion. All these changes were independent of sex.

(Table 1).

For scent marking and body weight, sexual dimorphic profiles of variation were

observed, where only males showed increased scent marking and reduced body weight

(Table 1). No significant statistical variations were observed in locomotion and

individual piloerection with social isolation (Table 1).

After ayahuasca treatment (PH), but not after vehicle (VE), males decreased

scratching with respect to the IC. Such reduction in the PH lasted more 7 days, being

also observed in the tPE (Figure 2A and Table 2). Again, only males showed an

increase in feeding after treatment with ayahuasca, which was sustained until tPE, and

did not vary after treatment with vehicle (Figure 2B and Table 2). Both sexes increased

body weight after ayahuasca, but not after vehicle, and was sustained throughout tPE

(Figure 2C and Table 2). Body weight gain, induced by ayahuasca, allowed the recovery

to baseline weight levels (PT: t= -1.68, p=0.09 / tPE: t = -1.33, p = 0.18).

No significant alterations in response to both treatments, vehicle and ayahuasca,

were observed in fecal cortisol (Figure 3A and Table 2), as well as in autogrooming,

scent marking, locomotion, ingestion of sucrose and somnolence (Table 2). With

respect to individual piloerection, it was not possible to perform GLM analyzes due to

the large number of zero frequencies and low variation of data.

74

Figure 2 - Means + SEM of behaviors: (A) scratching, (B) feeding, (C) body weight in

male and female juveniles C. jacchus along IC= isolated context, VE = Vehicle

treatment, PH = Pharmacological treatment and tPE = tardive-Pharmacological Effects.

* = statistically significant difference between respective phase and phase indicated (s)

next to the symbol. GLM test and post hoc Fisher, p < 0.05.

75

Table 1 - Statistical values, GLM test and LSD post-hoc, and direction of alterations of

physiologic and behavior parameters in response to social isolated context, compared to

baseline.

VARIABLES STATISTICAL VALUES ALTERATION

Cortisol F= 4.38 p= 0.03 df= 1

P

F= 0.00 p=0.98 df = 1 P/S

↓

---

Autogrooming F= 15.31 p= 0.00 df= 1

P

F= 0.42 p= 0.73 df= 1 P/S

↑

---

Somnolence F= 3.73 p= 0.05 df= 1

P

F= 1.47 p= 0.22 df= 1 P/S

↑

---

Feeding F= 18.10 p= 0.00 df= 1

P

F= 0.90 p= 0.34 df= 1 P/S

↓

---

Ingestion of sucrose (frequency)

F= 22.61 p= 0.00 df= 1 P

F= 5.69 p= 1.16 df= 1 P/S

↓

---

(duration) F= 5.55 p= 0.02 df= 1

P

F= 0.12 p= 0.72 df= 1 P/S

↓

---

Scratching

F=4.72 p= 0.03 df= 1P

F=30.14 p= 0.00 df= 1 P/S

Male p= 0.00 ↑

Female p= 0.02 ↑

Scent-marking

F= 5.49 p= 0.02 df= 1 P

F= 9.68 p= 0.00 df= 1 P/S

Male p= 0.00 ↑

Female p= 0.15 ---

Body weight

F= 9.12 p= 0.00 df= 1 P

F= 11.27 p= 0.00 df= 1 P/S

Male p= 0.00 ↓

Female p= 0.81 ---

Locomotions

F= 0.03 p= 0.85 df= 1 P

F= 0.32 p= 0.57 df= 1 P/S

---

---

Individual piloerection

F= 0.03 p= 0.08 df= 1 P

F= 0.07 p= 0.78 df= 1 P/S

---

---

Acronyms: P = statistical analyze for phase,

P/S = statistical analyze for interaction

between phase and sex.

76


physiologic and behavior parameters in response to pharmacological treatments,

compared with isolated context.

VARIABLES STATISTICAL VALUES VE PT tPE

Cortisol

F= 1.18 p=0.31 df = 3 P

F= 0.42 p=0. 37 df = 3 P/S

--- --- ---

Autogrooming

F= 0.93 p=0.42 df = 3 P

F= 1.45 p=0. 22 df = 3 P/S

--- --- ---

Scratching F= 1.31 p=0.27 df = 3

P

F= 3.59 p=0.01 df = 3 P/S

Male p= 0.27 p= 0.00 ↓ p= 0.00 ↓

Female p= 0.19 p= 0.53 p= 0.16

Somnolence F= 0.57 p=0.63 df = 3

P

F= 0.77 p=0.51 df = 3 P/S

--- --- ---

Feeding F=2.23 p=0.08 df = 3

P

F = 3.55, p = 0.01, df = 3 P/S

Male p=0.81 p=0.02 ↑ p=0.00 ↑

Female p=0.29 p=0.69 p=0.57

Ingestion of sucrose (frequency)

F= 13.28 p=0.80 df = 3 P

F= 0.78 p=0.50 df = 3 P/S

--- --- ---

Scent-marking F= 3.14 p=0.06 df = 3

P

F= 5.30 p=0. 18 df = 3 P/S

--- --- ---

Body weight F = 15.35, p= 0.00, df = 3

P

F= 2.00 p=0.11 df = 3 P/S

p=0.02 ↓ p=0.00↑ p=0.00↑

Locomotion F= 1.01 p=0.38 df = 3

P

F= 1.80 p=0.14 df = 3 P/S

--- --- ---

Acronyms: P = statistical analyze for phase,

P/S = statistical analyze for interaction

between phase and sex.

Cortisol levels increased 24 hours (D1) and 48 hours (D2) after ayahuasca

ingestion, but not after vehicle, (Figure 3B and Table 3) Moreover, cortisol levels

observed at D1 and D2 returned to similar values found in BL (D1: t = -0.55, p=0.58 /

D2: t= -1.30, p= 0.21). No significant alterations were found in all behaviors at D1 and

77

D2 in response to vehicle or ayahuasca (Table 3). The GLM analysis of individual

piloerection and sucrose ingestion carried out due a large number zero’s and the low

variability of the data.

Figure 3 – Means + SEM of fecal cortisol: A) along IC= isolated context, VE = Vehicle

treatment, PH = Pharmacological treatment and tPE = tardive-Pharmacological Effects,

and B) At D1 (24h) and D2 (48h) after treatment with Vehicle (VE) and ayahuasca =

PH. * = statistically significant difference between respective phase and phase (s)

indicated (s) next to the symbol. GLM test and post hoc Fisher, p< 0.05.


acute cortisol in response to pharmacological treatments, in D1 (24h) and D2 (48h) after

treatment with Vehicle (VE) and ayahuasca (PH).

TREATMENT STATISTICAL VALUES ALTERATION

Vehicle

F= 3.19, p= 0.05, df = 2 D/T

F = 0.54, p = 0.58, df = 2 D/T/S

D1 (24h)

p = 0.21

---

D2 (48h) p = 0.56 ---

Ayahuasca

F= 3.19, p= 0.05, df = 2 D/T

F = 0.54, p = 0.58, df = 2 D/T/S

D1 (24h)

p = 0.03

↑

D2 (48h) p = 0.02 ↑

Acronyms: D/T

= statistical analyze for interaction between day and treatment; D/T/S

=

statistical analyze for interaction among day, treatment and sex

78

DISCUSSION

This study found a rapid antidepressant effect of ayahuasca on behavioral

expression in males and females juvenile common marmosets presenting depressive-

like state induced by chronic social isolation. In addition, both fecal cortisol and body

weight returned to baseline levels, when the animals were living in their family groups.

Moreover, some behavioral alterations indicative of depression-like state was reduced

mainly in males.

After being moved from the family group and remain socially isolated during

eight weeks (IC), marmosets increase auto-direct stereotypic behaviors as scratching

and autogrooming. In non-human primates such behaviors have been expressed during

psychosocial stress [44, 45]. Feeding reduction, increases in somnolence and anhedonia,

inferred here by reduction sucrose ingestion, for both sexes were also observed after

social isolation. Males also showed body weight loss and increase in scent marking,

which in context of stress is considered as an anxious behavior, because it occurs

without a defined interest, such as those involved in territorial defense and reproductive

signalization [46]. The presence of anhedonia, somnolence, reduction in feeding and

body weight changes are consistent with symptoms observed in patients with depression

and are used as guidelines to perform the diagnostic of this pathology as described in

diagnostic and Statistical Manual of Mental Disorders (DSM- 5).

After treatment with the vehicle, no changes were recorded for any behaviors or

body weight. However, a single dose of ayahuasca improved some of these symptoms

of the depressive-like state, mainly in males. In this case, a significant reduction of

scratching and increased feeding indicates a positive effect on the recovery of such as

functions. Although in females no changes in feeding have been observed, body weight

regulation to baseline levels occurs for both sexes. In a previous study where the

antidepressant nortriptyline was used, in similar protocol and animal model, only

females showed reductions in scratching after treatment [34]. The different action of

ayahuasca and nortriptyline to improve depressive symptoms in male and female

common marmosets points out to the importance of studying both sexes in translational

studies of depression. Furthermore, despite in humans sex difference in response to

antidepressants has been recorded, males of animal models continue to be more

frequently used in experimental protocols for depression studies [47, 48].

79

The positive ayahuasca modulation observed in feeding behavior is potentially

important for patients with depression with loss of appetite and body weight. The

anorectic effect of tricyclic antidepressants observed by Galvão-Coelho et al. [34] after

treatment with nortriptyline in common marmosets is also perceived in patients with

depression and it is considered a side-effect that induces a minor tolerance.

Available studies with ayahuasca and animal models of depression until the

present moment did not use non-human primates as animal models and also did not use

juvenile animals, this is the first one. Normally, the studies use adult rodents as animal

models of depression [25, 49], species phylogenetically more distant of humans than

common marmosets. Despite these studies also observed positive antidepressant effects

with the use of ayahuasca, or it´s specific components, differently of the present study,

they used 14 days of treatment and did not observe the continued response after

treatment stopped. For instance, rats treated with Harmine (5, 10 and 15 mg/kg) for 14

days showed improvements in forced swimming and open-field tests [25]. Wistar

female rats treated with ayahuasca for 14 days also presented better performance in

forced swimming test, when compared to a group treated with fluoxetine [49]. On the

other side, this study showing improvement in body weight and depressed-like

behaviors that remained until 14 days after one single-dose ayahuasca treatment.

Besides of the well-known pharmacological action of ayahuasca, such as

antagonist of MAO and the transporter of serotonin and agonist of the 5-HT-2 receptor,

others pharmacological targets can be involved in this rapid antidepressant effect of

ayahuasca observed in juvenile’s marmosets. Recently, some antidepressant effects in

rodents, as a reduction of anhedonia, were associated with Sigma-1 receptors activation

[20, 21]. Moreover, some indirect pathways modulate by DMT agonist action on 5-

HT2a and sigma-1 receptors can stimulate molecular and cellular events involved in

neural and synaptic plasticity, such as encoding of transcription factors (c-fos, egr1, egr-

2), synthesis of brain-derived neurotrophic factor (BDNF), enhances of CaMKII

/CaMKIV and protein kinase B (Akt) activities in hippocampus, all compatible with

antidepressant action [50, 51, 20].

Ayahuasca treatment did not induced alterations in autogrooming, scent marking,

somnolence and ingestion of sucrose solution similar to that results observed after

treatment with nortriptyline [34]. The absence of drug modulation in these behaviors

might be related with the dose and duration of ayahuasca treatment, which might be not

enough to promote a stronger antidepressant effect, suggesting that alternatives

80

protocols should be tested in order to verify a more robust behavioral improvement in

juveniles’ marmosets.

Regarding cortisol variation across the study’ phases, after chronic social isolation

of eight weeks, common marmosets showed significantly low levels of fecal cortisol.

Low levels of cortisol have been reported after strong stressors both in humans and in

small animals [52, 53, 54]. For instance, in juvenile common marmosets exposed to the

repeated separation of their families in infancy [55] or chronic social isolation along the

juvenile stage [56, 34]. A recent study with common marmosets found that 21 days of

social isolation in juvenile stage is enough to reduced cortisol to levels below baseline

[56]. Hypocortisolemia also was correlated to depression-like state in adult females of

Macaca fascicularis [54]. Moreover, previous studies have reported hypocortisolemia in

patients with atypical unipolar major depression and major depression with remittent

conditions [57, 58, 33].

During prolonged stress response, the complex systems of the interaction of

negative feedbacks of hypothalamus-pituitary-adrenal (HPA) axis could turn

imbalanced and changes adrenal function, which in turns reduces cortisol synthesis [59].

A sustention of cortisol at low levels deregulates all system of adaptation since cortisol

is a pleiotropic hormone that regulates hormonal, neural and immune system responses

to challenging situations [60, 61]. Individuals that show a chronic decrease in cortisol

levels normally present weakness, weight loss and immunological dysfunction [62].

Low levels of cortisol observed after isolation started rising already at 24 and 48

after treatment with ayahuasca, recovering cortisol to baseline levels. The observed

homeostatic regulation was fast and did not extend into the later phases (PT and tPE). A

previous study with the same animal model of juvenile depression, but treated with

nortriptyline chloride (PamelorTM

), revealed that nortriptyline increases cortisol

occurred after one week, and the raise overpassed baseline levels. These suggest that

ayahuasca induced faster and more adjusted regulation in cortisol levels than

nortriptyline. Furthermore, previous studies with ayahuasca have been suggesting

antidepressant effects of ayahuasca in a treatment resistant depression already one day

after a single dosing session with ayahuasca [9, 32]. It is interesting, however, to note

that the rapid antidepressant effect observed in both cases of use of ayahuasca, classic

antidepressants usually take at least two weeks to reach the desired therapeutic response

[63, 64].

81

The effects of antidepressants on the HPA axis depend on the class of the drug

(MAOi, tricycle, Selective Serotonin Reuptake Inhibitors, or others) [65, 66].

Serotoninergic agonists drugs, such as ayahuasca and nortriptyline, might modulate

both secretion of corticotropin-releasing hormone (CRH) and/or adrenocorticotropic

hormone (ACTH), at the hypothalamic and pituitary gland, respectively [67, 68].

Moreover, the duration of treatment also is an important issue in HPA axis modulation.

Normally, acute treatment increases cortisol levels and long-term ones, in the opposite,

induces a reduction. Probably, reduced cortisol secretion by adrenals is due to an up-

regulation of glucocorticoids receptors (GR and MR) in the brain, which in turn can

increase negative feedback [69, 70].

The differences in the modulation of cortisol levels by ayahuasca and nortriptyline

might be in part due to its distinct chemical proprieties and duration of treatment.

Ayahuasca was administrated only once to animals, whereas in our previous study

nortriptyline was injected during seven consecutive days [34]. Some anterior studies

have proposed that a single dose of ayahuasca in humans is enough to improve clinical

symptoms for seven days [9, 32] and to regulate salivary and plasmatic cortisol to

homeostatic levels [33].

In summary, behavioral symptoms, body weight and cortisol profiles of

depression found in male and female juvenile common marmosets exposed to chronic

social isolation (8 weeks) were large part reverted by ayahuasca treatment, more

prominently in males. Nevertheless, when the effectiveness of ayahuasca was compared

with nortriptyline, ayahuasca, apparently, showed more remarkable antidepressant

results than nortriptyline, since the start of improvements of physiological alterations

and behavioral symptoms of depression were faster, long lasting and adjusted.

Therefore, this study carries significant additional evidences that support the

antidepressant action of ayahuasca using a depression animal model phylogenetically

more closely of humans than rodents, a non-human primate. Moreover, for the first

time, the therapeutic value of ayahuasca as an effective antidepressant drug in juvenile

age was demonstrated. All anterior studies with ayahuasca were developed with adult

animal models or human, this is the first study with juveniles. As puberty is an

important ontogenetic period of brain plasticity, a treatment presenting fast

antidepressant effects, emerges for the first time, to our knowledge, as a good

alternative. Further studies may evaluate the safety and tolerability of the use of

ayahuasca as an antidepressant treatment at a young age.

https://en.wikipedia.org/wiki/Corticotropin-releasing_hormone

82

Conflict of Interest Statement

This research was conducted in the absence of any commercial or financial relationships

that could be construed as a potential conflict of interest.

Author and Contributors

Designed the experiment: Galvão-Coelho N. L., Maia-de-Oliveira J.P, De Araujo D.B.,

Soares, B.L, Rachetti V.P.S. and Sousa M.B.C.;

Collected experimental data: Da Silva F. S.; Silva, E. A. S.; Sousa Junior, G. M.;

Carried out statistical analysis: Da Silva F. S.;

Arranged figures: Da Silva F. S., Silva E. A. S and Soares, B.L.;

Prepared manuscript: Da Silva F.S., Galvão-Coelho N.L., Sousa M.B.C., Soares B.L,

De Araujo D.B., Rachetti V.P.S. and Maia-de-Oliveira J.P.

Acknowledgments

We would like to thank Edinolia Câmera, Antonio B. da Silva, Geniberto C. dos Santos

and Janaína Nitta for animal and veterinary care and Raíssa Nóbrega de Almeida for

hormonal measurements.

Abbreviations:

BL, Baseline; IC, Isolated context; VE, Vehicle Treatment; PH, Pharmacological

treatment; tPE, tardive-Pharmacological Effects. D1, Day 1; D2, Day 2.

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89

CONCLUSÃO GERAL

A abundante e crescente incidência do Transtorno de Depressão Maior (TDM)

entre a população jovem está associada a um conjunto de fatores que inclui desde os

genéticos até os psicossociais, como problemas acadêmicos, financeiros, familiares,

abuso físico ou sexual. Essa fase é um período ontogenético caracterizado por mudanças

biológicas muito significativas para o desenvolvimento do indivíduo, tendo em vista

que os hormônios sexuais promovem efeitos organizacionais, modulando a plasticidade

e o funcionamento dos circuitos cerebrais envolvidos com as respostas sexuais, mas

também com as respostas de recompensa, de estresse, dentre outras. Constata-se, por

tanto, que as experiências negativas vivenciadas durante essa fase claramente podem

induzir alterações mal adaptativas, podendo promover prejuízos permanentes,

persistindo inclusive na fase adulta.

A farmacoterapia é a medida mais empregada no tratamento dessa patologia, no

entanto, ainda existem muitas limitações a serem vencidas, limitações essas que

compreendem desde atraso para iniciar os efeitos desejados até uma série de efeitos

colaterais.

Na busca por uma melhor compreensão do TDM e por tratamentos

farmacológicos alternativos, mais eficazes que os atuais e que possam aliviar de

maneira mais satisfatória os sintomas dessa patologia, este estudo inicialmente validou

o Callithrix jacchus, como modelo de primata translacional de depressão juvenil e

apontou relevantes ações antidepressivas do chá da ayahuasca. Apesar de algumas

limitações, como: a duração do tratamento da nortriptilina, o modelo aqui apresentado

atendeu a todos os critérios de validação, sendo o primeiro modelo translacional de

depressão juvenil, com primata não- humano, que possui validade etiológica, de face,

funcional, preditiva, inter-relacional, evolutiva e populacional, inclusive utilizando

machos e fêmeas, o que não é rotineiro, possibilitando assim sua utilização em áreas

complementares de investigações.

Este estudo também apontou evidências adicionais à literatura atual, que

suportam o uso do chá de ayahuasca como medicamento antidepressivo, apresentando

ainda uma ação mais eficaz quando comparado com um antidepressivo clássico

(nortriptilina) de uso comum em pacientes com depressão. Os resultados

90

comportamentais e fisiológicos (cortisol fecal) obtidos com o grupo tratado pela

ayahuasca permitiu inferir que um fármaco produzido a partir deste chá apresentará

ações mais rápidas, sendo assim mais interessante do ponto de vista clínico, uma vez

que a velocidade para iniciar melhora dos sintomas é um fator limitante do tratamento

atual com antidepressivos. Ademais, os usuários do chá em contextos religiosos não

demosntram tolerância à dose, nem efeitos colaterais crônios. Todas essas

características fazem da ayahuasca uma potencial droga antidepressiva, inclusive para

utilização em adolescentes. Sendo assim, estes resultados prósperos estimulam maiores

investigações sobre a ação antidepressiva da ayahuasca, podendo inclusive observar-se

o efeito desta droga sobre a plasticidade neuronal no modelo animal aqui validado.

91

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