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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
2
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.
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
3
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, 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
5
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
8
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
9
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.
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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.
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
3.2.1 Hormonal profile
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.
3.2.2 Behavioral profile
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
3.3.1 Hormonal profile
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).
3.3.2 Behavioral profile
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.
Reference
1. WHO. Depression and Other Common Mental Disorders: Global Health Estimates.
(2017). Available from: http://www.who.int/mental_health/management/
depression/prevalence_global_health_estimates/en/
2. Ramaker MJ, Dulawa SC. Identifying fast-onset antidepressants using rodent
models. Mol Psychiatry (2017) 22:10. doi:10.1038/mp.2017.36
3. Drysdale AT, Grosenick L, Downar J, Dunlop K, Mansouri F, Meng Y, et al.
Resting-state connectivity biomarkers define neurophysiological subtypes of
depression. Nat Med (2017) 23:11. doi:10.1038/nm.4246
4. Belzung C. Innovative drugs to treat depression: did animal models fail to be
predictive or did clinical trials fail to detect effects? Neuropsychopharmacology.
(2014) 39:11. doi:10.1038/npp.2013.342
5. Pence BW, O’Donnell JK, Gaynes BN. The depression treatment cascade in primary
care: a public health perspective. Curr Psychiatry Rep (2012) 14:8.
doi:10.1007/s11920-012-0274-y
6. Warden D, Rush AJ, Trivedi MH, Fava M, Wisniewski SR. The STAR*D project
results: a comprehensive review of findings. Curr Psychiatry Rep (2007) 9:11.
doi:10.1007/s11920-007-0061-3
7. Willner P, Belzung C. Treatment-resistant depression: are animal models of
depression fit for purpose? Psychopharmacology (2015) 232:23.
doi:10.1007/s00213-015-4034-7
8. Willner P, Muscat R, Papp M. Chronic mild stress-induced anhedonia: a realistic
animal model of depression. Neurosci Biobehav Rev (1992) 16:10.
doi:10.1016/S0149-7634(05)80194-0
58
9. Dedic N, Walser SM, Deussing JM. Mouse models of depression. In: Uehara T,
editor. Psychiatric Disorders–Trends and Developments. (Vol. 8), Croatia:
Intechopen (2011). 37 p.
10. Krishnan V, Nestler EJ. Animal models of depression: molecular perspectives. Curr
Top Behav Neurosci (2011) 7:26. doi:10.1007/7854_2010_108
11. Li X, Xu F, Xie L, Ji Y, Cheng K, Zhou Q, et al. Depression-like behavioural
phenotypes by social and social plus visual isolation in the adult female Macaca
fascicularis. PLoS One (2013) 8:9. doi:10.1371/journal.pone.0073293
12. Rollin MD, Rollin BE. Crazy like a fox. Camb Q Healthc Ethics (2014) 23:11.
doi:10.1017/S0963180113000674
13. Anisman K, Matheson K. Stress, depression, and anhedonia: caveats concerning
animal models. Neurosci Biobehav Rev (2005) 29:21. doi:10.1016/j.
neubiorev.2005.03.007
14. Razafsha M, Behforuzi H, Harati H, Wafai RA, Khaku A, Mondello S, et al. An
updated overview of animal models in neuropsychiatry. Neuroscience (2013) 240:15.
doi:10.1016/j.neuroscience.2013.02.045
15. Hua-Cheng Y, CAO X, Das M, Xin-Hong Z, Tian-Ming G. Behavioral animal
models of depression. Neurosci Bull (2010) 26:10. doi:10.1007/s12264-010-0323-7
16. Boyko M, Kutz R, Grinshpun Y, Zvenigorodsky V, Gruenbaum SE, Zlotnik A.
Establishment of an animal model of depression contagion. Behav Brain Res (2014)
15:358–63. doi:10.1016/j.bbr.2014.12.017
17. Hammels C, Pishva E, De Vry J, van den Hove DLA, Prickaerts J, van Winkel R, et
al. Defeat stress in rodents: from behavior to molecules. Neurosci Biobehav Rev
(2015) 59:29. doi:10.1016/j.neubiorev.2015.10.006
18. Vetulani J. Early maternal separation: a rodent model of depression and a prevailing
human condition. Pharmacol Rep (2013) 65:10. doi:10.1016/S1734-1140(13)71505-
6
19. Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature (2008)
455:8. doi:10.1038/nature07455
20. Abelaira HM, Réus GZ, Quevedo J. Animal models as tools to study the
pathophysiology of depression. Rev Bras Psiquiatr (2013) 35:8. doi:10.1590/1516-
4446-2013-1098
21. Willner P, Mitchell PJ. The validity of animal models of predisposition to
depression. Behav Pharmacol (2002) 13:19. doi:10.1097/00008877-200205000-
00001
59
22. Biesmans S, Bouwknecht JA, Donck LV, Langlois X, Acton PD, De Haes P, et al.
Peripheral administration of tumor necrosis factor-alpha induces neuroinflammation
and sickness but not depressive-like behavior in mice. Biomed Res Int (2015)
2015:716920. doi:10.1155/2015/716920
23. Belzung C, Lemonie M. Criteria of validity for animal models of psychiatric
disorders: focus on anxiety disorders and depression. Biol Mood Anxiety Disord
(2011) 1:1–14. doi:10.1186/2045-5380-1-9
24. Balmus IM, Ciobica A, Antioch I, Dobrin R, Timofte D. Oxidative stress
implications in the affective disorders: main biomarkers, animal models relevance,
genetic perspectives, and antioxidant approache. Oxid Med Cell Longev (2016)
2016:1–26. doi:10.1155/2016/3975101
25. Stewart AM, Kalueff AV. Developing better and more valid animal models of brain
disorders. Behav Brain Res (2015) 276:4. doi:10.1016/j.bbr.2013.12.024
26. Stewart AM, Kalueff AV. Anxiolytic drug discovery: what are the novel approaches
and how can we improve them? Exp Opin Drug Discov (2014) 9:12.
doi:10.1517/17460441.2014.857309
27. Pryce CR, Feldon J, Fuchs E, Knuesel I, Oertle T, Sengstag C, et al. Postnatal
ontogeny of hippocampal expression of the mineralocorticoid and glucocorticoid
receptors in the common marmoset monkey. Eur J Neurosci (2005) 21:14.
doi:10.1111/j.1460-9568.2005.04003.x
28. Cacioppo JT, Hawkley LC, Norman GJ, Berntson GG. Social isolation. Ann N Y
Acad Sci (2011) 1231:6. doi:10.1111/j.1749-6632.2011.06028.x
29. Shively CA, Willard SL. Modeling depression in adult female cynomolgus monkeys
(Macaca fascicularis). Am J Primatol (2012) 74:14. doi:10.1002/ajp.21013
30. Cinini SM, Barnabe GF, Galvão-Coelho N, de Medeiros MA, Perez-Mendes P,
Souza MBC, et al. Social isolation disrupts hippocampal neurogenesis in young non-
human primates. Front Neurosci (2014) 8:9. doi:10.3389/fnins.2014.00045
31. Hankin BJ. Adolescent depression: description, causes and intervention. Epilepsy
Behav (2006) 8:13. doi:10.1016/j.yebeh.2005.10.012
32. Ganzel BL, Morris PA. Allostasis and the developing human brain: explicit
consideration of implicit models. Dev Psychopathol (2011) 23:19.
doi:10.1017/S0954579411000447
33. Thapar A, Collishaw S, Pine DS, Thapar AK. Depression in adolescence. Lancet
(2012) 379:11. doi:10.1016/S0140-6736(11)60871-434.
34. Qin D, Chu X, Feng X, Li Z, Yang S, Lü L. The first observation of sensonal
affective disorder symptoms in rhesus macaque. Behav Brain Res (2015) 292:6.
doi:10.1016/j.bbr.2015.07.005
60
35. Abbott DH, Hearn JP. Physical, hormonal and behavioral aspects of sexual
development in the marmoset monkey, Callithrix jacchus. J Reprod Fertil (1978)
53:11. doi:10.1530/jrf.0.0530155
36. Dixson AF, Lunn SF. Post-partum changes in hormones and sexual behaviour in
captive groups of marmosets (Callithrix jacchus). Physiol Behav (1987) 41:6.
doi:10.1016/0031-9384(87)90314-3
37. Miller CT, Freiwald WA, Leopold DA, Mitchell JF, Silva AC, Wang X. Marmosets:
a neuroscientific model of human social behavior. Neuron (2016) 90:219–33.
doi:10.1016/j.neuron.2016.03.018
38. Galvão-Coelho NL, Silva HPA, Leão AC, Sousa MBC. Common marmosets
(Callithrix jacchus) as a potential animal model for studying psychological disorders
associated with high and low responsiveness of hypothalamicpituitary-adrenal axis.
Rev Neurosci (2008) 19:14. doi:10.1515/REVNEURO. 2008.19.2-3.187
39. Galvão-Coelho NL, Silva HPA, Sousa MBC. The influence of sex and relatedness
on stress response in common marmosets (Callithrix jacchus). Am J Primatol (2012)
74:8. doi:10.1002/ajp.22032
40. de Sousa MB, Galvão AC, Sales CJ, de Castro DC, Galvão-Coelho NL. Endocrine
and cognitive adaptations to cope with stress in immature common marmosets
(Callithrix jacchus): sex and age matter. Front Psychiatry (2015) 6:11.
doi:10.3389/fpsyt.2015.00160
41. Bales K, Dietz J, Baker A, Miller K, Tardif SD. Effects of allocare-givers on fitness
of infants and parents in callitrichid primates. Folia Primatol (2000) 71:11.
doi:10.1159/000021728
42. Tardif S, Bales K, Williams L, Moeller E, Abbott D, Schultz-Darken N, et al.
Preparing New World monkeys for laboratory research. ILAR J (2006) 47:307–15.
doi:10.1093/ilar.47.4.307
43. Phillips KA, Bales KL, Capitanio JP, Conley A, Czoty PW, ’t Hart BA, et al. Why
primate models matter. Am J Primatol (2014) 76:26. doi:10.1002/ajp.22281
44. Stevenson MF, Poole TB. An ethogram of common marmoset (Callithrix jacchus)
general behavioral repertoire. Anim Behav (1976) 24:23. doi:10.1016/S0003-
3472(76)80053-X
45. Sousa MBC, Ziegler T. Diurnal variation on the excretion patterns of steroids in
common marmoset (Callithrix jacchus) females. Am J Primatol (1998) 46:105–17.
doi:10.1002/(SICI)1098-2345(1998)46:2<105:AID-AJP1>3.0.CO;2-#
46. Castro DC, Sousa MBC. Fecal androgen levels in common marmoset (Callithrix
jacchus) males living in captive family groups. Braz J Med Biol Res (2005) 38:7.
doi:10.1590/S0100-879X2005000100011
61
47. Leão AC, Neto ADD, Sousa MBC. New developmental stages for common
marmosets (Callithrix jacchus) using mass and age variables obtained by k-means
algorithm and self-organizing maps (som). Comput Biol Med (2009) 39:7.
doi:10.1016/j.compbiomed.2009.05.009
48. Lewis HM, Gluck JP, Petitto JM, Hensley LL, Ozer H. Early social deprivation in
nonhuman primates: long-term effects on survival and cell-mediated immunity. Biol
Psychiatry (2000) 47:7. doi:10.1016/S0006-3223(99)00238-3
49. Willard S, Shively CA. Modeling depression in adult female cynomolgus monkeys
(Macaca fascicularis). Am J Primatol (2012) 74:14. doi:10.1002/ajp.21013
50. Moura JC, Noroes MM, Rachetti VDPS, Soares BL, Preti D, Nassini R, et al. The
blockade of transient receptor potential ankirin 1 (TRPA1) signalling mediates
antidepressant-and anxiolytic-like actions in mice. Br J Pharmacol (2014) 171:10.
doi:10.1111/bph.12786
51. Altmann J. Observational study of behavior: sampling methods. Behaviour (1974)
49:40. doi:10.1163/156853974X00534
52. Erkert HG. Characteristics of the circadian activity rhythm in common marmosets
(Callithrix jacchus). Am J Primatol (1989) 17:15. doi:10.1002/ajp.1350170403
53. Barros M, Tomaz C. Non-human primate models for investigating fear and anxiety.
Neurosci Biobehav Rev (2002) 26:14. doi:10.1016/S0149-7634(01) 00064-1
54. Dettling AC, Feldon J, Pryce CR. Early deprivation and behavioral and
physiological responses to social separation/novelty in the marmoset. Pharmacol
Biochem Behav (2002) 73:10. doi:org/10.1016/S0091-3057(02)00785-2
55. DeVries A, Craft TKS, Glasper ER, Neigh GN, Alexander JK. Social influences on
stress responses and health. Psychoneuroendocrinology (2007) 32:16.
doi:10.1016/j.psyneuen.2007.04.007
56. Wittig RM, Crockford C, Lehmann J, Whitten PL, Seyfarth RM, Cheney DL.
Focused grooming networks and stress alleviation in wild female baboons. Horm
Behav (2008) 54:7. doi:10.1016/j.yhbeh.2008.02.009
57. Vasconcellos M, Rocha MCDO, Maciel VH. Revisão teórica sobre depressão pela
análise do comportamento e por alguns manuais psiquiátricos. ConScientiae Saúde
(2010) 9:6. doi:10.1146/annurev-clinpsy-050212-185606
58. Paul IA, English JA, Halaris A. Sucrose and quinine intake by maternallydeprived
and control rhesus monkeys. Behav Brain Res (2000) 112:7. doi:10.1016/S0166-
4328(00)00173-X
59. Raminelli JLF, Sousa MBC, Cunha MS, Barbosa MFV. Morning and afternoon
patterns of fecal cortisol excretion among reproductive and non-reproductive male
and female common marmosets, Callithrix jacchus. Biol Rhythm Res (2001) 32:8.
doi:10.1076/brhm.32.2.159.1357
62
60. Steptoe A, Shankar A, Demakakos P, Wardle J. Social isolation, loneliness, and all-
cause mortality in older men and women. Proc Natl Acad Sci U S A (2013) 110:3.
doi:10.1073/pnas.1219686110
61. Cacioppo JT, Cacioppo S, Capitanio JP, Cole SW. The neuroendocrinology of
social isolation. Annu Rev Psychol (2015) 66:34. doi:10.1146/annurevpsych-010814-
015240
62. Cohen S, Wills TA. Stress, social support, and the buffering hypothesis. Psychol
Bull (1985) 98:310. doi:10.1037/0033-2909.98.2.310
63. Taylor JH, Mustoe AC, Hochfelder B, French JA. Reunion behavior after social
separation is associated with enhanced HPA recovery in young marmoset monkeys.
Psychoneuroendocrinology (2015) 57:8. doi:10.1016/j. psyneuen.2015.03.019
64. Gerber P, Schnell CR, Anzenberger G. Behavioral and cardiophysiological
responses of common marmosets (Callithrix jacchus) to social and environmental
changes. Primates (2002) 43:15. doi:10.1007/BF02629648
65. Shively CA, Register TC, Friedman DP, Morgan TM, Thompson J, Lanier T. Social
stress-associated depression in adult female cynomolgus monkeys (Macaca
fascicularis). Biol Psychol (2005) 69:17. doi:10.1016/j.biopsycho. 2004.11.006
66. Cubicciotti DI, Mendoza SP, Mason WA, Sassenrath EN. Differences between
Saimiri sciureus and Callicebus moloch in physiological responsiveness:
implications for behavior. J Comp Psychol (1986) 100:6. doi:10.1037/0735-
7036.100.4.385
67. Dufour V, Sueur C, Whiten A, Buchanan-Smith HM. The impact of moving to a
novel environment on social networks, activity and wellbeing in two new world
primates. Am J Primatol (2011) 73:9. doi:10.1002/ajp.20943
68. Young RJ. Environmental Enrichment for Captive Animals. Oxford: Blackwell
Science (2003).
69. Yamamoto ME, Volpato GL. Percorrendo a história do estudo do comportamento
animal: origens e influências. Natal: EDUFRN (2006).
70. Detillion CE, Craft TKS, Glasper ER, Prendergast BJ, Devries AC. Social
facilitation of wound heating. Psychoneuroendocrinology (2004) 29:7.
doi:10.1016/j.psyneuen.2003.10.003
71. Pizzagalli DA. Depression, stress, and anhedonia: toward a synthesis and integrated
model. Annu Rev Clin Psychol (2014) 10:30. doi:10.1146/annurev-clinpsy-050212-
185606
72. Heshmati M, Russo SJ. Anhedonia and the brain reward circuitry in depression.
Curr Behav Neurosci Rep (2015) 2:7. doi:10.1007/s40473-015-0044-3
63
73. Matthews K, Christmas D, Swan J, Sorrell E. Animal models of depression:
navigating through the clinical fog. Neurosci Biobehav Rev (2005) 29:10.
doi:10.1016/j.neubiorev.2005.03.005
74. Dean J, Keshavan M. The neurobiology of depression: an integrated view. Asian J
Psychiatry (2017) 27:10. doi:10.1016/j.ajp.2017.01.025
75. Frazer A, Morilak DA. What should animal models of depression model? Neurosci
Biobehav Rev (2005) 29:8. doi:10.1016/j.neubiorev.2005.03.006
76. Koenig B, Lindholm AK, Lopes PC, Dobay A, Steinert S, Bruschmann FJU. A
system for automatic recording of social behavior in a free-living wild house mouse
population. Anim Biotelemetry (2015) 3:12. doi:10.1186/s40317-015-0069-0
77. Pryce CR, Rüedi-Bettschen D, Dettling AC, Feldon J. Early life stress: longterm
physiological impact in rodents and primates. Physiology (2002) 17:5.
doi:10.1152/nips.01367.2001
78. Wang Z, Insel TR. Parental behavior in voles. Adv Study Behav (1996) 25:23.
79. Young L, Wang Z. The neurobiology of pair bonding. Nat Neurosci (2004) 7:9.
doi:10.1038/nn1327
80. Beery AK, Lacey EA, Francis FD. Oxytocin and vasopressin receptor distributions
in a solitary and a social species of tuco-tuco (Ctenomys haigi and Ctenomys
sociabilis). J Comp Neurol (2008) 507:12. doi:10.1002/cne. 21638
81. Faulkes CG, Bennett NC, Cotterill FPD, Stanley W, Mgode GF, Verheyen E.
Phylogeography and cryptic diversity of the solitary-dwelling silvery mole-rat, genus
Heliophobius (family: Bathyergidae). J Zool (2011) 47:24. doi:10.1111/j.1469-
7998.2011.00863.x
82. Anacker AMJ, Beery AK. Life in groups: the roles of oxytocinin mammalian
sociality. Front Behav Neurosci (2013) 7:10. doi:10.3389/fnbeh.2013.00185
83. Faulkers CG, Bennett NC. Plasticity and constraints on social evolution in African
mole-rats: ultimate and proximate factors. The Royal Society. Philos Trans R Soc
Lond B Biol Sci (2013) 368:10. doi:10.1098/rstb.2012.0347
84. Levine S, Mody T. The long-term psychobiological consequences of intermittent
post natal separation in the squirrel monkey. Neurosci Biobehav Rev (2003) 27:6.
doi:10.1016/S0149-7634(03)00011-3
85. Ehlert U, Gaab J, Heinrichs M. Psychoneuroendocrinological contributions to the
etiology of depression, posttraumatic stress disorder, and stress-related bodily
disorders: the role of the hypothalamus-pituitary-adrenal axis. Biol Psychol (2001)
57:11. doi:10.1016/S0301-0511(01)00092-8
64
86. Oldehinkel AJ, Van den Berg MD, Flentge F, Bouhuys AL, Ter Horst GJ, Ormel J.
Urinary free cortisol excretion in elderly persons with minor and major depression.
Psychiatry Res (2001) 104:8. doi:10.1016/S0165-1781(01)00300-6
87. Bremmer MA, Deeg DJ, Beekman AT, Penninx BW, Lips P, Hoogendijk WJ.
Major depression in late life is associated with both hypo-and hypercortisolemia.
Biol Psychiatry (2007) 62:7. doi:10.1016/j.biopsych.2006.11.033
88. Penninx BWJH, Milaneschi Y, Lamers F, Vogelzangs N. Understanding the somatic
consequences of depression: biological mechanisms and role of depression symptom
profile. BMC Med (2013) 11:14. doi:10.1186/1741-7015-11-129
89. Kunugi H, Hori H, Numakawa T, Ota M. The hypothalamic-pituitaryadrenal axis
and depressive disorder: recent progress. Nihon Shinkei Seishin Yakurigaku Zasshi
(2012) 32:203–9.
90. Kunugi H, Hori H, Ogawa S. Biochemical markers subtyping major depressive
disorder. Psychiatry Clin Neurosci (2015) 69:11. doi:10.1111/pcn.12299
91. Siriram K, Rodriguez-Fernandez M, Doyle FJ III. Modeling cortisol dynamics in the
neuro-endocrine axis distinguishes normal, depression, and post-traumatic stress
disorder (PTSD) in humans. PLoS Comput Biol (2012) 8:15.
doi:10.1371/journal.pcbi.1002379
92. Yehuda R. Post-traumatic stress disorder. N Engl J Med (2002) 346:6.
doi:10.1056/NEJMra012941
93. Fries E, Hesse J, Hellhammer J, Hellhammer DH. A new view on hypocortisolism.
Psychoneuroendocrinology (2005) 30:6. doi:10.1016/j.psyneuen. 2005.04.006
94. McEwen BS, Bowles NP, Gra JD, Hill MN, Hunter RG, Karatsoreos IN, et al.
Mechanisms of stress in the brain. Nat Neurosci (2015) 18:10. doi:10.1038/nn.4086
95. Badanes LS, Watamura SE, Hankin BL. Hypocortisolism as a potential marker of
allostatic load in children: associations with family risk and internalizing disorders.
Dev Psychopathol (2011) 23:15. doi:10.1017/S095457941100037X
96. Schriber RA, Guyer AE. Adolescent neurobiological susceptibility to social context.
Dev Cogn Neurosci (2016) 19:1–18. doi:10.1016/j.dcn.2015.12.009
97. Walker EF, Walder DJ, Reynolds F. Developmental changes in cortisol secretion in
normal and at-risk youth. Dev Psychopathol (2001) 13:11.
doi:10.1017/S0954579401003169
98. Aalto-setälä T, Marttunen M, Tuulio-Henriksson A, Poikolainen K, Lönnqvist J.
Depressive symptoms in adolescence as predictors of adulthood depressive disorders
and maladjustment. Am J Psychiatry (2002) 159:3. doi:10.1176/appi.ajp.159.7.1235
99. Maxwell MA, Cole DA. Weight changes and appetite disturbance as symptoms of
adolescent depression: toward an integrative biopsychosocial model. Clin Psychol
Rev (2009) 29:260–73. doi:10.1016/j.cpr.2009.01.007
65
100. Adam TC, Epel ES. Stress, eating and reward system. Physiol Behav (2007) 91:41.
doi:10.1016/j.physbeh.2007.04.011
101. Maniam J, Morris MJ. The link between stress and feeding behaviour.
Neuropharmacology (2012) 63:13. doi:10.1016/j.neuropharm.2012.04.017
102. Chandolia RK, Luetjens CM, Wistuba J, Yeung CH, Nieschlag E, Simoni M.
Changes in endocrine profile and reproductive organs during puberty in the male
marmoset monkey (Callithrix jacchus). Reproduction (2006) 132: 355–63.
doi:10.1530/rep.1.01186
103. Kokras N, Dalla C. Preclinical sex differences in depression and antidepressant
response: implications for clinical research. J Neurosci Res (2017) 95:5.
doi:10.1002/jnr.23861
104. Khawam EA, Laurencic G, Malone DA. Side effects of antidepressants: an
overview. Cleve Clin J Med (2006) 73(4):351–3. doi:10.3949/ccjm.73.4.351
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
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
Table 2 - Statistical values, GLM test and LSD post-hoc, and direction of alterations of
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.
Table 3 - Statistical values, GLM test and LSD post-hoc, and direction of alterations of
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.
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.
REFERENCES
1. Berton O, Nestler EJ. (2006) New approaches to antidepressant drug discovery:
beyond monoamines. Nat Rev Neurosci. 7: 137-151. doi: 10.1038/nrn1846
2. Belmaker RH, Agam G. (2008) Major depressive disorder. N Engl J Med. 358:13.
doi: 10.1056/NEJMra073096.
3. WHO. (2017) Depression and Other Common Mental Disorders: Global Health
Estimates. Available from: http://www.who.int/mental_health/management/
depression/prevalence_global_health_estimates/en/
4. Wong M, Licinio J. (2001) Research and treatment approaches to depression.
Nature Reviews Neuroscience 2: 343-351. doi: 10.1038/35072566
5. Vismari L, Alves JG, Palermo-Neto J. (2008) Depressão, antidepressivos e sistema
imune: um novo olhar sobre um velho problema. Ver. Psiq. Clín. 35: 8. doi:
10.1590/s0101-60832008000500004
6. Warden D, Rush AJ, Trivedi MH, Fava M, Wisniewski SR. (2007) The STAR*D
project results: a comprehensive review of findings. Curr Psychiatry Rep 9:11.
doi:10.1007/s11920-007-0061-3
83
7. Zarate C. (2011) A randomized trial of an N-methyl-D-aspartate antagonist and
neural correlates of rapid antidepressant response in treatment-resistant bipolar
depression. Biological Psychiatry 69: 9. 360 PARK AVE SOUTH, NEW YORK,
NY 10010-1710 USA: ELSEVIER SCIENCE INC.
8. Zarate CA, Singh JB, Carlson PJ, Brutsche NE, Ameli R, Luckenbaugh DA, … &
Manji, HK. (2006) A randomized trial of an N-methyl-D-aspartate antagonist in
treatment-resistant major depression. Archives of General Psychiatry 63: 856-864.
doi:10.1001/archpsyc.63.8.856
9. Palhano-Fontes F, Barreto D, Onias H, Andrade KC, Novaes M, Pessoa J, ... &, de
Araujo DB. (2017) Rapid antidepressant effects of the psychedelic ayahuasca in
treatment-resistant depression: a randomised placebo-controlled trial. BioRxiv. 9
doi: https://doi.org/10.1101/103531
10. Hatzinger M, Brand S, Perren S, Stadelmann S, von Wyl A, von Klitzing K,
Holsboer-Trachsler E. (2010) Sleep actigraphy pattern and behavioral/emotional
difficulties in kindergarten children: association with hypothalamic-pituitary-
adrenocortical (HPA) activity. Journal of psychiatric research 44: 8.
https://doi.org/10.1016/j.jpsychires.2009.08.012
11. Malcolm BJ, Lee KC. (2017) Ayahuasca: An ancient sacrament for treatment of
contemporary psychiatric illness? Mental Health Clinician. 7: 6.
10.9740/mhc.2017.01.039
12. Halpern BS, Walbridge S, Selkoe KA, Kappel CV, Micheli F, D'agrosa C, ... &
Fujita R. (2008) A global map of human impact on marine
ecosystems. Science. 319: 948-952. doi: 10.1126/science.1149345
13. Fabregas JM, Gonzalez D, Fondevila S, Cutchet M, Fernandez X, Barbosa PCR, ...
& Bouso JC. (2010) Assessment of addiction severity among ritual users of
ayahuasca. Drug and Alcohol Dependence In Press. 111,257-261. doi:
10.1016/j.drugalcdep.2010.03.024
14. Barbosa PCR, Cazorla IM, Giglio JS, Strassman R. (2009) A six-month prospective
evaluation of personality traits, psychiatric symptoms and quality of life in
ayahuasca-naïve subjects. J. Psychoactive Drugs. 41: 777-780.
doi.org/10.1080/02791072.2009.10400530
15. Barbosa PCR, Mizumoto S, Bogenschutz MP, Strassman RJ. (2012) Health status
of ayahuasca users. Drug Test Anal. 4:9. doi: 10.1002/dta.1383.
16. Baumeister D, Barnes G, Giaroli G, Tracy D. (2014). Classical hallucinogens as
antidepressants? A review of pharmacodynamics and putative clinical roles. Ther.
Adv. Psychopharmacol. 4: 156-169. doi: 10.1177/2045125314527985
17. Carbonaro TM, Gatch MB. (2016). Neuropharmacology of N, N-
dimethyltryptamine. Brain Res Bull. 126: 74-88. doi:
10.1016/j.brainresbull.2016.04.016
84
18. Frecska E, Bokor P, Winkelman M. (2016). The therapeutic potentials of
ayahuasca: possible effects against various diseases of civilization. Front.
Pharmacol. 7. doi: 10.3389/fphar.2016.00035
19. Kulkarni SK, Dhir A. (2009) Current investigational drugs for major
depression. Expert opinion on investigational drugs, 18: 767-788. doi:
10.1517/13543780902880850.
20. Fontanilla D, Johannessen M, Hajipour AR, Cozzi NV, Jackson MB, Ruoho AE.
(2009). The hallucinogen N, N-dimethyltryptamine (DMT) is an endogenous
sigma-1 receptor regulator. Science. 323. doi: 10.1126/science.1166127
21. Fukunaga K, Moriguchi S. (2017) Stimulation of the Sigma-1 Receptor and the
Effects on Neurogenesis and Depressive Behaviors in Mice. In Sigma Receptors:
Their Role in Disease and as Therapeutic Targets. 201-211. Springer International
Publishing. doi: 10.1007/978-3-319-50174-1_14.
22. Santos RG. (2007). Ayahuasca: neurochemistry and pharmacology. SMAD 3, 1-11.
23. Callaway JC, McKenna DJ; Grob CS, Brito GS, Raymon LP, Poland RE, Andrade
EN, Andrade, EO, Mash DC. (1999). Pharmacokinetics of Hoasca alkaloids in
healthy humans. Journal of Ethnopharmacology 65: 243-256.
doi.org/10.1016/S0378-8741(98)00168-8
24. Buckholtz NS, Boggan WO. (1977). Inhibition by β-carbolines of monoamine
uptake into a synaptosomal preparation: structure-activity relationships. Life
sciences, 20: 2093-2100. doi: 10.1016/0024-3205(77) 90190-4
25. Fortunato JJ, Réus GZ, Kirsch TR, Stringari RB, Fries GR, Kapczinski F, ... &
Quevedo J. (2010). Chronic administration of harmine elicits antidepressant-like
effects and increases BDNF levels in rat hippocampus. Journal of neural
transmission, 117(10), 1131-1137. doi: 10.1007/s00702-010-0451-2.
26. Tadic A, Wagner S, Gorbulev S, Dahmen N, Hiemke C, Braus DF, Lieb K. (2011).
Peripheral blood and neuropsychological markers for the onset of action of
antidepressant drugs in patients with Major Depressive Disorder. BMC Psychiatry
11: 16. doi: 10.1186/1471-244X-11-16.
27. Dziurkowska E, Wesolowski M, Dziurkowski M. (2013). Salivary cortisol in
women with major depressive disorder under selective serotonin reuptake inhibitors
therapy. Archives of women's mental health, 16: 139-147. doi: 10.1007/s00737-
013-0329-z.
28. Burgese DF, Bassitt DP. (2015). Variation of plasma cortisol levels in patients with
depression after treatment with bilateral electroconvulsive therapy. Trends in
psychiatry and psychotherapy, 37: 27-36. doi.org/10.1590/2237-6089-2014-0031
29. Santos RG, Valle M, Bouso JC, Nomdedéu JF, Rodrıguez-Espinosa J, McIlhenny
EH, … & Riba, J. (2011). Autonomic, Neuroendocrine, and DosImmunological
Effects of Ayahuasca. J Clin Psychopharmacol. 31:717-26 doi:
10.1097/JCP.0b013e31823607f6
85
30. Santos RG, Landeira-Fernandez J, Strassman RJ, Motta V, Cruz APM. (2007)
Effects of ayahuasca on psychometric measures of anxiety, panic-like and
hopelessness in Santo Daime members. Journal of Ethnopharmacology 112:507-
513. doi.org/10.1016/j.jep.2007.04.012
31. Bouso JC, González D, Fondevila S, Cutchet M, Fernández X, Barbosa P, … &
Riba J. (2012). Personality, psychopathology, life attitudes and neuropsychological
performance among ritual users of ayahuasca: a longitudinal study. PLoS One. 7.
doi: 10.1371/jornal.pone.0042421.
32. Osório FDL, Sanches RF, Macedo LR, dos Santos RG, Maia-de-Oliveira JP,
Wichert-Ana L, ... & Hallak JE. (2015). Antidepressant effects of a single dose of
ayahuasca in patients with recurrent depression: a preliminary report. Revista
Brasileira de Psiquiatria, 37: 13-20. doi.org/10.1590/1516-4446-2014-1496
33. Galvão ACM, de Almeida RN, Silva EAS, Freire FAM, Palhano-Fontes F, Onias
H, … & Galvão-Coelho NL. (2017) A Single Dose of
Ayahuasca Regulates Salivary and Plasmatic Cortisol in Treatment-Resistant
Depression. Submitted.
34. Hankin BL. (2006). Adolescent depression: Description, causes, and interventions.
Epilepsy & Behavior 8: 102-114. doi.org/10.1016/j.yebeh.2005.10.012
35. Blakemore SJ. (2008). The social brain in adolescence. Nature Reviews
Neuroscience, 9: 267-277. Doi:10.1038/nr2353
36. Ganzel BL, Morris PA. (2011). Allostasis and the developing human brain: Explicit
consideration of implicit models. Development and psychopathology, 23: 955-974.
https://doi.org/10.1017/S0954579411000447
37. Thapar A, Collishaw S, Pine DS, Thapar AK. (2012). Depression in adolescence.
Lancer 379:1056-1067. doi.org/10.1016/S0140-6736(11)60871-4
38. Galvão-Coelho NL, Galvão ACDM, Silva FS, Sousa MBCD. (2017). Common
marmosets: a potential translational animal model of juvenile depression. Frontiers
in psychiatry, 8:175. doi.org/10.3389/fpsyt.2017.00175
39. Sharma V, McNeill JH. (2009). To scale or not to scale: the principles of dose
extrapolation. Journal of Pharmacology 157: 907-21. doi: 10.1111/j.1476-
5381.2009.00267.x
40. Savoldi R, Polari D, Pinheiro-da-Silva J, Silva PF, Lobao-Soares B, Yonamine M,
... & Luchiari AC. (2017). Behavioral changes over time following ayahuasca
exposure in zebrafish. Frontiers in behavioral neuroscience, 11: 139. doi:
10.3389/fnbeh.2017.00139
41. Altmann J. (1974). Observational study of behaviour: sampling methods.
Behaviour 49: 227-267. doi: 10.1163/156853974X00534
86
42. Raminelli JLF, Sousa MBC, Cunha MS, Barbosa MFV. (2001) Morning and
afternoon patterns of fecal cortisol excretion among reproductive and non-
reproductive male and female common marmosets, Callithrix jacchus. Biol Rhythm
Res 32:8. doi:10.1076/brhm.32.2.159.1357
43. Sousa MBC, Ziegler T. (1998) Diurnal variation on the excretion patterns of
steroids in common marmoset (Callithrix jacchus) females. Am J Primatol 46:105-
17. doi:10.1002/(SICI)1098-2345(1998)46:2<105:AID-AJP1>3.0.CO;2-#
44. Polizzi di Sorrentino E, Schino G, Tiddi B, Aureli F. (2012). Scratching as a
window into the emotional responses of wild tufted capuchin
monkeys. Ethology, 118: 1072-1084. doi.org/10.1016/j.anbehav.2011.11.016
45. Ferreira RG, Mendl M, Wagner PGC, Araujo T, Nunes, D, Mafra AL. (2016).
Coping strategies in captive capuchin monkeys (Sapajus spp.). Applied Animal
Behaviour Science, 176: 120-127. doi.org/10.1016/j.applanim.2015.12.007
46. Barros M, Tomaz C. (2002). Non-human primate models for investigating fear and
anxiety. Neuroscience and biobehavioural reviews 26: 14. doi.org/10.1016/S0149-
7634(01)00064-1
47. Harro J. (2012). Animal models of depression vulnerability. In Behavioral
Neurobiology of Depression and Its Treatment. 25. Springer Berlin Heidelberg. doi:
10.1007/7854_2012_221.
48. Kokras N, Dalla C. (2017) Preclinical sex differences in depression and
antidepressant response: implications for clinical research. J Neurosci Res 95:5.
doi:10.1002/jnr.23861
49. Pic-Taylor A, Motta LG, Morais JA, Melo-Junior W, Santos AFA, Campos LA, ...
& Caldas ED. (2015). Behavioral and neurotoxic affects of ayahuasca infusion
(Banisteriopsis caapi and Psychotria viridis) in female Wistar rat. Behav
Processes. 118:102-10. doi: 10.1016/j.beproc.2015.05.004
50. Frankel PS, Cunningham KA. (2002). The hallucinogen d-lysergic acid
diethylamide (d-LSD) induces the immediate-early gene c-Fos in rat
forebrain. Brain research, 958: 251-260. doi.org/10.1016/S0006-8993(02)03548-5
51. González-Maeso J, Weisstaub NV, Zhou M, Chan P, Ivic L, Ang R, ... & Sealfon
SC. (2007). Hallucinogens recruit specific cortical 5-HT 2A receptor-mediated
signaling pathways to affect behavior. Neuron, 53: 439-452.
doi.org/10.1016/j.neuron.2007.01.008
52. Moreira CM, Peternelli dos Santos L, de Sousa MBC, Izar P. (2016). Variation in
glucocorticoid levels: survival and reproductive demands in wild black capuchins
(Sapajus nigritus). International Journal of Psychological Research, 9: 20-29.
doi:10.21500/20112084.2303
53. Tu MT, Zunzunegui MV, Guerra R, Alvarado B, Guralnik J M. (2013). Cortisol
profile and depressive symptoms in older adults residing in Brazil and in
87
Canada. Aging clinical and experimental research, 25: 527-537. DOI
10.1007/s40520-013-0111-0
54. Shively CA, Willard SL. (2012) Modeling depression in adult female cynomolgus
monkeys (Macaca fascicularis). Am J Primatol 74:14. doi:10.1002/ajp.21013
55. Dettling AC, Feldon J, Pryce CR. Early deprivation and behavioral and
physiological responses to social separation/novelty in the marmoset. Pharmacol
Biochem Behav (2002) 73:10. doi:org/10.1016/S0091-3057(02)00785-2
56. de Sousa MB, Galvão AC, Sales CJ, de Castro DC, Galvão-Coelho NL. (2015)
Endocrine and cognitive adaptations to cope with stress in immature common
marmosets (Callithrix jacchus): sex and age matter. Front Psychiatry 6: 11.
doi:10.3389/fpsyt.2015.00160
57. Fries E. (2008). 5. Hypocortisolemic Disorders. In Stress 174: 60-77. Karger
Publishers. DOI:10.1159/000119047
58. Kunugi H, Hori H, Ogawa S. (2015) Biochemical markers subtyping major
depressive disorder. Psychiatry Clin Neurosci 69:11. doi:10.1111/pcn.12299
59. Boonen E, Van den Berghe G. (2016). MECHANISMS IN ENDOCRINOLOGY:
New concepts to further unravel adrenal insufficiency during critical
illness. European journal of endocrinology, 175: 1-9. doi: 10.1530/EJE-15-1098
60. Maletic V, Robinson M, Oakes T, Iyengar S, Ball SG, Russell J. (2007).
Neurobiology of depression: an integrated view of key findings. Int. J. Clin. Pract.
61. doi: 10.1111/j.1742-1241.2007.01602.x
61. McEwen BS, Bowles NP, Gra JD, Hill MN, Hunter RG, Karatsoreos IN, Nasca C.
(2015) Mechanisms of stress in the brain. Nat Neurosci 18:10. doi:10.1038/nn.4086
62. Castro M, Elias LL. (2003). Insuficiência adrenal crônica e aguda. Medicina
(Ribeirao Preto. Online). 36. doi: http://dx.doi.org/10.11606/issn.2176-
7262.v36i2/4p375-379
63. Benbouzid, M., Choucair‐Jaafar, N., Yalcin, I., Waltisperger, E., Muller, A.,
Freund‐Mercier MJ, Barrot M. (2008). Chronic, but not acute, tricyclic
antidepressant treatment alleviates neuropathic allodynia after sciatic nerve cuffing
in mice. European journal of pain, 12: 1008-1017.
doi: 10.1016/j.ejpain.2008.01.010
64. Levinstein MR, Samuels BA. (2014). Mechanisms underlying the antidepressant
response and treatment resistance. Behavioral neuroscience. 8: 1-
12. doi: 10.3389/fnbeh.2014.00208
65. Baghai TC, Binder EB, Schule C, Salyakina D, Eser D, Lucae S, ... & Deiml T.
(2006). Polymorphisms in the angiotensin-converting enzyme gene are associated
with unipolar depression, ACE activity and hypercortisolism. Molecular
psychiatry, 11: 1003-1015. doi:10.1038/sj.mp.4001884
88
66. Schüle C. (2007). Neuroendocrinological mechanisms of actions of antidepressant
drugs. Journal of neuroendocrinology, 19: 213-226. doi: 10.1111/j.1365-
2826.2006.01516.x
67. Jørgensen EH, Vijayan MM, Aluru N, Maule AG. (2002). Fasting modifies Aroclor
1254 impact on plasma cortisol, glucose and lactate responses to a handling
disturbance in Arctic charr. Comparative Biochemistry and Physiology Part C:
Toxicology & Pharmacology, 132: 235-245. doi.org/10.1016/S1532-
0456(02)00069-8
68. Jørgensen HS. (2007). Studies on the neuroendocrine role of serotonin. Dan Med
Bull, 54: 266-288.
69. Budziszewska B, Siwanowicz J, Przegaliński E. (1994). The effect of chronic
treatment with antidepressant drugs on the corticosteroid receptor levels in the rat
hippocampus. Polish journal of pharmacology, 46: 147-152.
doi: 10.1038/sj.bjp.0704368
70. Eiring A; Sulser F. (1997). increased synaptic availability of norepinephrine
following desipramine is not essential for increases in gr mrna. J neural transm
104: 1255-1258. doi: 10.1007/bf01294725
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
REFERENCIAS GERAIS
1. American psychiatric association. 2013. Diagnostic and statistical manual of mental
disorders (5th ed.). Wasshington, dc: author.
2. Anacker C, Zunszain P. A., Carvalho La, Pariante CM. (2011a). The glucocorticoid
receptor: pivot of depression and of antidepressant treatment?
Psychoneuendocrinology, 36, 415-4
3. Anacker C, Zunszain PA, Cattaneo A, Carvalho LA, Garabedian MJ, Thuret S, Price
J, Pariante CM. (2011b). Antidepressants increase human hippocampal neurogenesis
by activating the glucocorticoid receptor. Molecular psychiatry 7, 738-750.
4. Badanes LS, Watamura SE, Hankin BL. (2011). Hypocortisolism as a potential
marker of allostatic load in children: associations with family risk and internalizing
disorders. Development and psychopathology, 23, 881-896.
5. Barbosa PCR, Cazorla IM, Giglio JS, Strassman, R. (2009). A six-month prospective
evaluation of personality traits, psychiatric symptoms and quality of life in
ayahuasca-naïve subjects. J. Psychoactive Drugs. 41, 205-212.
6. Belzung C. (2014). Innovative drugs to treat depression: did animal models fail to be
predictive or did clinical trials fail to detect effects? Neuropsychopharmacology, 39,
1041-1051.
7. Blakemore SJ. (2008). The social brain in adolescence. Neurocience. 9, 267-277.
8. Boland RJ, Keller MB. (2004). Antidepressants. In schatzber a. F., nemeroff, c.b.
(eds.), textbook of psychopharmacology. American psychiatric press, 3, 847-864.
9. Boletim Brasileiro de avaliação de tecnologias em saúde. (2012). Antidepressivos no
transtorno depressivo maior em adultos. 18, 1-35. Author.
10. Bouso JC, González D, Fondevila S, Cutchet M, Fernández X, Barbosa PCR, ...,
Riba J. (2012). Personality, psychopathology, life attitudes and neuropsychological
performance among ritual users of ayahuasca: a longitudinal study. PLoS One. 7,
e42421.
11. Bouso, J.C., Fábregas, J.M., Antonijoan, R.M., Rodríguez-Fornells, A., Riba, J.
(2013). Acute effects of ayahuasca on neuropsychological performance: differences
in executive function between experienced and occasional
users. Psychopharmacology. 230, 415-424.
12. Bouso J, Palhano-Fontes F, Rodríguez-Fornells A, Ribeiro S, Sanches R, Crippa
J. (2015). Long-term use of psychedelic drugs is associated with differences in brain
structure and personali0074y in humans. Eur Neuropsychopharmacol 25, 483-492.
13. BRASIL. Conselho Nacional Antidrogas. Resolução nº 4 - CONAD, de 4 de
Novembro de 2004. Dispõe sobre o uso religioso e sobre a pesquisa da ayahuasca
92
que o informam [on-line]. [consulta 24 de novembro de 2017]. Disponível em:
http://www.cesnur.org/2004/daime1.htm
14. BRATS. (2012) Antidepressivos no Transtorno Depressivo Maior em Adultos.
Disponível em: http://bvsms.saude.gov.br/bvs/periodicos/brats_18.pdf
15. Butelman ER, Kreek MJ. (2017). Medications for substance use disorders
(SUD): Emerging approaches. Expert Opinion on Emerging Drugs.30, 1-15.
16. Callaway JC, Mckenna DJ, Grob CS, Brito GS, Raymon LP, Poland RE,
Andrade EN, Andrade EO, Mash, DC. (1999). Prarmacokinetics of hoasca alkaloids
in healthy humans. Jornal of ethnopharmacology. 65, 243-256.
17. Callaway JC. (2005). Fast and slow metabolizers of hoasca. Jornal of
psychoactive drugs. 37, 1-5.
18. Campbell S, Marriott M, Nahmias C, Macqueen GM. (2004). Lower
hippocampal volume in patients suffering from depression: a meta-analysis. Am j.
Psychiatry. 161, 598-607.
19. Canale A, Furlan MMDP. (2006). Depressão. Arq mudi. 10, 23-31.
20. Cazenave SO. (2000). Banisteriopsis caap: acao alucinogena e uso ritual. Rev.
psiquiatr. clín. 27, 32-5.
21. Clark LA, Watson D. (1991). Tripartite model of anxiety and depression:
psychometric evidence and taxonomic implications. Journal of abnormal
psychology, 100, 316-36.
22. Costa MCM, Figueiredo MC, Cazenave SOS. (2005). Ayahuasca: uma
abordagem toxicológica do uso ritualístico. Rev. Psiq. Clín. 32, 310-318.
23. Coutinho T. (2017). A questão da legitimidade e da legalidade dos usos
contemporâneos da ayahuasca: Um estudo de caso. Dilemas-Revista de Estudos de
Conflito e Controle Social, 6, 331-355.
24. Cunha JA. (2009). Psicodiagnóstico-V. Artmed Editora.
25. De Sousa, MBC, de Menezes-Galvão AC, Sales CJR, de Castro DC, Galvão-
Coelho NL. (2015). Endocrine and cognitive adaptations to cope with stress in
immature common Marmosets (Callithrix jacchus): sex and age Matter. Frontiers in
psychiatry, 6, 160.
26. De Souza PA. (2011). Alcaloides e o chá de ayahuasca: uma correlação dos
“estados alterados da consciência” induzido por alucinógenos. Revista brasileira. 13,
349-358.
27. Dixson AF, Lunn SF. (1987). Post- partum changes in hormones and sexual
behaviour in captive groups of marmosets (callithrix jacchus). Physiol behav, 41,
577-583.
93
28. Doris A, Ebmeier K, Shajahan P. (1999). Depressive illness. The Lancet, 354,
1369-1375.
29. Dos Santos, R.D., Landeira-Fernandez, J., Strassman, R.J., Motta, V., Cruz, A.P.M.
(2007). Effects of ayahuasca on psychometric measures of anxiety, panic-like and
hopelessness in Santo Daime members. J. Ethnopharmacol. 112, 507-513.
30. Dos Santos RG, Valle M, Bouso JC, Nomdedéu JF, Rodríguez-Espinosa J,
McIlhenny EH,..., Riba J. (2011). Autonomic, neuroendocrine, and immunological
effects of ayahuasca: a comparative study with d-amphetamine. Journal of clinical
psychopharmacology, 31, 717-726.
31. Fava M, Kendler KS. (2000). Major depressive disorder. Neuron. 28, 335–341.
32. Fletcher J. (2010). Adolescent depression and educational attainment: results
using sibling fixed effects. Health economics. 19, 855–871.
33. França ISX, Souza JA, Baptista RS, Britto VRS. (2008). Medicina popular:
benefícios e malefícios das plantas medicinais. Revista brasileira de enfermagem. 61,
201-208.
34. Friborg O, Martinsen EW, Martinussen M, Kaiser S, Øvergård KT, Rosenvinge
JH. (2014). Comorbidity of personality disorders in mood disorders: a meta-analytic
review of 122 studies from 1988 to 2010. Journal of affective disorders, 152, 1-11.
35. Frodl T, Schaub A, Banac S, Charypar M, Jager M, Kummler P, ..., Meisenzahl
EM. (2006). Reduced hippocampal volume correlates with executive dysfunctioning
in major depression. J psychiatry neuroscienc. 31, 316-325.
36. Galvão ACDM. (2015). Modulação do estresse social sobre parâmetros
fisiológicos, comportamentais, cognitivos e plasticidade neuronal em saguis
(Callithrix jacchus) juvenis: um modelo psiquiátrico e cognitivo (Tese de mestrado,
Universidade Federal do Rio Grande do Norte).
37. Galvão-Coelho NL, Silva HPA, Sousa MBC. (2012). The influence of sex and
relatedness on stress response in common marmosets (callithrix jacchus). American
journal of primatology, 74, 819-827.
38. Guimaraes F, Graeff f. (2000). Fundamentos de psicofarmacologia. Editora
atheneu.
39. Hodgson K, Tansey KE, Uher R, Dernovsek MZ, Mors O, Huser J, ..., Mcguffin
P. (2015). Exploring the role of drugmetabolising enzymes in antidepressant side
effects. Psychopharmacology (berl). 232, 2609-2617
40. Jacob MS, Presti DE. (2005). Endogenous psychoactive tryptamines
reconsidered: an anxiolytic role for dimethyltryptamine. Medical hypotheses. 64,
930-937.
94
41. Joca SR, Padovan CM, Guimarães FS. (2003). Stress, depression and the
hippocampus. Revista brasileira de psiquiatria, 25, 46-51.
42. Kavan MG, Barone EJ. (2014). Grief and Major Depression-Controversy Over
Changes in DSM-5 Diagnostic Criteria. American family physician, 90, 690-694.
43. Kessler RC, Berglund P, Demler O, Jin R, Koretz D, Merikangas K.R, ..., Wang
PS. (2003). The epidemiology of major depressive disorder: results from the National
Comorbidity Survey Replication (NCS-R). Jama, 289, 3095-3105.
44. Kupfer DJ, Frank E, Phillips ML. (2012). Major depressive disorder: new
clinical, neurobiological, and treatment perspectives. Lancet. 379, 1045-1055.
45. Labigaline EJ. (1998). O uso de ayahuasca em um contexto religioso por ex-
dependentes de álcool. (Tese de Mestrado), São paulo: escola paulista de medicina.
46. Lacreuse A, Chang J, Metevier CM, LaClair M, Meyer JS, Ferris CM. (2014).
Oestradiol modulation of cognition in adult female marmosets (Callithrix
jacchus). Journal of neuroendocrinology, 26, 296-309.
47. Leuner B, Glasper ER, Mirescu C. (2007). Acritical time for new neurons in the
adult hippocampus. The journal of neuroscience. 27, 5845-584.
48. Lima D. (2004). Depressão e doença bipolar na infância e adolescência. Jornal
de pediatria. 80, 11-20.
49. Lima IVM, Sougey EB, Vallada-Filho HP. (2004). Farmacogenética do
tratamento da depressão: busca de marcadores moleculares de boa resposta aos
antidepressivos. Revista de psiquiatria clínica. 31, 40-43.
50. Lupien SJ, Mcewen BS, Gunnar MR, Heim C. (2009). Effects of stress
throughout the lifespan on the brain, behaviour and cognition. Nature reviews
neuroscience, 10, 434-445.
51. Magness CL, Fellin PC, Thomas MJ, Korth MJ, Agy MB, Proll C, …, Iadonat
SP. (2005). Analysis of the macaca mulatta transcriptome and the sequence
divergence between macaca and human. Genome biology. 6, 1-16.
52. Marques AH, Cizza G, Sternberg E. (2007). Interações imunocerebrais e
implicações nos transtornos psiquiátricos Brain-immune interactions and
implications in psychiatric disorders. Rev Bras Psiquiatr. 29, 27-32.
53. Martinez, GB, Silva CDP. (2010). Ayahuasca–aspectos botânicos e
farmacológicos Ayahuasca–botanical and pharmacological aspects. Revista de
Atenção à Saúde (antiga Rev. Bras. Ciên. Saúde), 7.
54. Maughan B, Collishaw S, Stringaris A. (2013). Depression in childhood and
adolescence. J can acad child adolesc psychiatry, 22.
55. Mckenna DJ, Callaway JC, Grob CS. (1998). The scientific investigation of
ayahuasca. The heffter review of psychedelic research. 1, 1-12.
95
56. Meneguetti DUO, Meneguetti NFSP. (2014). Benefícios a saúde ocasionados
pela ingestão da ayahuasca: contexto social e ação neuropsicológica,
fisioimunológica, microbiológica e parasitária. Cadernos brasileiros de saúde
mental. 6,104-121.
57. Moica T, Gligor A, Moica S. (2016). The relationship between cortisol and the
hippocampal volume in depressed patients–a MRI pilot study. Procedia
Technology, 22, 1106-1112.
58. Palhano-Fontes F, Alchieri JC, Oliveira JPM, Soares BL, Hallak JEC, Galvão-
Coelho NL, Araujo DB. (2014). The therapeutic potentials of ayahuasca in the
treatment of depression. In: the therapeutic use of ayahuasca, pp. 23-39. Springer
berlin heidelberg.
59. Payne JD, Nadel L. (2004). Sleep, dreams, and memory consolidation: the role
of the stress hormone cortisol. Learning & Memory, 11, 671-678.
60. Pryce C, Rüedi-Bettschen C, Dettling AC, Weston A, Russig H, Ferger B, Feldon
J. (2005). Long-term effects of early-life environmental manipulations in rodents
and primates: potential animal models in depression research. Neurosci biobehav rev
29, 649-674.
61. Pryce C, Ruedi-Bettschen D, Dettling AC, Feldon J. (2002). Early life stress:
long-term physiological impact in rodents and primates. News physiol sci 17, 150-
155.
62. Romero CEC. (2011). Estrés y cortisol: implicaciones en la memória y el sueño.
Elementos. 82, 33-38.
63. Saltzman W, Hogan BK, Abbott DH. (2006). Diminished cortisol levels in
subordinate female marmosets are associated with altered central drive to the
hypothalamic-pituitary-adrenal axis. Biological psychiatry. 60, 843-849.
64. Santos RG. (2007). Ayahuasca: neuroquímica e farmacologia. Revista brasileira
saude mental álcool e drogas. 3, 1-11.
65. Sapolsky RM. (2004). Is impaired neurogenesis relevant to the affective
symptoms of depression? Biol psychiatry 56, 137-9.
66. Schmidt, H. D., & Duman, R. S. (2007). The role of neurotrophic factors in adult
hippocampal neurogenesis, antidepressant treatments and animal models of
depressive-like behavior. Behavioural pharmacology, 18(5-6), 391-418.
67. Sen S, Duman R, Sanacora G. (2008). Serum brain-derived neurotrophic factor,
depression, and antidepressant medications: meta-analyses and implications.
Biological psychiatry. 64, 527-532.
68. Sims, BE, Nottelmann E, Koretz D, Pearson J. (2006). Prevention of depression
in children and adolescents. American journal of preventive medicine. 31, 99-103.
69. Sousa MBC, Galvão ACM, Sales CJR, Galvão-Coelho NL. (2015). Endocrine
and cognitive adaptations to cope with stress in immature male and female common
marmosets (callithrix jacchus). Frontiers. 6, 1-11.
96
70. Stevenson MF, Poole TB. (1976). An ethogram of common marmoset (callithrix
jacchus): general behavioral repertoire. Animal behavior. 24, 428-451.
71. Stevenson MF, Rylands AB. (1988). The marmosets, genus Callithrix. In: R. A.
Mittermeier, A. B. Rylands, A. F. Coimbra-Filho & G. A. B. Fonseca (eds.), Ecology
and behavior of Neotropical primates, 2, 131-222.
72. Taylor JH, Mustoe AC, Hochfelder B, French JA. (2015) reunion behavior after
social separation is associated with enhanced hpa recovery in young marmoset
monkeys. Psychoneuroendocrinol. 57, 93-101.
73. Thapar A, Collishaw S, Pine DS, Thapar AK. (2012). Depression in
adolescence. Lancet. 379, 1056-1067.
74. Trivedi MH, Rush AJ, Wisniewski SR, Nierenberg AA, Warden D, Ritz L, …,
Fava M. (2006). Evaluation of outcomes with citalopram for depression using
measurement-based care in star*d: implications for clinical practice. Am j.
Psychiatry. 163, 28-40.
75. Vismaril L, Alves GJ, Palermo-Neto J. (2008). Depressão, antidepressivos e
sistema imune: um novo olhar sobre um velho problema. Rev psiq clín. 35, 196-204.
76. Warden D, Rush AJ, Trivedi MH, Fava M, Wisniewski SR.(2007). The star*d
project results: a comprehensive review of findings. Current psychiatry reports, 9,
449-459.
77. WFMH. (2012) DEPRESSION: A Global Crisis. Disponível em:
http://www.who.int/mental_health/management/depression/wfmh_paper_depression
_wmhd_2012. Acesso em: 20 de novembro de 2017.
78. WHO. (2017) Depression and Other Common Mental Disorders: Global Health
Estimates. Available from: http://www.who.int/mental_health/management/
depression/prevalence_global_health_estimates/en/
79. Willard SL, Shively CA. (2012). Modeling depression in adult female
cynomolgus monkeys (macaca fascicularis). Am j primatology 74, 528-542.
80. Young CC. (2012). Screening for depression in adolescents. The journal for
nurse practitioners 8, 73-74.
81. Zunszain PA, Anacker C, Cattaneo A, Carvalho LA, Pariante CM. (2011).
Glucocorticoids, cytokines and brain abnormalities in depression. Progress in Neuro-
Psychopharmacology and Biological Psychiatry, 35, 722-729.
82. Zverová M, Fisar Z, Jirák R, Kitzlerová E, Hroudová J, Raboch J. (2013).
Plasma cortisol in alzheimer’s disease with or without depressive symptoms. Medical
science monitor. 19, 681-689.
97
Callithrix jacchus, Apolo (1341).
98