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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

INSTITUTO DO CÉREBRO

PROGRAMA DE PÓS-GRADUAÇÃO EM NEUROCIÊNCIAS

Aron de Miranda Henriques Alves

INVESTIGANDO FENÓTIPOS COMPORTAMENTAIS E

ELETROFISIOLÓGICOS ASSOCIADOS AO ESTRESSE

SOCIAL

Natal - RN

Dezembro de 2015

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Aron de Miranda Henriques Alves

Investigando fenótipos comportamentais e eletrofisiológicos

associados ao estresse social

Tese apresentada à Universidade Federal do Rio

Grande do Norte como requisito parcial para

obtenção do título de Doutor em Neurociências.

Orientador: Claudio Marcos Teixeira de Queiroz

Natal - RN

Dezembro de 2015

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Henriques-Alves, A.M. Investigando fenótipos comportamentais e eletrofisiológicos

associados ao estresse social. [Tese] Natal: Universidade Federal do Rio Grande do Norte,

Instituto do Cérebro, 2015.

Tese apresentada ao Programa de Pós-Graduação em Neurociências da Universidade

Federal do Rio Grande do Norte para a obtenção do título de doutor em

Neurociências. Orientador: Prof . Dr. Claudio Marcos Teixeira de Queiroz

Aprovado em: ____ / ____ / ____

Banca examinadora

Prof. Dra.: Isabel Marian Hartmann de Quadros

Instituição: UNIFESP

Assinatura: ____________________________________________________________

Prof. Dr.: Wilfredo Blanco Figuerola

Instituição: UERN

Assinatura: ____________________________________________________________

Prof. Dr.: Diego Andrés Laplagne

Instituição: UFRN

Assinatura: ____________________________________________________________

Prof. Dr.: Richardson Naves Leão

Instituição: UFRN

Assinatura: ____________________________________________________________

Prof. Dr.: Claudio Marcos Teixeira de Queiroz (Orientador)

Instituição: UFRN

Assinatura: ____________________________________________________________

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Índice

ABSTRACT..........................................................................................................................................................9 1. INTRODUÇÃO...........................................................................................................................................10

1.1. A resposta de estresse e o desenvolvimento dos transtornos psiquiátricos ............ 12 1.2. Modelos animais para o estudo de transtornos psiquiátricos ................................. 15

1.2.1. O modelo de derrota social para estudo dos transtornos de humor e ansiedade..18 1.2.2. Comportamentos de defesa como indicadores de fenótipos associados à ansiedade e depressão.................................................................................................................................................20

1.3. Estresse social e os sistemas dopaminérgicos mesolímbico e mesocortical em modelos animais .................................................................................................................. 23

2. OBJETIVOS.................................................................................................................................................27 3. CAPÍTULO 1................................................................................................................................................28 Ethological evaluation of the effects of social defeat stress in mice: beyond the social interaction ratio................................................................................................................................................28

3.1. Abstract ........................................................................................................................ 28 3.2. Introduction ................................................................................................................. 28 3.3. Material and Methods ................................................................................................. 31

3.3.1. Animals............................................................................................................................................31 3.3.2. Social defeat stress......................................................................................................................31 3.3.3. Social interaction test.................................................................................................................32 3.3.4. Ethological analysis of social interaction............................................................................34 3.3.5. Sucrose-preference test..............................................................................................................36 3.3.6. Statistical analysis.......................................................................................................................36

3.4. Results ........................................................................................................................... 37 3.4.1. Defeated mice exhibited initially transient period of social avoidance during extended sessions of social interaction.............................................................................................37 3.4.2. Delayed expression of social investigation behaviors in defeated mice....................39 3.4.3. Risk assessment behavior during social investigation.....................................................40 3.4.4. Flight behavior after social investigation............................................................................41 3.4.5. Defeated mice showed distinct patterns of flight occurrence over time....................44 3.4.6. Segregation of defeated mice into susceptible and resilient subpopulations...........45

3.5. Discussion ..................................................................................................................... 48 3.5.1. Social avoidance behavior after social defeat...................................................................49 3.5.2. Disentangle anxiety- and depression-related behaviors during social interaction........................................................................................................................................................................50 3.5.3. Learning to be fearless...............................................................................................................51 3.5.4. Short sessions restrict temporal unfolding of social behaviors....................................52

3.6. Conclusions .................................................................................................................. 53 4. CAPÍTULO 2................................................................................................................................................54 Opposite modulation of VTA dopamine neurons during social investigation behavior in susceptible and resilient mice after repeated social defeat stress...............................................54

4.1. Abstract ........................................................................................................................ 54 4.2. Introduction ................................................................................................................. 55

4.3.1. Animals............................................................................................................................................56

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4.3.2. Manufacture of microelectrode arrays.................................................................................56 4.3.3. Surgery.............................................................................................................................................56 4.3.4. Social defeat stress......................................................................................................................57 4.3.5. Social interaction test.................................................................................................................58 4.3.6. Electrophysiological recordings and spike sorting..........................................................58 4.3.7. Classification of VTA waveforms............................................................................................59 4.3.8. Analysis of object and social investigation-triggered single unit activity................60 4.3.9. Unit firing rate spatial maps....................................................................................................60 4.3.10. Tissue preparation and histological analysis...................................................................61

4.4. Results ........................................................................................................................... 61 4.4.1. Electrophysiological recording and classification of mice behavioral phenotype in the social interaction test.......................................................................................................................61 4.4.2. Classification of putative DA and non-DA neurons.........................................................62 4.4.3. Putative DA neurons activity during social investigation behavior in resilient and susceptible mice........................................................................................................................................63 4.4.4. Putative non-DA neurons activity during social investigation behavior in resilient and susceptible mice................................................................................................................................67

4.5. Discussion ..................................................................................................................... 69 4.5.1. Methodological caveats and study limitations...................................................................71 4.5.2. Concluding remarks....................................................................................................................72

5. DISCUSSÃO GERAL................................................................................................................73 6. CONCLUSÕES...........................................................................................................................77 7. REFERÊNCIAS..........................................................................................................................78

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Índice de figuras

Figura 1 | Sistemas dopaminérgicos mesolímbico e mesocortical ………………………13 Figure 2 | Experimental design ………………………………………………………………….32 Figure 3 | Transient social avoidance after social defeat stress ……….…………………37 Figure 4 | Temporal evolution of social investigation behavior revealed delayed motivation for social interaction in defeated mice ….………………….………….…..……39 Figure 5 | Repeated social defeat stress leads to sustained stretched-attend posture during social investigation bouts ...………………………………………………….…………41 Figure 6 | Increased flight behavior occurrence after social defeat stress ..………...…42 Figure 7 | Defeated mice display distinct patterns of flight occurrence over time .......45 Figure 8 | Segregation of defeated mice into susceptible and resilient subpopulations using different indexes …………………..…………………………………………..…….….…47 Figure 9 | Tetrode recordings and spike sorting of VTA units ..……………..……………58 Figure 10 | Classification of putative DA neurons ………………………………..…………63 Figure 11 | General activity of putative DA neurons of susceptible and resilient mice during free object and social exploration ..…………………………………………...………64 Figure 12 | Putative DA neuron activity during social investigation behavior in resilient and susceptible mice …………...……………………………………………………...…………65 Figure 13 | Putative DA neuron activity is differentially modulated during social investigation behavior in resilient and susceptible mice ...……………………..…………66 Figure 14 | General activity of putative non-DA neurons of susceptible and resilient mice during free object and social exploration …...…………………………………………67 Figure 15 | Putative non-DA neuron activity during social investigation behavior in resilient and susceptible mice ..…………………………………………………………...……68 Figure 16 | Putative non-DA neuron activity is differentially modulated during social investigation behavior in resilient and susceptible mice …...……………………..………69

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RESUMO Os objetivos desta tese foram os de investigar padrões comportamentais e eletrofisiológicos associados

à resiliência e suscetibilidade ao estresse social induzido em camundongos. Para isso, utilizamos um

protocolo de indução de estresse crônico contínuo a partir de derrotas sociais baseado no paradigma

residente-intruso. Os resultados da tese são apresentados em dois estudos. No primeiro estudo,

camundongos C57BL/6J submetidos a episódios repetidos de derrota social apresentaram motivação

tardia para interagir com um camundongo desconhecido em sessões prolongadas (10 min) do teste de

interação social. Utilizando uma abordagem etológica associada à análise computacional de vídeos foi

possível rastrear precisamente a posição dos camundongos durante a realização de comportamentos de

investigação social. Analisamos ainda a expressão detalhada de comportamentos defensivos, tais como

investigação em postura estendida e fugas, ambos associados ao comportamento de investigação

social. A partir dessas análises demonstramos que a realização do comportamento de investigação

social em postura estendida era significativamente maior para o grupo derrotado comparado ao grupo

controle. Ainda, um subgrupo de camundongos derrotados apresentou investigação social em postura

estendida de forma persistente e sem habituação. Utilizando uma medida da distância de investigação

durante as investigações sociais calculamos um índice de aproximação (IA) para cada animal e

separamos um subgrupo apresentando fenótipo relacionado à ansiedade. A incidência de fugas

também foi maior no grupo derrotado em comparação com os controles. A persistência na ocorrência

desse comportamento foi observada em um subgrupo de camundongos submetidos às derrotas sociais.

Calculamos então um índice de fugas (IF) que se correlacionou inversamente com a preferência por

sacarose, sendo útil para identificar animais anedônicos. No segundo estudo, foram combinados

análise etológica e registros eletrofisiológicos com tetrodos na área tegmentar ventral de camundongos

submetidos à derrotas sociais. Utilizando critérios eletrofisiológicos e farmacológicos classificamos

unidades na área tegmentar ventral como supostos neurônios dopaminérgicos e não-dopaminérgicos.

Durante o comportamento de investigação social foi observado que a modulação da taxa de disparo

dessas subpopulações neuronais distintas ocorreu de maneira oposta em animais suscetíveis e

resilientes ao estresse social. Em suma, propomos que sessões prolongadas associadas à análise

etológica detalhada durante os testes de interação social podem prover informação para classificação

de camundongos em resilientes e susceptíveis após repetidas derrotas sociais. Ainda, a expressão do

fenótipo suscetível parece estar associada ao comprometimento do sistema dopaminérgico

mesolímbico na atribuição de valor de incentivo às interações sociais normalmente associadas ao

aumento da atividade neuronal mesolímbica.

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ABSTRACT

The aims of this thesis were to investigate behavioral and electrophysiological patterns associated to

resilience and susceptibility to social stress in mice. For this, we used a chronic social defeat stress

protocol based on the resident-intruder paradigm. The results are presented here in two studies. In the

first study, C57BL/6J mice submitted to repeated social defeat episodes showed delayed motivation to

interact with an unfamiliar conspecific in long duration (10 min) sessions of the social interaction test.

By using an ethological approach combined with computational video analysis, it was possible to track

precisely the mouse position during social investigation behavior performance. With that approach, it

was analyzed the detailed expression of defensive behaviors, such as stretched attended postures and

flights, both associated to social investigation behaviors. From these analyzes, it was demonstrated

that social investigation behaviors based on stretched attend postures were significantly higher in

defeated mice in comparison to controls. Still, a subpopulation of defeated mice showed persistently

and non-habituating stretched attend postures during social investigation. By using a measure based on

the investigation distance during social investigations, it was possible to compute an approach index

(AI) to each animal and separate a subpopulation showing an anxiety-related phenotype. The flight

incidence was also increased in defeated group as compared with controls. The persistent occurrence

of this behavior was observed in a subpopulation of defeated mice. We calculated a flight index (FI)

that inversely correlated with sucrose preference, showing to be useful to identify anhedonic animals.

In the second study, we combined ethological approach and electrophysiological recordings in the

ventral tegmental area of mice submitted to chronic social defeat stress. By using electrophysiological

and pharmacological criteria, single-units recorded from the ventral tegmental area were classified as

putative dopaminergic and non-dopaminergic neurons. During the social investigation behavior it was

observed that firing rate modulations of distinct neuronal subpopulations occurred in opposite manner

in social defeat susceptible and resilient mice. In summary, this work proposes that longer sessions of

the social interaction test associated to ethological approach can provide information for the

behavioral classifications of resilient and susceptible mice after social defeat stress. Furthermore, the

expression of susceptible phenotype could be related to the midbrain dopaminergic system impairment

in the incentive value assignment to social interactions normally associated with increased mesolimbic

neuronal activity.

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1. INTRODUÇÃO

Em uma definição ampla, nos referimos ao estresse social como as mudanças

fisiológicas que ocorrem em resposta a interações sociais (Miczek, 2015). Quando se

manifesta de uma forma branda (e.g., discussões em família, durante uma abordagem por

pessoa desconhecida) tais mudanças são geralmente transitórias e raramente apresentam

consequências deletérias para o organismo (Almeida, 2005). Entretanto, quando representa

ameaça à integridade física do indivíduo, como em um assalto (Boudreaux et al., 1998) ou

humilhação pública (Carleton et al., 2011), ou quando é prolongado, como nos casos de

racismo (Williams, 1999) e xenofobia (Crocker, 2015), o estresse social se torna fator de risco

para o desenvolvimento de transtornos psiquiátricos.

A relação entre estresse social e transtornos psiquiátricos é conhecida na cultura

ocidental há pelo menos 200 anos (Rosen, 1959). Atualmente, sabemos que transtornos de

humor e ansiedade (Carleton et al., 2011; Eaton et al., 2001), bem como a esquizofrenia

(Haddad et al., 2015; Lederbogen et al., 2013) e abuso de drogas (Sinha, 2001) estão

fortemente relacionados ao estresse de natureza social, sobretudo nos grandes centros urbanos

(Abbott, 2012; Lederbogen et al., 2011; Steinheuser et al., 2014).

De acordo com a organização mundial de saúde, somente a depressão atinge

atualmente 5% da população mundial, sendo uma das principais causas de morbidez no

mundo e fator de risco para o desenvolvimento de doenças cardiovasculares, distúrbios

metabólicos e para o suicídio (WHO | Depression, 2015). A ampla diversidade no que diz

respeito ao desenvolvimento e resposta a tratamentos farmacológicos sugere que a depressão

possui distintas etiologias (Charney & Manji, 2004; Krishnan & Nestler, 2008; Nestler et al.,

2002). Na maioria dos casos, os sintomas da depressão se manifestam juntamente com sinais

e sintomas tradicionalmente associados à outros distúrbios psiquiátricos, como os transtornos

de ansiedade (Gorman, 1996; Kessler et al., 1999; Mineka et al., 1998). A alta taxa de

comorbidade entre os distúrbios psiquiátricos e a ausência de critérios objetivos capazes de

caracterizar essas patologias (Gorman, 1996; Mineka et al., 1998) dificultam seu correto

diagnóstico e consequentemente, seu tratamento adequado. De fato, a falta de marcadores

específicos para os transtornos associados ao estresse social é considerado um dos fatores

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determinantes para os baixos índices de eficácia dos medicamentos usados no tratamento da

ansiedade e depressão (Krishnan & Nestler, 2008; Waugh et al., 2012).

É importante ressaltar que a vulnerabilidade aos transtornos e patologias relacionadas

ao estresse pode variar consideravelmente em indivíduos expostos aos fatores de risco

descritos acima (Southwick et al., 2005). Nos anos 1970, a psicologia começa a empregar o

termo resiliência, que em Latim significa recuo 1 e na física de materiais refere-se à

capacidade de um material elástico retornar a sua forma original após sofrer deformação, para

descrever a capacidade que alguns indivíduos tem para se recuperar após traumas e situações

de estresse extremas (para revisão, ver Russo 2012). O conceito de resiliência será

amplamente empregado nessa tese para se referir à resistência relativa que indivíduos

possuem frente à adversidade (Feder et al., 2009; Southwick & Charney, 2012). Assim,

indivíduos que se adaptam bem e não desenvolvem distúrbios fisiológicos ou psicológicos

após situações estressoras são considerados resilientes, e os que sucumbem à doença,

suscetíveis. Estudos recentes sugerem que tal variação individual está relacionada à aspectos

funcionais e estruturais do sistema nervoso central que ainda permanecem em sua maior parte

desconhecidos (Hariri & Holmes, 2015). Na última década, uma série de evidências, tanto a

partir de pesquisas em humanos (Dillon et al., 2014; Russo & Nestler, 2013) como em outros

animais (Berton et al., 2006; Chaudhury et al., 2013; Tye et al., 2013), sugere a importância

das vias dopaminérgicas mesolímbica e mesocortical no desenvolvimento dos quadros

patológicos crônicos associados ao estresse social. A compreensão desses processos e de

como se desenvolvem possui importantes implicações terapêuticas e socioeconômicas. Nesse

contexto, fundamental importância é dada ao desenvolvimento e aprimoramento dos modelos

animais para o estudo dos transtornos psiquiátricos (Hariri & Holmes, 2015; Nestler &

Hyman, 2010). Nesta tese apresentamos os resultados de dois estudos baseados no modelo

residente-intruso para investigação das consequências do estresse de natureza social sobre o

comportamento de camundongos e sobre a atividade de neurônios dos sistemas

dopaminérgicos nesses animais.

1Online Etymology Dictionary. Retrieved December 07, 2015 from Dictionary.com website http://dictionary.reference.com/browse/resiliency

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1.1. A resposta de estresse e o desenvolvimento dos transtornos psiquiátricos

Os sintomas comportamentais dos transtornos psiquiátricos relacionados ao estresse

compreendem principalmente os domínios afetivo, cognitivo e motivacional (Hollon et al.,

2015). Pacientes diagnosticados com depressão e ansiedade geralmente apresentam anedonia

e aversão social (Pizzagalli, 2014). Esse sintomas também são compartilhados por indivíduos

durante períodos agudos de abstinência farmacológica (Lüthi & Lüscher, 2014). Estudos

sugerem que o desenvolvimento desses sintomas está relacionado a alterações nos sistemas

dopaminérgicos mesolímbico e mesocortical - bem como em estruturas límbicas que fazem

parte dos seus alvos de projeção - durante a exposição prolongada ao estresse (Russo &

Nestler, 2013).

Por estresse, compreendemos o conjunto de respostas fisiológicas adaptativas frente a

qualquer ameaça, com o objetivo de manter o equilíbrio do meio interior (homeostase) e

consequentemente garantir a sobrevivência do organismo (Ulrich-Lai & Herman, 2009).

Essas alterações fisiológicas são organizadas pelo encéfalo e deflagradas por meio de dois

sistemas efetores primários, o componente simpático-adrenomedular do sistema nervoso

autônomo e o eixo hipotálamo-pituitária-adrenocortical (HPA; Ziegler & Herman, 2002). O

componente simpático-adrenomedular do sistema nervoso autônomo é responsável pelo

aumento imediato na força de contração cardíaca e nos níveis de adrenalina, que é liberada a

partir da medula da glândula adrenal e possui efeitos sobre o tônus vascular em vasos no

músculo esquelético e na pele e também sobre a regulação do metabolismo energético

(Ulrich-Lai & Herman, 2009). A ativação do eixo HPA tem como resultado o aumento dos

níveis sistêmicos de hormônios corticosteroides, os quais sinalizam respostas mais lentas

(Droste et al., 2008), e possuem consequências para a ativação subsequente da resposta de

estresse (Tsigos & Chrousos, 2002).

Liberados na corrente sanguínea a partir das glândulas adrenais, os corticosteroides se

ligam a receptores intracelulares que atuam como fatores de transcrição (Tsai & O’Malley,

1994) regulando a expressão gênica (Yamamoto, 1985) em estruturas cerebrais envolvidas no

controle da resposta de estresse, como o hipotálamo (Itoi et al., 2004), hipocampo (Jacobson

& Sapolsky, 1991), córtex pré-frontal (Diorio et al., 1993) e amígdala (Tasker & Herman,

2011). Essas estruturas estão envolvidas em processos cognitivos como memória e tomada de

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decisão (Cahill et al., 1995; Roozendaal et al., 2001), bem como em comportamentos

motivados (Hollon et al., 2015; Russo & Nestler, 2013). Dessa forma, a resposta de estresse

permite a antecipação do impacto do estresse sobre o organismo, favorecendo sua

sobrevivência a um menor custo metabólico.

Nesse contexto, os sistemas dopaminérgicos mesolímbico e mesocortical, que agem

no entroncamento de processos neurobiológicos relacionados ao aprendizado e tomada de

decisão durante as interações sociais, seriam fundamentais (Gunaydin et al., 2014; Rilling et

al., 2002). Tais sistemas compreendem os neurônios dopaminérgicos originados na área

tegmentar ventral (VTA, do inglês ventral tegmental area; Lammel et al., 2008) (Figura 1).

Esses neurônios são a fonte primária da dopamina, que por sua vez é importante na

modulação da excitabilidade neuronal (Gulledge & Jaffe, 1998; Nitsche et al., 2010) e para

processos de plasticidade celular em estruturas límbicas, como o núcleo acumbens (Sim et al.,

2013), hipocampo (Jenson et al., 2015), amigdala (Li & Rainnie, 2014) e córtex pré-frontal

(Otani et al., 2003). O conjunto de circuitos influenciados por essas estruturas possui função

na codificação e regulação do valor de incentivo2 de estímulos no ambiente (Salamone &

Correa, 2012; Schultz, 2006).

Figura 1 | Sistemas dopaminérgicos mesolímbico e mesocortical. Representação de um cérebro de roedor demonstrando as vias dopaminérgicas originadas na área tegmentar ventral VTA e seus alvos de projeções eferentes. hipocampo (Hipp), núcleo accumbens (NAc), hipotálamo lateral (LH), habenula lateral (LHb), amígdala (Amy) e córtex pré-frontal medial (mPFC). As projeções dopaminérgicas são representadas em verde, as glutamatérgicas em vermelho e as GABAérgicas em azul (Adaptado a partir de Russo & Nestler, 2013).

2 A literatura se refere ao valor de incentivo associado à determinado estímulo como uma espécie de atributo motivacional, dado pelo cérebro, à representação neural desse estímulo. Assim, estímulos associados a recompensas teriam valência positiva, e estímulos aversivos, valência negativa (Berridge & Robinson, 1998).

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Alguns autores sugerem que o sistema mesolímbico em particular possui função

fundamental para a detecção de estímulos salientes no ambiente e para o aprendizado das

consequências dos comportamentos em relação a esses estímulos (Berridge & Robinson,

1998; Schultz, 2006). Assim, seu funcionamento adequado seria importante para a expressão

de respostas comportamentais adaptativas subsequentes (Russo & Nestler, 2013).

Estudos sugerem a participação dos corticosteroides, bem como de outros

sinalizadores da resposta de estresse, como o fator liberador de corticotropina (CRF, do inglês

corticotropin releasing factor; Lemos et al., 2012) e o fator neurotrófico derivado do cérebro

(BDNF, do inglês brain derived neurotrophic factor; Berton et al., 2006) nas mudanças de

longo prazo na expressão gênica e na conectividade das estruturas límbicas. Essas mudanças

contribuem para processos de memória, bem como afetam a fisiologia do sistema

neuroendócrino (Koolhaas et al., 2006).

Sabemos que a exposição crônica ao estresse altera a estrutura e a função de regiões

cerebrais envolvidas no controle do eixo HPA e nas respostas autonômicas (Herman et al.,

1995). Assim, a circuitaria neural envolvida na deflagração e controle das respostas de

estresse é reorganizada no cérebro cronicamente estressado, o que envolve o recrutamento de

algumas regiões e atividade reduzida em outras (McEwen et al., 2015; Ulrich-Lai & Herman,

2009). Entretanto, para alguns indivíduos, dependendo de sua predisposição genética, bem

como da modalidade e duração das respostas de estresse, esses mecanismos de controle

podem sofrer alterações persistentes que levam a respostas fisiológicas exacerbadas ou

desajustadas no indivíduo, característica de transtornos psiquiátricos (de Kloet et al., 2005).

Transtornos psiquiátricos relacionados ao estresse social, como a depressão e a

ansiedade, possuem em comum alterações morfológicas e funcionais no sistema mesolímbico

(McEwen, 2012; Russo & Nestler, 2013). Retração de dendritos apicais e redução na

densidade das espículas dendríticas no córtex pré-frontal (Radley et al., 2008) e no hipocampo

(Magariños & McEwen, 1995) foram descritos em animais submetidos ao estresse crônico.

Em humanos, a análise histológica post mortem sugere que o volume dessas regiões está

reduzido em pacientes depressivos, em grande parte devido à morte de células gliais e redução

do número de sinapses (Czéh & Lucassen, 2007). Essa observação foi confirmada por

ressonância magnética funcional em pacientes depressivos (Pizzagalli, 2014). Entretanto, não

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se sabe se essas alterações estavam presentes a priori nos pacientes com depressão, se são o

resultado acumulado de anos da patologia ou ainda, se refletem o uso crônico de fármacos

antidepressivos.

Atualmente pouco se sabe a respeito dos mecanismos fisiológicos envolvendo a

transição de respostas adaptativas em alterações cronicamente patológicas (de Kloet et al.,

2005; Koolhaas et al., 2006). Parte desse problema reside na determinação de quando as

respostas fisiológicas, inicialmente adaptativas, passam a integrar respostas comportamentais

patológicas (de Kloet et al., 2005). Avanços nesse sentido podem ser realizados a partir da

utilização de modelos animais para estudo dos transtornos psiquiátricos (Nestler & Hyman,

2010), associadas a uma análise detalhada do comportamento animal (Fonio et al., 2012b). As

justificativas, contribuições e limitações dessa abordagem experimental são apresentadas a

seguir.

1.2. Modelos animais para o estudo de transtornos psiquiátricos

O uso de modelos animais no estudo dos transtornos psiquiátricos é fundamental para

a compreensão da fisiopatologia, bem como relevante para o desenvolvimento de novos

tratamentos e fármacos (Koolhaas et al., 2006; Nestler & Hyman, 2010). Para isso, um bom

modelo animal deve reproduzir na espécie experimental as principais características da

condição humana (Nestler & Hyman, 2010), incluindo testes de validade de face, preditiva e

de constructo (Belzung & Lemoine, 2011). Entretanto, muitos estudos utilizam espécies

filogeneticamente distantes dos seres humanos, como os roedores. Assim, não se espera que

modelos animais reproduzam por completo as características de transtornos psiquiátricos

(Nestler & Hyman, 2010; Ward et al., 2011). Alguns sintomas, como tristeza, sentimento de

culpa e alucinações são de natureza subjetiva e tipicamente humanos, sendo impossíveis de

reproduzir em animais atualmente (Nestler & Hyman, 2010). Outra limitação é a forma como

os fenótipos comportamentais são identificados nos animais e como se relacionam aos

sintomas observados em humanos (Fonio et al., 2012b). Por exemplo, a aversão social

observada em camundongos após um determinado tratamento pode estar relacionada a

processos neurobiológicos diferentes da aversão social observada em humanos diagnosticados

com depressão (Toth & Neumann, 2013). Entretanto, isso não significa que seja impossível o

16

desenvolvimento de modelos animais válidos, apenas que as observações comportamentais

devem ser feitas com cautela, considerando determinados critérios de validade (Chaouloff,

2013; Koolhaas et al., 2006; Nestler & Hyman, 2010).

Os modelos animais devem atender a critérios de validade de constructo, de face e

farmacológica (Belzung & Lemoine, 2011). Na validade de constructo, os métodos utilizados

para produzir os sintomas nos animais devem ser relevantes para o desenvolvimento das

psicopatologias humanas (Yanagi et al., 2012). Idealmente, o agente causador ou

procedimento utilizado no desenvolvimento do modelo recriaria nos animais os processos

etiológicos relevantes para a condição humana. Pelo uso desse método seria atingida a

validade de face, ou seja, a reprodução de características neurobiológicas estruturais e

fisiológicas, bem como de características comportamentais semelhantes à condição humana

(Goswami et al., 2013). Na validade farmacológica, a resposta fisiológica e comportamental

do modelo à administração de um determinado fármaco seria preditiva do tratamento desse

fármaco na condição humana (Kaiser & Feng, 2015; Nestler & Hyman, 2010).

Entre os mais bem sucedidos modelos animais encontram-se os modelos para doenças

neurodegenerativas, tais como a doença de Alzheimer (Götz & Ittner, 2008; Morrissette et al.,

2009) e determinados tipos de autismo (Etherton et al., 2009; Shahbazian et al., 2002), onde a

inserção ou inativação no genoma de roedores de determinados alelos relacionados à doença

humana reproduz nesses animais as características da doença de uma forma bastante

semelhante. No entanto, transtornos psiquiátricos como a depressão e a ansiedade possuem

arquitetura genética mais complexa (Smoller, 2015), onde tem fundamental importância a

interação entre genes e a influência sobre estes de fatores ambientais durante o

desenvolvimento (Mullins et al., 2015; Nievergelt et al., 2015). Outro ponto relevante é que a

própria classificação e diagnóstico dos transtornos de ansiedade e depressão humanos é

confusa (Angst et al., 2015), dificultando a aplicação dos critérios de validade acima

descritos. Para reproduzir as características desses transtornos em animais, os modelos mais

satisfatórios levam em consideração o estresse como fator de risco.

A indução de estresse crônico moderado em animais tem sido aplicado com relativo

sucesso na reprodução dos comportamentos patológicos e alterações fisiológicas observadas

nos transtornos relacionados ao estresse humano (Conway et al., 2015; Zhu et al., 2014).

17

Nesses protocolos, roedores são submetidos repetidamente à estímulos estressores variados,

tais como choque nas patas, ruído intenso, contenção e baixas temperatura, que podem ou não

ser apresentados de forma imprevisível (Willner, 2005). Em alguns estudos, os animais

desenvolvem sinais de anedonia após 8 semanas (validade de face; Li et al., 2010), um dos

principais sintomas da depressão. Esse quadro pode ser revertido pela administração crônica

de antidepressivos, como os inibidores seletivos de recaptação de serotonina (e.g., imipramina

e fluoxetina; validade farmacológica; Grippo et al., 2006).

Modelos animais de estresse crônico moderado possuem a vantagem de permitir o

teste de hipóteses utilizando estressores controlados experimentalmente (e.g. choque, ruído

intenso, contenção). No entanto, esses estímulos certamente induzem respostas fisiológicas e

comportamentais que não possuem relação com a biologia da espécie (Koolhaas et al., 2006;

Korte et al., 2005). Assim, pode haver incompatibilidade no que diz respeito ao estímulo

estressor e os mecanismos adaptativos que a espécie animal em questão possui (de Kloet et

al., 2005; Koolhaas et al., 2006). Nesses casos, os animais podem não se adaptar devido a

limitações intrínsecas de sua biologia e o desenvolvimento de sintomas patológicos ser

inevitável (McEwen, 1998).

Com o objetivo de contornar a inespecificidade da resposta ao estresse à estímulos

antinaturais, diversos pesquisadores se dedicaram a identificar agentes indutores com maior

significado ecológico. O objetivo explícito é o de induzir respostas adaptativas em

consonância com a fisiologia da espécie estudada (Koolhaas et al., 2006). Aqui é importante

ressaltar o conceito de validade ecológica, ou seja, entender como os limites dos mecanismos

fisiológicos da espécie em questão funcionam em resposta a estressores naturais (Koolhaas et

al., 1999; McEwen, 1998).

Dado que muitas espécies de mamíferos, incluindo os seres humanos, mantém algum

tipo de organização social (Crook et al., 1976), conflitos e perturbações em relações

intraespecíficas representam fonte importante de estresse. O estresse de natureza social é,

algumas vezes, de alta severidade, como pode ser mensurado por meio da quantificação da

magnitude e da duração das respostas fisiológicas (Kirschbaum et al., 1995; Sapolsky, 1982,

1990). Isso implica que a intensidade das respostas de estresse não é necessariamente

relacionada de forma direta à natureza física do estímulo estressor (e.g. intensidade do choque

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nas patas ou do ruído sonoro), mas ao grau com o qual os mecanismos adaptativos de defesa

são desafiados (Koolhaas et al., 2006; McEwen, 1998).

Modelos de estresse social foram aplicados com sucesso utilizando várias espécies,

incluindo roedores (Berton et al., 2006; Miczek, 1979), primatas (Meyer & Hamel, 2014;

Sanchez et al., 2015), porcos (Rault et al., 2015) e peixes (Jeffrey et al., 2014; Verbeek et al.,

2008). Um modelo animal amplamente utilizado para estudo das consequências do estresse

social no desenvolvimento de sintomas de ansiedade e depressão é o modelo de derrota social,

baseado no paradigma residente-intruso, o qual discutirei a seguir.

1.2.1. O modelo de derrota social para estudo dos transtornos de humor e ansiedade

No modelo de derrota social baseado no paradigma intruso-residente, animais, no

nosso caso, camundongos, são submetidos a repetidos confrontos contra um camundongo

maior e mais agressivo, chamado de residente (Kudryavtseva et al., 1991; Miczek, 1979).

Especificamente, o camundongo experimental (intruso) é colocado dentro da caixa do

camundongo residente, que ataca o intruso quase imediatamente, motivado pela defesa de seu

território. Após alguns minutos o residente derrota o intruso que, por sua vez, passa a

demonstrar sinais comportamentais de submissão. Durante as 24 horas seguintes, residente e

intruso são mantidos na mesma caixa, separados fisicamente por uma tela, porém sob as pistas

visuais, auditivas e olfativas um do outro. Depois disso, o camundongo intruso é submetido a

um novo ciclo de subordinação social e agressões por parte de outro animal residente. Esse

protocolo pode ser repetido por vários dias, normalmente de 5 a 10 dias (Golden et al., 2011).

O caráter crônico do estresse de natureza social é geralmente considerado um fator importante

no desenvolvimento das patologias (Keeney & Hogg, 1999; Kinsey et al., 2007). Assim, ao

final do procedimento, os camundongos intrusos desenvolvem uma série de alterações

comportamentais e fisiológicas semelhantes aos quadros de depressão e ansiedade em

humanos (Iñiguez et al., 2014; Kudryavtseva et al., 1991). Os principais sintomas incluem

aversão à interação social e anedonia, bem como elevação nas taxas de hormônios associados

à resposta de estresse (Iñiguez et al., 2014; Keeney & Hogg, 1999; Krishnan et al., 2007),

como a corticosterona e adrenalina. É importante mencionar que nem todos os animais

submetidos ao modelo de derrota social desenvolvem os principais sintomas que caracterizam

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patologia (Feder et al., 2009; Krishnan et al., 2007). Alguns animais desenvolvem sintomas

relacionados à ansiedade, mas não anedonia (Krishnan et al., 2007). A constatação de que há

variabilidade individual em relação a vulnerabilidade ao estresse no modelo de derrota social

faz deste um modelo útil para o estudo da resiliência ao estresse social.

Normalmente, após os procedimentos de indução do estresse, o comportamento social

dos animais é avaliado em testes de interação social. Na variação mais utilizada desses testes,

chamado teste de evitação-preferência social (Berton et al., 2006), os camundongos são

submetidos à exploração de um campo aberto em duas sessões. Na primeira sessão, há apenas

uma gaiola de contenção vazia, que serve como um objeto inanimado. Na segunda sessão, há

um camundongo desconhecido preso dentro da gaiola de contenção. Podemos então,

quantificar o tempo ocupação da zona de interação social ao redor da caixa de contenção e da

área dos cantos da arena (Golden et al., 2011; Venzala et al., 2012). Dado o caráter saliente e

apetitivo dos estímulos sociais (Gunaydin et al., 2014), animais naive geralmente passam a

maior parte do tempo investigando o animal desconhecido no interior da caixa de contenção e

assim apresentam maior tempo de ocupação da zona de interação durante a sessão social. Esse

comportamento também é verdadeiro para os animais resilientes que foram submetidos ao

estresse social. Entretanto, animais susceptíveis terão preferência pela ocupação dos cantos da

arena e evitarão a zona de interação social, como um sinal claro de aversão social

desenvolvido em decorrência do estresse das derrotas sociais (Toth & Neumann, 2013). Como

há um continuum nos parâmetros de tempo de ocupação da zona de interação social e cantos

da arena durante as sessões, muitos pesquisadores usam uma razão de interação social

(Krishnan et al., 2007; Yin et al., 2015), calculada a partir dos tempos de ocupação da zona de

interação durante as sessões objeto e social, para estabelecer um limiar e separar animais com

preferência por interação social (i.e. resilientes; maior tempo na zona de interação durante a

sessão social) de animais com aversão social (i.e. suscetíveis; maior tempo na zona de

interação durante a sessão objeto). O problema associado ao uso de medidas de ocupação é

que informações importantes a respeito do comportamento de investigação social nos animais

é substituída por uma única medida simples (Fonio et al., 2012b). Isso compromete uma

identificação mais refinada a respeito do fenótipo dos animais, já que o comportamento de

aversão social pode estar relacionado a diferentes estados fisiológicos (e.g. perda de interesse

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em interações sociais ou ativação das respostas fisiológicas relacionadas ao medo de interagir;

Toth & Neumann, 2013).

Outro fator importante é a duração dos testes de interação social. Estudos recentes

sugerem que a identificação do fenótipo comportamental realizada em testes de curta duração

e em ambientes novos podem induzir ao erro na fenotipagem devido à ansiedade induzida por

novidade (i.e., neofobia; Fonio et al., 2012a; Hager et al., 2014). Em experimentos realizados

com diferentes linhagens de camundongos, Fonio et al., (2012a) mostrou que linhagens

usadas como padrão para ansiedade crônica (e.g. BALB/c), apresentam maior tempo de

exploração de áreas abertas após 30 minutos em comparação com camundongos selvagens e

C57BL/6J. Vale mencionar que 30 minutos é o tempo máximo utilizado na maioria dos

estudos que se propõem investigar ansiedade crônica em modelos animais (Fonio et al.,

2012b).

O problema na utilização de testes de curta duração também pode valer para testes de

interação social na tentativa de identificar animais apresentando fenótipo relacionado à

depressão. Nesse caso, podemos tomar animais apresentando neofobia como apresentando

fenótipo depressivo, o que certamente produziria uma subpopulação possuindo diferentes

alterações neurobiológicas relacionadas à aversão social. Por essa razão, abordagens mais

sofisticadas são necessárias para estudar como a síndrome comportamental de camundongos

se relaciona com os transtornos de humor e ansiedade humanos.

1.2.2. Comportamentos de defesa como indicadores de fenótipos associados à ansiedade e

depressão

Foi provavelmente a partir das observações de Charles Darwin descritas em 1882 em

The expression of emotions in man and animals que surgiram as primeiras associações entre

transtornos emocionais humanos e padrões comportamentais de outros animais em resposta a

ameaças (Snyder & Pearn, 2007). Essa visão foi bastante desenvolvida pelo casal Blanchard

da Universidade do Havaí - USA a partir da década de 1970, que descreveu aspectos comuns

entre padrões de comportamento defensivo de roedores e respostas comportamentais à

ameaças em humanos (Blanchard et al., 1998, 2001b, 2001c; Blanchard & Blanchard, 1988).

Em um estudo publicado em 2001, Blanchard e colaboradores, utilizando situações

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imaginárias onde as características das ameaças variavam de acordo com a sua iminência e

severidade, encontraram correlações significativas entre padrões defensivos de

comportamento humano e aqueles observados em experimentos com roedores (Blanchard et

al., 2001b).

Em especial, comportamentos de avaliação de risco observados em ratos e

camundongos possuem importância para o estudo da ansiedade (Blanchard et al., 1990;

Graeff, 1994). O grupo de Robert John Rodgers, demonstrou que comportamentos de

avaliação de risco em testes padrão para ansiedade fornecem mais informação do que alguns

índices clássicos (e.g., percentual de entradas nos braços abertos no labirinto em cruz elevado;

Holmes et al., 2000; Rodgers et al., 1999). Comportamentos de avaliação de risco são

descritos como comportamento exploratório, porém realizados em postura defensiva

(exploração em postura estendida, do inglês stretched attend posture; Kaesermann, 1986). A

exploração em postura estendida é caracterizada pelo alongamento do pescoço e

posicionamento da cabeça em direção ao estímulo a ser investigado enquanto o animal

mantêm os membros flexionados em posição favorável à fuga (Blanchard & Blanchard, 1988;

Eilam, 2005; Grant & Mackintosh, 1963). Em geral, ratos e camundongos realizam

exploração em postura estendida quando há incerteza sobre a possibilidade de um

determinado ambiente ou estímulo apresentar ameaças (e.g., predadores, animais da mesma

espécie motivados para agressão territorial; Campos et al., 2013; Reis et al., 2012). Nesse

contexto a exploração em postura estendida possui função adaptativa já que permite o animal

obter informação a respeito da localização de uma possível ameaça antes de adotar uma

manobra evasiva na direção errada (Blanchard et al., 2011). Entre outras características

importantes, vários estudos demonstraram que a exploração em postura estendida possui

validade farmacológica para comportamentos relacionados à ansiedade (Blanchard et al.,

2001a; Sorregotti et al., 2013). Portanto, fármacos que reduzem estados de ansiedade em

humanos, tais como os benzodiazepínicos, também reduzem a exploração em postura

estendida em ratos (Albrechet-Souza et al., 2007) e camundongos (Campos et al., 2013).

O comportamento de exploração em postura estendida tem sido usado para determinar

o grau de ansiedade de animais submetidos ao condicionamento aversivo com choque elétrico

(Hager et al., 2014). Embora reconhecido como de grande relevância para a compreensão dos

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mecanismos neurobiológicos relacionados à ansiedade, os comportamentos de avaliação de

risco ainda são pouco descritos com medidas quantitativas no contexto das interações social.

Outro comportamento estudado em camundongos são as fugas (Dixon, 1998). Fugas

geralmente são caracterizadas como respostas de medo (Blanchard et al., 2005), na qual o

animal se retira em alta velocidade para longe do estímulo ameaçador (Grant & Mackintosh,

1963; Yilmaz & Meister, 2013).

A potencial relação das fugas com transtornos psiquiátricos humanos se dá no

contexto dos transtornos de pânico (Graeff & Del-Ben, 2008). O transtorno de pânico é

definido no DSM-V principalmente pela ocorrência recorrente de ataques de pânico, que por

sua vez são caracterizados pelo surgimento súbito e intenso do sentimento de medo e

desconforto, atingindo o pico em poucos minutos (American Psychiatric Association, 2013).

Relatos clínicos também relacionam a sensação de pânico ao desejo urgente de fugir

(American Psychiatric Association, 2013; Blanchard et al., 2001a). Geralmente, pacientes

diagnosticados com transtorno de pânico podem apresentar sintomas de depressão, como

aversão social e perda de interesse em atividades lúdicas, devido ao medo de ter novos

ataques de pânico e se sentirem envergonhados em ambiente social (Altınbaş et al., 2015;

Breier et al., 1984).

Diversos estudos demonstraram validade farmacológica dos comportamentos de fuga

em camundongos, exacerbando a ocorrência desse comportamento pelo uso de fármacos

panicogênicos (e.g., yoimbina, bicuculinha; Blanchard et al., 2001a; Shekhar & DiMicco,

1987) e reduzindo sua ocorrência diante da administração de panicolíticos (e.g., alprazolam,

clonazepam; Blanchard et al., 2001a; Griebel et al., 1998).

Em nosso primeiro artigo (Capítulo 1), utilizamos os comportamentos de exploração

em postura estendida e fugas durante o teste de interação social em animais submetidos ao

modelo de derrota social. Nesse estudo apresentamos dois índices baseados nesses

comportamentos para melhor caracterização de fenótipos de ansiedade e depressão.

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1.3. Estresse social e os sistemas dopaminérgicos mesolímbico e mesocortical em modelos animais

A literatura sobre os sistemas dopaminérgicos mesolímbico e mesocortical é vasta e

muitas vezes contraditória (Salamone & Correa, 2012). Talvez esse fato seja reflexo da

natureza intrincada dos sistemas dopaminérgicos do cérebro dos animais mamíferos. Apenas

recentemente, com a popularização das técnicas de recombinação de DNA e ferramentas

optogenéticas, foi possível mapear subpopulações distintas de neurônios dopaminérgicos e

dar início à caracterização de suas atividades, padrões de conectividade e funções específicas

em circuitos neuronais (Chaudhury et al., 2013; Gunaydin et al., 2014; Krishnan et al., 2007;

Lammel et al., 2008, 2011, 2012; Lemos et al., 2012; Tye et al., 2013).

Diversos estudos demonstram a participação dos neurônios dopaminérgicos da área

tegmentar ventral (VTA) na expressão de comportamentos de interação social (Gunaydin et

al., 2014; Tidey & Miczek, 1996). Como mencionado anteriormente, esses neurônios fazem

parte das vias mesolímbica e mesocortical, as quais apresentam modificações

neurofisiológicas, neuroquímicas e estruturais nas patologias associadas ao estresse social

(Berton et al., 2006; Cao et al., 2010; Chaudhury et al., 2013; Friedman et al., 2014; Walsh et

al., 2014).

Neurônios dopaminergicos da VTA são naturalmente ativados por experiências

psicossociais positivas, como afiliação e cooperação (Iwasaki et al., 2014; Rilling et al., 2002;

Robinson et al., 2002). De acordo com essa afirmação, o aumento do tônus dopaminérgico

durante interações sociais é evidenciado pelo aumento tanto na atividade de neurônios DA da

VTA (Gunaydin et al., 2014) como na concentração de dopamina no núcleo accumbens

(Robinson et al., 2002; 2011). Esses resutados estão de acordo com a noção clássica,

amplamente caracterizada em estudos relacionados às drogas de abuso, de que neurônios DA

são ativados em resposta a estímulos de recompensa (Schutz, 2006). Entretanto, ao contrário

do que se acreditou durante décadas, existe atualmente considerável corpo de evidências

demonstrando que neurônios dopaminérgicos da VTA também aumentam sua atividade em

resposta à estímulos aversivos (Brischoux et al., 2009; Bromberg-Martin et al., 2010; Lammel

et al., 2011; Matsumoto and Hikosaka, 2009; Schultz, 2010), incluíndo estímulos sociais

como agressão e subordinação social (Anstrom et al., 2009).

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Diante desse novo panorama, surgiram na última década hipóteses nas quais a

reorganização aberrante de circuitos neuronais associados às vias mesolímbica e mesocortical

seria a causa dos comportamentos patológicos observados em transtornos relacionados ao

estresse social (Berton et al., 2006; Cao et al., 2010; Hollon et al., 2015; Lemos et al., 2012;

Russo & Nestler, 2013). Em uma dessas hipóteses, o estresse social induziria formas

específicas de plasticidade sináptica em circuitos neuronais relacionados à atribuição do valor

de incentivo a estímulos sociais (Berton et al., 2006; Hollon et al., 2015; Krishnan et al.,

2007). Essas mudanças se tornariam persistentes em indivíduos susceptíveis o que levaria aos

sintomas observados na depressão e ansiedade, como anedonia e aversão à interação social

(Berton et al., 2006; Hollon et al., 2015; Krishnan et al., 2007; Lüthi & Lüscher, 2014). A

literatura recente, no entanto, apresenta resultados conflitantes e muitas vezes opostos acerca

da função dos neurônios dopaminérgicos no desenvolvimento do quadro patológico

decorrentes do estresse social. Por um lado, estudos associam a redução da atividade

dopaminérgica em fenótipos relacionados à depressão (Tye et al., Dunlop & Nemeroff, 2007).

Por exemplo, Tye e colaboradores (2013), utilizando técnicas de optogenética, demonstraram

que o controle bidirecional (estimulação ou inibição) da atividade de neurônios

dopaminérgicos da VTA modula, também de forma bidirecional (induz ou atenua),

comportamentos relacionados à depressão causada por estresse crônico (Tye et al., 2013).

Por outro lado, há estudos que demonstram aumento do tônus dopaminérgico em

resposta ao estresse social crônico. Nessa linha de evidências, Krishnan e colaboradores

(2007), utilizando fatias isoladas do cérebro de camundongos, demonstraram que animais

suscetíveis ao estresse social apresentavam aumento da taxa de disparo espontânea de

neurônios dopaminérgicos da VTA. Nesse estudo, camundongos resilientes ao estresse social

desenvolveram adaptações caracterizadas por aumento na expressão de canais para potássio e

a consequente redução da excitabilidade neuronal, o que resultava na preservação da taxa de

disparo espontânea em valores semelhantes ao grupo controle (Krishnan et al., 2007).

Adicionalmente, em estudos utilizando camundongos anestesiados, foi demonstrado que o

estresse social crônico aumenta tanto a taxa de disparo tônica como fásica dos neurônios

dopaminérgicos apenas nos camundongos susceptíveis (Cao et al., 2010; Razzoli et al., 2011).

Em um estudo com ratos se comportando livremente, Anstrom e colaboradores (2009)

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demonstraram que neurônios dopaminérgicos da VTA respondem à agressão subordinação

social aumentando sua taxa de disparo e ocorrência de disparos em salvas. Em outro estudo,

Chaudhury e colaboradores (2013), utilizando técnicas de optogenética, demonstraram que a

ativação fásica de neurônios dopaminérgicos que se projetam pra o núcleo accumbens

promove aversão social em animais submetidos ao estresse social. De maneira oposta, a

supressão da atividade desses neurônios produziu nos animais a expressão do fenótipo

resiliente durante os testes de interação social. Esses resultados se opõem à visão clássica de

que o aumento da atividade de neurônios dopaminérgicos da VTA está relacionada à

estímulos de valência positiva (Gunaydin et al., 2014; Berridge & Robinson, 1998; Schultz,

2006), relatados também em relação a comportamentos de interação social (Robinson et al.,

2002, 2011).

Talvez a aparente contradição presente na litertura sobre a neurofisiopatologia dos

transtornos relacionados ao estresse esteja relacionada à determinadas inadequações

metodológicas . Por exemplo, alguns estudos classificam o fenótipo comportamental a priori

em seguida a atividade neuronal é registrada em preparações ex vivo ou em animais

anestesiados (Krishnan et al., 2007; Cao et al., 2010; Razzoli et al., 2011). Nesses casos, é

possível que as variações observadas na fisiologia neuronal não corresponda diretamente à

síndrome comportamental desencadeada pela exposição crônica ao estresse. Aqui, afirmamos

a importância da investigação da atividade neuronal durante a expressão de comportamentos

motivados naturalmente, e executados livremente (e.g. interações sociais), para se estabelecer

uma relação com o fenótipo comportamental.

Em outros estudos, a atividade neuronal é registrada em ensaios comportamentais de

curta duração e correlacionada à medidas comportamentais cumulativas (e.g. tempo total

gasto na zona de interação social; Chaudhury et al., 2013), as quais desperdiçam a dinâmica

de desenvolvimento do comportamento exploratório dos animais, caracterizada por um

transiente de habituação natural (i.e. neofobia). Devemos notar que as medidas cumulativas

são muitas vezes inadequadas, visto que os processos dinâmicos relacionados a atividade

neuronal e ao comportamento ocorrem em janelas de tempo relativamente curtas, como em

milissegundos ao invés de minutos ou horas (Buzsáki & Watson, 2012). Além disso, nenhum

dos estudos citados acima investiga a atividade dos neurônios dopaminérgicos da VTA de

26

animais resilientes e susceptíveis durante a expressão natural de comportamentos de

investigação social. Portanto, para entender melhor os fenômenos neurobiológicos associados

aos fenótipos resiliente e susceptível ao estresse social, utilizamos em nosso segundo estudo

registros eletrofisiológicos de neurônios da área tegmentar ventral durante testes de interação

social em camundongos submetidos ao estresse de derrotas sociais.

27

2. OBJETIVOS

O objetivo geral da presente tese foi o de investigar como os padrões de ativação de

neurônios da área tegmentar ventral se relacionam com o comportamento associado ao

estresse social. Nossa hipótese é que animais susceptíveis ao estresse social apresentam

redução da função de neurônios da via dopaminérgica mesolímbica durante a atribuição de

valor de incentivo a interações sociais.

Nossos objetivos específicos foram:

1. Implementar um modelo animal para avaliação das consequências do estresse social

utilizando o paradigma residente-intruso;

2. Identificar padrões comportamentais associados a susceptibilidade e resiliência ao

estresse social em camundongos;

3. Registrar, por meio de implantes crônicos, o padrão de disparo neuronal na área

tegmentar ventral de camundongos em livre comportamento durante testes de

interação social após estresse social crônico.

28

3. CAPÍTULO 1

Ethological evaluation of the effects of social defeat stress in mice: beyond the social

interaction ratio

3.1. Abstract

In rodents, repeated exposure to unavoidable aggression followed by sustained

sensory threat can lead to prolonged social aversion. The chronic social defeat stress model

explores that phenomenon and it has been used as an animal model for human depression.

However, some authors have questioned whether confounding effects may arise as the model

also boosts anxiety-related behaviors. Despite its wide acceptance, most studies extract

limited information from the behavior of the defeated animal. Often, the normalized

occupancy around the social stimulus, the interaction zone, is taken as an index of depression.

We hypothesized that this parameter is insufficient to fully characterize the behavioral

consequences of this form of stress. Using an ethological approach, we showed that repeated

social defeat delayed the expression of social investigation in long (10 min) sessions of social

interaction. Also, the incidence of defensive behaviors, including stretched-attend posture and

high speed retreats, was significantly higher in defeated mice in comparison to controls.

Interestingly, a subpopulation of defeated mice showed recurrent and non-habituating

stretched-attend posture and persistent flights during the entire session. Two indexes were

created based on defensive behaviors to show that only recurrent flights correlate with sucrose

intake. Together, the present study corroborates the idea that this model of social stress can

precipitate a myriad of behaviors not readily disentangled. We propose that long sessions (>

150 s) and detailed ethological evaluation during social interaction tests are necessary to

provide enough information to correctly classify defeated animals in terms of resilience and

susceptibility to social defeat stress.

3.2. Introduction

Social stress is considered a major risk factor for the onset and development of

neuropsychiatric disorders (Charney and Manji, 2004; Sayed et al., 2015). It has been

29

suggested that genetics and developmental factors can contribute to determine whether the

individual will develop depression, anxiety, bipolar disorder, or schizophrenia (or

comorbidity between them) after stressful events (Connor-Smith and Compas, 2002;

Southwick et al., 2005; Turner et al., 1995; Vidal et al., 2007; Zelena et al., 1999). Despite

being often treated as single and separate clinical entities, such disorders share some

important pathological behaviors, including reduced social interaction (social phobia,

aversion or withdrawal) and anhedonia (Gorman, 1996; Kessler et al., 1999; Pizzagalli, 2014).

In fact, comorbidity rates are found to be high among stress-related psychopathologies, for

example, in mood and anxiety disorders (Gorman, 1996; Mineka et al., 1998; Waugh et al.,

2012). Interestingly, not all stress-suffering individuals develop one or another pathology and,

in fact, most of them are resilient to a certain degree of stress (Southwick et al., 2005;

Southwick and Charney, 2012), suggesting that genetic background and family history can

help to cope with social stress (Feder et al., 2009).

Most of our understanding of the mechanisms involved in the vulnerability to social

stress has come from studies performed in animals (Nestler and Hyman, 2010). Animal

models of social stress involve subjecting rodents to brief episodes of social subordination

and aggression by a larger and more aggressive conspecific (Koolhaas et al., 1997;

Kudryavtseva et al., 1991; Miczek, 1979). After repeated exposure to confrontations, rats and

mice show a wide range of depression-like symptoms, including anhedonia and social

avoidance (Krishnan et al., 2007; Kudryavtseva et al., 1991). Like humans, anxiety-like

symptoms are also observed in a subgroup of individuals (Krishnan et al., 2007). Importantly,

these stress-induced behaviors develop differently in subjects, which makes the social defeat

model useful to study resilience and susceptibility to stress (Krishnan et al., 2007; Nestler and

Hyman, 2010).

It has been argued that understanding human psychiatric disorders depends on better

animal models (Nestler and Hyman, 2010), not only through the development of new

experimental approaches but mainly by improving the characterization of the pathological

behavior (Fonio et al., 2012b; Toth and Neumann, 2013). Traditionally, social behaviors are

commonly evaluated by using social interaction tests (File and Seth, 2003; Golden et al., 2011)

which are intended to measure the amount of time the experimental animal has spent

30

interacting with an unfamiliar conspecific (Berton et al., 2006; Krishnan et al., 2007;

Kudryavtseva et al., 1991; Venzala et al., 2012). However, an indirect index of social

interaction is used instead. In most studies, it is calculated as a ratio between the amount of

time spent in the close vicinity of the social stimulus (i.e., the interaction zone) and the time

spent in the same area in the absence of any social cue during short-duration (~ 150 s)

sessions (Krishnan et al., 2007; Yin et al., 2015). Also, recent work has suggested that the

identification of behavioral phenotypes in relatively short duration tests can have confounding

effects due to acute experimental procedures and/or novelty-induced anxiety (Fonio et al.,

2012a; Hager et al., 2014). This is especially true when the emotional state of the animals is

under scrutiny. Another important issues include an almost completely absence of information

regarding defeat variables (i.e., latency to attack and number of attacks) which could add to

the inter-individual variability in the behavioral outcome (Chaouloff, 2013) and lack of

specificity of some antidepressants for depressive- and anxiety-related behaviors induce by

the defeat (Berton et al., 1999; Venzala et al., 2012). For these reasons, more sophisticated

behavioral approaches are necessary to evaluate how changes in animal behavior triggered by

social defeat relates to human neuropsychiatric disorders (Peters et al., 2015).

In this context, many research groups have been using ethologically-oriented measures

to enhance biological significance of behavioral tests (Blanchard and Blanchard, 1988; Hager

et al., 2014; Kshama et al., 1990; Peters et al., 2015; Rodgers and Johnson, 1995; Sorregotti et

al., 2013). In this respect, quantification of defensive behaviors, such as stretched-attend

postures and flights, are quite informative in social interaction tests. Stretched-attend posture

occurs during risk assessment and is often observed when the animal is not sure about the

presence and/or location of the threat source (Blanchard and Blanchard, 1988; C and H, 1963).

In mice, stretched-attend posture is characterized by the elongation of the forepart of the

animals' body towards unknown stimuli while the animal keeps a relative safe distance from

the possible threat (Augustsson and Meyerson, 2004; Hager et al., 2014) - a highly adaptive

behavior - relevant for the correct choice of defensive possibilities such as fleeing (flight),

freezing or attacking defensively (Blanchard et al., 2011; Eilam, 2005; Stankowich and

Blumstein, 2005). However, behavioral defensive states are, metabolically speaking, costly

(McEwen et al., 2015) and therefore, correct identification of possible threats is crucial to

31

allow the return to non-defensive behaviors (Blanchard et al., 2011; Blanchard and Blanchard,

1988). Our working hypothesis states that depression- and anxiety-related behaviors in mice

reflect the difficulty that the experimental animal has in evaluating reward and threat

respectively, and ethologically-oriented measures can be used to disentangled these profiles

(Peters et al., 2015). To test that, we have performed ten-minute long sessions of the social

interaction tests to characterize the time-course of social investigation and risk assessment

behavior in repeated (5 days) socially defeated mice. We have introduced novel indexes based

on defensive behaviors to segregate and differentiate subpopulations of defeated mice

showing depression- and anxiety-like phenotypes. The present study highlights the

importance of quantifying species' typical behaviors to better understand the mechanisms of

social avoidance after repeated social stress.

3.3. Material and Methods

3.3.1. Animals

Male C57BL/6J (12-20 wk, 25-35 g) and retired breeder Swiss (16-25 wk, 35-45 g)

mice were used as intruder (experimental) and resident (aggressor), respectively. All animals

were provided by the animal facility of the Brain Institute of the Federal University of Rio

Grande do Norte (Natal, Brazil) and were single- (Swiss) or group-housed (C57BL/6J;

maximum of six mice per cage) at standard conditions (23±2 ºC, 12-h light/dark cycle, lights

on at 7am). Food and water were available ad libitum. All procedures were in accordance with

the international guidelines and the Institutional Animal Care and Use Committee at the

Federal University of Rio Grande do Norte, and all protocols were approved before the

beginning of the experiments (Protocol #38/2011).

3.3.2. Social defeat stress

We used a modified version of the resident-intruder paradigm as reported previously

(Krishnan et al., 2007). The model consisted of placing an intruder (C57BL/6J) mouse inside

the home cage of a heavier and more aggressive resident (Swiss). Before the start of the

32

experiments, Swiss mice were selected based on their aggressive behavior (Miczek et al.,

2001). Only animals displaying attack latencies shorter than 30 s in at least two consecutive

sessions (out of four screening tests) were included in the experiments. Briefly, intruder mice

were exposed to a different resident mouse for no longer than 3 min each day, during 5

consecutive days (Figure 2A). During confrontation, resident mouse attacked the intruder

within the first 30 seconds (average and standard deviation latency to attack: 13.5 ± 19.7 s; n

= 81 confrontations). Preliminary experiments from our lab showed that 3 minutes were

sufficient to allow over 30 bites from the resident and to induce sustained subordination

behavior from the intruder (i.e., submissive upright, vocalization and flight). After each

confrontation, a perforated plexiglass partition was used to separate the resident's cage in two

halves and the animals were in sensory contact for 24 hours, until the next confrontation

session (Figure 2B). Control mice experienced similar experimental condition but no physical

contact occurred. Animals were handled daily and inspected for health conditions. If severe

wounds were detected, animals were removed from the experiment. After the last

confrontation session (5th day), animals were single-housed for the rest of the experiment.

3.3.3. Social interaction test

The social interaction test used was based on the social approach-avoidance test

previously described by Berton et al, (2006). All experiments took place 24 hours after the

last defeat during the daylight period and in a different environment of the confrontation

sessions. First, animals were transferred to the new, quiet and dimly lit room 1 h before the

beginning of the test. After habituation, each animal was placed in the center of a square arena

(white plexiglass open field, 37 cm each side and 30 cm high) and behavior was monitored by

video (Cineplex Studio, 50 fps, camera placed above the arena). Animals were allowed to

fully explore the arena twice, for 600 seconds in each session, under two different

experimental sessions. In the first (“object” session), an empty perforated plexiglass cage (10

x 6.5 x 30 cm) was placed in the middle of one wall of the arena (Figure 2C). In the second

session (“social” session), an unfamiliar Swiss male mouse was introduced into the cage as a

social stimulus. Although it can be argued that the probe mouse used in the social interaction

test resembles the aggressor, and this could foster social aversion, this possibility is unlikely,

33

since previous experiments demonstrate similar amounts of social investigation irrespective to

the strain (i.e., C57BL/6J; Berton et al., 2006). Before each session, the arena was cleaned

with 5 % alcohol solution to minimize odor cues. Between both sessions, the experimental

mouse was removed from the arena, and returned to its home cage for two minutes.

Figure 2 | Experimental design. (A) Timeline showing all the steps of the experimental manipulations during 15 days. Animals were individually housed on day 0. (B) Resident-intruder paradigm was used as repeated social defeat stress (see Methods for a detailed description). (C) Scheme of the social interaction test (object and social sessions) showing the position of the restraining cage and the interaction zone (light-gray) and corners (dark-gray), as well as its respective dimensions.

34

3.3.4. Ethological analysis of social interaction

Locomotion and arena occupancy during object and social sessions were determined

using animals' horizontal position extracted by a custom-made video tracking software

(MouseLabTracker; Tort et al., 2006). Conventional measures of arena occupancy, like time

spent in the interaction zone and corners were quantified. The former is commonly used as

social preference-avoidance index and is calculated by measuring the time spent in an 8 cm

wide corridor surrounding the restraining cage. The corners were defined as two squares of

similar areas in the opposite wall of the arena (Figure 3.1C). The Social Interaction ratio (SIr)

was calculated as:

objectsocial

social

TIZTIZTIZ

SIr (1)

where TIZsocial is the time spent in the interaction zone during the social session and TIZobject is

the time spent in the interaction zone during the object session. This SIr is a slightly modified

version of the ratio used previously (Krishnan et al., 2007). Values vary from 0 to 1, where

SIr > 0.5 indicates preference for social interaction and SIr < 0.5 indicates social avoidance.

Further ethological analyses were performed with the assistance of custom-made

routines written in Matlab (Mathworks) and commercial software (Cineplex, Plexon Inc.).

Occurrence, start and end, duration, frequency, and time course of investigative and defensive

behaviors were determined. Social investigation bouts started when, within the interaction

zone, animals' snouts got in contact with the surface of the perforated plexiglass cage while

maintaining their heads directed to the inside of the cage and they ended when animals faced

another direction. We used the stretched-attend posture evolution during social investigation

to calculate an Approach Index. This index uses the investigation distance, i.e., the distance

between the cage and animal's center of mass during social investigation, to infer the level of

anxiety during investigation. For that, we first averaged the onset distance to the cage (Donk)

for all K investigation bouts of animal i:

35

K

kkii Don

KavgDon

1

1 (2)

Then, we normalized the avgDoni for all animals (N) from control and defeated groups:

N

iiNi avgDon

NavgDonNormDon

1

1 (3)

The Approach Index (AI) for each mouse i was calculated as:

NormDonDoffDonK

AIK

kkikii

1

1 (4)

where Donki and Doffki are the investigation distances at the onset and offset of the

investigation bouts, respectively, for animal i. Approaches during social investigation bouts

(i.e., reduction in the stretched-attend posture) yields high values of AI. Based on population

distribution, we post-hoc defined AI = 0.6 as the threshold to separate subpopulations of

defeated animals. Thus, AI < 0.6 was interpreted as sustained anxiety-like state during social

investigation.

Flight behaviors were manually scored when the animal suddenly retreats from the

social interaction zone and runs towards the corners, as previously described (Blanchard and

Blanchard, 1988; C and H, 1963; Eilam, 2005). Averaged animal's velocity in 200 ms bins

was used to assist the scoring procedure. To determine whether flight behavior increases or

decreases in the 10-min long session, we applied a linear fit model to flight occurrences in

150-s bins and determined the slope (coefficient of regression) of flight dynamics within a

session. This value was used as a Flight Index (FI). A FI < 0 indicates that flight occurrence

decreases over time, while a FI > 0 indicates sustained flight occurrence throughout the

session (i.e., no habituation or adaptation).

The time spent in the center of the arena during social investigation was used as an

36

indirect measure of anxiety (Belzung and Griebel, 2001) and sucrose intake (see below) was

used as a measure of anhedonia (Papp et al., 1991). Both measures were used to compare

defeated animals with low (< 0.6) and high (> 0.6) AI and positive and negative value of FI.

This analysis was used to cross-validate our indexes. All behavioral measures were averaged

for four consecutive epochs of 150-s, for both object and social sessions (chunks of 30-, 50-

and 300-s duration yielded similar results and are not shown).

3.3.5. Sucrose-preference test

Twenty four hours after the social interaction test, all animals were allowed to choose

between 1% sucrose solution and water for 48 h. Sucrose preference was calculated as a ratio

of sucrose intake to the total amount of liquid intake. It was used as an index of the hedonic

state (Papp et al., 1991) and compared between conditions and according to the behavioral

indexes described above. Solutions were filled in 50 ml tubes, renewed and weighed daily, at

8 am. Animals were not food deprived before or during the experiment and the positions of

the tubes in the cage were interchanged at each 12 h to account for drinking place preferences.

3.3.6. Statistical analysis

All behavioral and statistical analyses were performed using custom-made routines in

Matlab (Mathworks). Normality and variance homogeneity were verified using Kolmogorov-

Smirnov' and Bartlett’s tests, respectively. Levene's test was used to compare the variability

of distributions (represented as the coefficient of variation) between groups. Statistical

analyses of behavioral parameters were performed using one- or two-way ANOVA with

repeated measures considering group (control vs defeat) as the independent factor, and session

(object vs social) and time (0-150, 150-300, 300-450, 450-600 bins) as the within-groups

factors. This approach allowed the investigation of possible interactions between factors.

Non-parametric comparisons were made using Mann-Whitney U-test. Chi-square was used to

compare proportions. Statistical significance was set at 5 % and Bonferroni correction for

multiple comparisons was applied. Data are expressed as mean ± S.E.M unless otherwise

specified.

37

3.4. Results

3.4.1. Defeated mice exhibited initially transient period of social avoidance during extended

sessions of social interaction

It was recently argued that behavioral measures can change significantly in time

(Fonio et al., 2012b). Here, time-dependent variation in social behavior induced by repeated

social defeat stress was assessed in extended, 10-min long sessions, of the social interaction

test. During the object session, defeated mice did not differ from controls in the general

pattern of arena occupancy (Figures 3.2A). However, in the presence of an unfamiliar

conspecific (social session), animals from the control group spent more time in the interaction

zone, whereas defeated mice spent less time in the interaction zone (Figure 3A). This

difference was statistically significant in the first 150-s bin for the time in the interaction zone

(Figure 3B; Time vs Group interaction: F(3,512) = 3.72; P < 0.01) and corners (Figure 3C;

Time vs Group interaction: F(3,512) = 3.69; P < 0.01). After 150 s, the occupation of the

interaction zone and corners were similar for controls and defeated mice (Figure 3B and 2C).

The initial transient difference in arena occupation was observed only in the presence of an

unfamiliar conspecific, but not during object session (Figure 3B; Time vs Session interaction:

F(3,512) = 2.07; P = 0.19, and Figure 3C; Time vs Session interaction: F(3,512) = 0.61; P =

0.60).

38

Figure 3 | Transient social avoidance after social defeat stress. Heat map of arena occupancy for representative control (upper panels) and defeated (lower panels) mice at two different time points (after 150 s and 600 s) of the object (A) and social (B) sessions. Warm and cold colors represent high and low occupancy rates, respectively (same color bars for all figures). Note that repeated social defeat stress modifies occupancy maps (lower panels) leading to avoidance of the interaction zone during social session, mainly in the first time bin (150-s). The time spent in the interaction zone (C) and corners (D), as well as the locomotion (E) and immobility (F) during object and social sessions for controls (N = 30) and defeated (N = 36) mice. IZ: interaction zone. (*P < 0.05 control vs defeated in the same time bin of the social session. Bonferroni post hoc test. Data points of the control and defeated groups are slightly shifted within each time bin for clarity purposes.

Repeated social defeat stress induced a markedly decrease in locomotor activity

(Figure 3D, main effect for Group: F(1,512) = 154.9; P < 0.001) and increased immobility

39

(Figure 3E; main effect for Group: F(1,512) = 96.6, P < 0.001) during both object and social

sessions. Importantly, this behavior showed no adaptation in time (main effect for Time:

F(3,512) = 0.88, P < 0.44) during object and social sessions. Also, this change in exploratory

pattern cannot be explained by movement impairment since average speed of locomotion as

well as the highest speed achieved during object and social sessions were not different

between controls and defeated mice (data not shown). Together, these observations suggest

that social avoidance in defeated animals occurs mostly during the first 150 s of the test.

Below we explore whether this result reflects, at least in a certain extend, novelty-induced

anxiety.

3.4.2. Delayed expression of social investigation behaviors in defeated mice

To better understand how social interaction evolves in time we analyzed parameters of

object and social investigation behavior, specifically the investigation time, the number of

investigation bouts, the average duration of investigation bouts and the number of

investigation bouts per interaction zone entry (Figure 4). Both controls and defeated mice

showed increased investigation time of the social stimulus in comparison to the object

stimulus (Figure 4A; main effect for Session: F(1,512) = 195.02, P < 0.001). However, only

control mice showed habituation to the presence of the unfamiliar conspecific, since the time

spent investigating the social stimulus decreased after 300 s (Figure 4A, right panel; Time vs

Group interaction: F(3,512) = 4.70; P < 0.01). Despite the general increase in time investigating

the social stimulus in comparison to the object stimulus for both groups, only defeated mice

showed decreased number of social investigation bouts (Figure 4B, main effect for Group:

F(1,512) = 36.2, P < 0.001). This was particularly true for the initial 150-s bin of the social

session (Figure 4B; Time vs Group interaction: F(3,512) = 7.01; P < 0.001). Interestingly,

defeated mice showed increased duration (average) of social investigation bouts later in the

test in comparison to controls (300-s bin; Figure 4C, right panel; Time vs Group interaction:

F(3,512) = 2.7, P < 0.05). Further, longer sessions of social interaction revealed increased

variance for the number of bouts per interaction zone entry in defeated group, particularly in

the 300-s and 450-s bins (Figure 4D; coefficients of variation: 42 % and 49 % for controls

and 108 % and 112 % for defeated group in the 150-300 s and 300-450 s time bins,

40

respectively; P < 0.001 Levene's test). These results suggest that longer (10min) sessions of

social interaction can reveal subtle differences between control and defeated animals than the

standard parameter of occupancy time of the interaction zone or corners. Next, we evaluated

whether the expression of defensive behaviors are also modified by repeated social defeat

stress.

Figure 4 | Temporal evolution of social investigation behavior revealed delayed motivation for social interaction in defeated mice. (A) Both controls and defeated mice spent more time investigating social than object stimuli, but only controls showed habituation of social investigation. (B) Defeated mice show initial suppression of the number of investigation bouts and delayed motivation to interact socially, as suggested by (C) the increased duration of social investigation bouts after 300 s. (D) Number of investigation bouts per interaction zone entry reveals large variation in socially motivated behavior of defeated group. # P < 0.05 in comparison to the 150-s bin of the social session, same group. P < 0.05 control vs defeated in the same time bin of the social session. Bonferroni post hoc test. Data points of the control and defeated groups are slightly shifted for clarity purposes.

3.4.3. Risk assessment behavior during social investigation

By measuring the distance of the animals' center of mass to the borders of the

restraining cage (i.e., investigation distance) during the onset until the offset of social

investigation bouts, we were able to infer the degree by which the animals approach the social

stimulus from the start to the end of the investigation bouts (Figure 5). Control mice

progressively reduced investigation distance from the onset to the offset of social

investigation bouts (upper panels in Figure 5A and Figure 5B, 3.4C). In contrast, the

41

approach during investigation was significantly smaller in defeated mice, especially in the

offset of social investigation bouts (Figure 5C). We then analyzed the extent of approach-

avoidance tendencies while mice were engaged in social investigation. For this, we created an

AI for social investigation (see Methods), where the value tends to be higher when the mouse

initiates the investigation bout from a short distance and when it approaches the social

stimulus from the onset to the offset of the social investigation bouts. If the animal initiates

the social investigation from a larger distance (i.e., when it investigates the social stimulus in

stretched-attend posture), or if it does not approach the restraining cage borders during the

investigation bout, the AI tends to be lower. As expected, control mice consistently

approached the social stimulus during social investigation, an effect evidenced by the high AI

values (upper histogram in Figure 5D; AI = 0.79 ± 0.04), while defeated mice did not (AI =

0.56 ± 0.06, P < 0.01; Mann-Whitney U-test). Furthermore, defeated mice showed higher

inter-individual variability in the AI (coefficients of variation: 29.2% and 53.3% for controls

and defeated groups, respectively; P < 0.05; Levene's test). For further analysis, a threshold of

AI = 0.6 was set according to its distribution and this value was used to separate defeated

animals with low and high levels of anxiety during social investigation (see below).

3.4.4. Flight behavior after social investigation

Flight behavior was characterized by abrupt and high speed ambulation away from the

interaction zone towards one of the corners. Animals' speed and selected behavior timestamps

over time during social session are shown in Figures 3.5A and 3.5B, for representative

control and defeated mice, respectively. Bursts of fast locomotion after brief forays into the

interaction zone (blue tags) and social investigation bouts (green tags) followed by corners

occupation (yellow tags) can be clearly identified in the video-tracking data (right panels in

42

Figure 5 | Repeated social defeat stress leads to sustained stretched-attend posture during social investigation bouts. (A) Illustrative examples of risk assessment at the onset (left) and offset (right) of social investigation in control (upper panel) and defeated (lower panel) animals. White arrows represent the investigation distance, computed as the shortest distance between animal’s center of mass (circle) and the edges of the restraining cage (tip of the arrow). (B) Investigation distance from the onset to the offset of the social investigation bout shown in A (control and defeated animal represented by gray and black lines, respectively). (C) Grand averaged investigation distance at the onset and offset of social investigation bouts. Note that the distances were similar for both groups at the onset of the social investigation but defeated mice showed increased distance to cage at the offset. (D) Frequency distribution histograms of the Approach Index (AI) for control (top, gray) and defeated (bottom, black) mice. P < 0.01, Student's t-test.

Figures 3.5A and 3.5B). An illustrative flight behavior is shown in Figures 3.5C and 3.5D

(dashed lines in Figure 6B). In this example, the animal started (1) by moving from the left

corner towards the interaction zone where it engaged in social investigation. After the end of

the social investigation bout, the animal abruptly retreated and fled back towards the corner (2)

43

Figure 6 | Increased flight behavior occurrence after social defeat stress. Temporal evolution of speed superimposed with the raster plot of behavioral events in one representative control (A) and defeated (B) animals. Flight behavior was scored when an abrupt increase in animal's velocity take place when moving from the interaction zone to the corners. Right panels depict video tracking data for control (A) and defeated (B) animals during the social condition. The tracks within the corners and interaction zone areas are shown in yellow and blue, respectively. (C, D) Animal's positions and the respective time-course of a typical approach followed by flight behavior (dashed line in B). (E) Flight incidence in control and defeated animals. (F) Number of flights over time during the social session. Only animals with at least one flight were considered (Controls: N = 6; Defeated: N = 24). P < 0.001, Chi-square test. P < 0.01, control vs defeated in the same time bin; # P < 0.01, in comparison to the 150-s bin, same group. Data points of the control and defeated groups are slightly shifted for clarity purposes.

(Figure 6C and 3.5D). Although the maximum speed during flights did not differ between

controls and defeated mice (mean ± SEM: 34.0 ± 1.5 cm/s and 34.5 ± 0.7 cm/s,

respectively;P=0.84,Student’st‐test),theincidenceofflightswashigherinthe defeated

group (67% [24 out of 36]) in comparison to controls (20%, [6 out of 30]; P < 0.001, Chi-

Square Test; Figure 6E). Also, the total number of flights (considering only those animals

which presented it) was higher in defeated animals in comparison to controls (main effect for

Group: F(1,256)=39.32, P<0.001). Additionally, flight occurrence decreased in time (main

44

effect for Time: F(3,256) = 5.48, P<0.01) but it dropped faster in control than defeated

animals (Time vs Group interaction: F(3,256)=2.89, P < 0.05) (Figure 6F). As observed for

social investigation, the temporal dynamics of flight behavior occurrence was also highly

variable within the defeated group, and this observation is further explored below.

3.4.5. Defeated mice showed distinct patterns of flight occurrence over time

By visually inspecting the time-course of social investigation and flights, as well as

arena occupation, we noticed considerable inter-individual variation in coping strategies

among defeated animals. Although some individuals of this group initially showed active

avoidance responses (social investigation bouts followed by flights), in the second half of the

social session, they switched to a strategy of uninterrupted social interaction (Figure 7A).

Differently, some individuals displayed a recurrent pattern of social investigation followed by

flight, associated with increased time spent in corners (Figure 7B). We also observed

individuals with initially long lasting passive avoidance that switched to active responses only

in the second half of the social session (Figure 7C). To determine if the number of flights

was decreasing or increasing during the session, we linearly fit the total number of flights in

the 4 consecutive 150-s bins and calculated the slope of the fitted curve (right graphs in

Figures 3.6A, 3.6B and 3.6C). If the number of flights decreased during the 600 s session, we

expected a negative slope. Figures 3.6D and 3.6E depict the linear fitted curves for those

animals with two or more flights in the control (N=6) and defeated (N=24) groups,

respectively. Figure 7F shows the frequency distribution histogram for the fitted slopes. All

control mice decreased flight behavior during the social session. However, a considerable

heterogeneous pattern was observed for defeated animals, suggesting the existence of at least

two subpopulations of defeated mice regarding active strategies to cope with potential

stressful situations. For the following analysis, we considered the calculated slope of flight

occurrence as the FI and a value of 0 (zero) was established as the threshold to separate

defeated animals in two subpopulations.

45

3.4.6. Segregation of defeated mice into susceptible and resilient subpopulations

Traditionally, socially defeated mice are classified as resilient and susceptible using

the SIr, or some variation of the index, in 150-s long sessions (Berton et al., 2006; Golden et

al., 2011). This labeling can be further verified using the sucrose preference test. Here,

defeated mice drank 20% less sucrose than controls (absolute sucrose intake in 48h, in grams:

15.7 ± 0.8 and 12.5 ± 0.7 , for control and defeated groups, respectively; P < 0.05, Student t-

test). Importantly, no difference in water intake was observed (in grams, 4.8 ± 0.3 and 4.8 ±

0.3, for control and defeated groups, respectively; P = 0.86, Student t-test). We also computed

the SIr for the first 150-s of the sessions to show that 33% (N = 12/36) of defeated mice

displayed SIr < 0.5 and therefore, could be classified as susceptible (Figure 8A; left panel).

Using this classification, we compared the sucrose preference and the time spent in the center

of the arena in susceptible and resilient animals. Surprisingly, both subpopulations did not

differ in the sucrose preference test (Figure 8A; middle and right panels). Conversely, only

the resilient subgroup of defeated animals spent less time in the center of the arena during

social session (Figure 8A; right panel). This observation suggests that behavioral

phenotyping based exclusively on interaction zone occupancy during 150-s long sessions can

be prone to misclassification.

46

47

← Opposite page

Figure 7 | Defeated mice display distinct patterns of flight occurrence over time. Illustrative examples of the temporal evolution of exploratory behavior during the social interaction test (social session) for defeated animals showing attenuated (A), sustained (B) and delayed (C) flight occurrence. Fitted curves for the number of flight occurrences in the respective examples are shown in the right graphs (x-axis are in bins and insets show the R-square and fitted equations). Control animals (Figure 5A) show decreased flight occurrence after 150-s, while flight behavior decreases more slowly in socially defeated mice. Fitted curves for the number of flight occurrences in control (D) and defeated (E) mice revealed strong variation in flight behavior in defeated, but not control, mice. (F) Frequency distribution histograms of the fitted slope (Flight Index) of control (top) and defeated (bottom) mice.

We then tested whether the previous presented indexes, namely the AI and the FI,

could better separate subpopulations of resilient and susceptible mice of the defeated group.

Animals with an AI below 0.6 spent significantly less time in the center zone of the arena

(Figure 8B; right), but no differences in sucrose preference were observed (Figure 8B;

middle). This measure allowed the identification of a subpopulation of susceptible mice

(50%; 18 out of 36) only for anxiety-related behaviors. On the contrary, the FI permitted the

identification of a subgroup of anhedonic animals (25%; 9 out of 36) in defeated mice

(Figure 8C; middle). These animals did not differ in the amount of time spent in center of the

arena (Figure 8C; Right), suggesting that the time-dependent expression of flight behavior

during social interaction can be used as a reliable measure to classify depressive-like

behavioral traits. Finally, using both indexes, we observed that 14 % (5 out of 36) of the

defeated group expressed both depressive- and anxiety-related symptoms.

48

Figure 8 | Segregation of defeated mice into susceptible and resilient subpopulations using different indexes. Left panels show the frequency distribution histograms for the SIr (A), AI (B) and FI (C). Middle and right panels show the sucrose preference and the total time spent in the center of the arena during social session, respectively. For a detailed criteria used to determine resilient and susceptible mice, please refer to the text. # P < 0.05 vs control only. P < 0.05 vs both control and resilient groups. Bonferroni post hoc test.

3.5. Discussion

Previous studies have reported significant changes in social interaction after social

defeat stress (Challis et al., 2013; Friedman et al., 2014; Krishnan et al., 2007; Kudryavtseva

et al., 1991; Razzoli et al., 2011; Venzala et al., 2012). Here, we corroborate and extend these

findings by showing that repeated (5 days) defeat interspersed by continuous sensory contact

with the resident-aggressor led to social avoidance. Surprisingly, the pattern of social

49

investigation changed during our 10-min long session; while control animals reduced

investigative behavior along the session, defeated mice increased it. Also, variability in

behavior investigation patterns was much higher in the defeated group in comparison to

controls. Quantifying the temporal evolution of risk-assessment and flight behaviors during

social investigation allowed us to grasp part of the myriad of exploratory repertoire after

social stress. Using these ethological tools, we present two new indexes that can be of use to

separate anxiety- and depressive-related phenotypes after social stress. The indexes can have

important consequences in the interpretation of biological data regarding resilience and

susceptibility to stress. Below, we discuss the implications of these ideas for the search of a

biological basis of human stress-related disorders.

3.5.1. Social avoidance behavior after social defeat

Chronic exposure to stress can lead to long-lasting changes in behavior (McEwen,

2012). Particularly, social defeat stress, which combines varied levels of both physical and

psychological stress, can result in anhedonia and social avoidance, as well as metabolic

disturbances in humans (Björkqvist, 2001; Hawker and Boulton, 2000) and rodents (Berton et

al., 2006; Bondar et al., 2009; Krishnan et al., 2007; Kudryavtseva et al., 1991; Walsh et al.,

2014). This has prompted the general idea that chronic social defeat stress in mice is a

suitable animal model to study stress-related psychiatric disorders, such as anxiety and

depression (Bartolomucci et al., 2009; Czéh et al., 2016; Hollis and Kabbaj, 2014; Nestler and

Hyman, 2010). In this respect, it has been extensively shown that social withdrawn and

decreased sucrose intake in socially defeated animals can be reversed by chronic treatment

with antidepressants (Berton et al., 2006; Krishnan et al., 2007; Rygula et al., 2006; Venzala

et al., 2012). However, it has been suggested that anxiety can modify the behavioral outcome

of both social interaction (Allsop et al., 2014) and sucrose preference tests (Bondar et al.,

2009), calling for new approaches towards social behavior quantification (Peters et al., 2015).

Social avoidance is a natural, adaptive and complex behavior which allows the

individual to deliberately withdraw from (potential) unpleasant situations (Blanchard et al.,

2005). Exaggerated and sustained avoidance has long been considered a pathological

symptom (Charney and Manji, 2004; Southwick et al., 2005). It may arise from the disrupted

50

motivational processes related to social interaction or from the activation of the neuronal

pathways associated with fear identification and responses towards social stimulus (Steimer,

2011; Toth et al., 2012; Toth and Neumann, 2013). Thus, social avoidance reflects both a

“depressive state” and an "anxiety state". Sucrose intake has also been prone to bias, since

gustatory and olfactory transduction, familiarity, context of liquid intake and group

comparisons can all modify the preference index (Bondar et al., 2009).

Despite these concerns, important contributions to the dopaminergic theory of stress-

related disorders have been put forwarded by the combination of social defeat stress model

and social interaction test (Berton et al., 2006; Krishnan et al., 2007; Walsh et al., 2014), but

not without contradictory results (Tye et al., 2013). In the following sessions, we argue that

careful quantification of defensive behaviors in long observational sessions can be of certain

value in the resolution of such contradictions.

3.5.2. Disentangle anxiety- and depression-related behaviors during social interaction

One obvious question when one considered disentangling anxiety- and depression-

related behaviors is whether they represent two distinct clinical entities or different symptoms

of a single, broad-spectrum, psychiatric illness. In this respect, comorbidity, the complex

overlap between manifestations of different disorders, has long been recognized in individuals

suffering from both anxiety and depression (Gorman, 1996; Mineka et al., 1998). Also,

prolonged stress can trigger both (Gorman, 1996; Mineka et al., 1998; Waugh et al., 2012).

Therefore, comorbidity introduces substantial difficulties in the diagnosis, treatment, and

understanding of the biological substrates of such pathologies. The ethological analysis

presented here suggests that intermingled anxiety- and depression-related behaviors can be

separated in mice exposed to social defeat by looking at defensive behaviors during social

interaction test (see Figure 8). This approach identified 50%, 25% and 14% of the defeated

animals with anxiety-, depression-related or both phenotypes, respectively, indicating that the

model of repeated social defeat stress used here is more likely to produce anxiety. Also, this

experimental approach can be used to model the clinical concept of "anxious depression", a

separate diagnostic class that combines symptoms of anxiety disorders and major depression

(Lydiard and Brawman-Mintzer, 1998). Further studies using antidepressants and anxiolytics

51

will be necessary to demonstrate the predictive validity of these indexes and to better

characterize the behavioral changes associated with social stress.

3.5.3. Learning to be fearless

Several lines of evidence suggest that during stretched-attend posture the animal

gathers information about impending stimulus (Blanchard et al., 2011). When the possible

menaces are explored or becomes familiar to the animal, they switch to non-defensive and

adaptive behaviors, like foraging and free exploration. Therefore, stretched-attend posture can

be interpreted as reflecting animal's state of apprehension which, at least from the

phenomenological point of view, would relate to human anxiety (Blanchard et al., 2001b). In

fact, stretched-attend postures are decreased after administration of anxiolytics, such as

diazepam and buspirone (Bilkei-Gorzo et al., 2002; Blanchard et al., 2001a), but not after

traditional antidepressants (Kaesermann, 1986; Molewijk et al., 1995; Varty et al., 2002). We

hypothesize that, during social investigation bouts, mice would express stretched-attend

posture as long as the social stimulus is perceived as a potential threat. Here, we showed that

defeated group displays two roughly defined distributions of the AI (see Figure 8B) while

expressing a sustained pattern of stretched-attend posture during social investigation bouts

(see Figure 5). Indeed, susceptible animal (AI < 0.6; Figure 8B) spent less time in the center

of the arena, a measure commonly associated with anxiety (Belzung and Griebel, 2001; Fonio

et al., 2012b). Notably, no difference in sucrose consumption was observed between resilient

and susceptible mice separated by this index. Together, this result suggests that AI is a

potential score to reliably segregate a subpopulation showing predominantly anxiety-like

phenotype in the social interaction test.

We also identified another defensive behavior during social interaction. Flight

behavior was scored when abrupt retreats and fast running soon after social investigation

bouts were observed (Blanchard et al., 1999; Blanchard and Blanchard, 1988). Under our

experimental conditions, this natural behavior (Calatayud et al., 2004; Yilmaz and Meister,

2013) was weakly triggered in controls (see Figure 6A). However, social defeat stress

increased its incidence and delayed its habituation (see Figure 6F). Again, we have noted a

large inter-individual difference in the pattern of flight occurrence (see Figure 7). Some

52

individuals displayed risk assessment and flight behaviors in the first tens of seconds of the

social session and soon ceased to flee, while others exhibited repeated behavioral sequences

of arrest followed by flight throughout the social session. Since behavioral flexibility in

coping with a stressor is a feature of resilience (Feder et al., 2009), we assert that FI is a

valuable index to identify susceptible animals to depressive-like state after social stress, as

suggested by the sucrose preference test (Figure 8C, but also (Koolhaas et al., 1999, 2006).

This idea resonates with earlier reports showing that mice exposed to context previously

associated with danger (e.g., predators, aggressive conspecifics) initially freeze, then display

risk assessment that gradually vanishes away giving rise to non-defensive behaviors

(Blanchard and Blanchard, 1988).

3.5.4. Short sessions restrict temporal unfolding of social behaviors

Increasing evidence demonstrate that short duration behavioral assays can be prone to

error (Fonio et al., 2012a, 2012b; Hager et al., 2014). In one study, the phenotypic

identification of anxiety-related behavior in two different strains of mice using the open-field

was dependent on the duration of the test (Fonio et al., 2012a). We have hypothesized that

novelty-induced anxiety can be an important factor determining social avoidance in shorter

sessions of social interaction. Long test durations can increase the opportunity to characterize

individual differences in coping strategies adopted by the experimental animals to overcome

behavioral challenges (Hager et al., 2014). By increasing the social interaction test sessions

four-fold, we demonstrated that social defeated mice showed a brief but transient period of

social avoidance at the beginning of the test (Figures 3.2 and 3.3). Notably, in most defeated

animals, this behavioral pattern was reversed after 150 s, which turns out to be the maximal

observational period used in many studies of social interaction (Chaudhury et al., 2013;

Friedman et al., 2014; Krishnan et al., 2007; Venzala et al., 2012). This observation favors the

interpretation that social avoidance in shorter sessions is more likely to reflect anxiety- than

depression-related symptoms. In fact, like controls, our group of defeated mice clearly prefer

social over object stimuli (Figure 4A). Interestingly, investigation decreases over the 600-s

long session only for the control group, suggesting that habituation took place (Figure 4A).

From the presented experiments, we could not determine whether defeated animals had some

53

sort of rebound effect produced by novelty-induced avoidance or whether the lack of

habituation relates to other cognitive alterations triggered by social defeat stress.

We also observed increased variance in the number of social investigation bouts per

interaction zone entry in defeated animals (see Figure 4D). This effect was only observed at

the second half (5 min) of the social interaction test, and it suggests that animals differently

habituate / recover from novelty-induced avoidance. These results suggest that the

heterogeneity in motivational processes associated to social behaviors can only be observed in

extended sessions of social interaction.

3.6. Conclusions

In conclusion, our results suggest that phenotypic classification based exclusively on

the amount of time spent in the interaction zone in short duration sessions may be

inappropriate, mainly when evaluating depression-like behaviors in socially defeated animals.

Behavioral measurements of social avoidance are influenced by novelty-induced anxiety and

therefore can bias quantification of the drive to social interaction. We propose that ethological

approach combined with longer sessions can overcome this limitation. Here, we have

introduced two new variables, namely the Approach Index (AI) and the Flight Index (FI), that

are easy to quantify and informative about the anxiety- and depression-like profile after

repeated social defeat stress. Further studies using pharmacological treatment are necessary to

completely demonstrate the full application of the presented indexes for the screening of

drugs for mood disorders.

54

4. CAPÍTULO 2

Opposite modulation of VTA dopamine neurons during social investigation behavior in

susceptible and resilient mice after repeated social defeat stress

4.1. Abstract

Animals respond differently to stress. While some of them are able to overcome the

stressor (resilience), others may develop persistent behavioral and physiological alterations,

characterizing psychopathology. Several lines of evidence suggest a link between behavioral

phenotype and long-term plasticity in the ventral tegmental area (VTA) dopaminergic (DA)

neurons after social defeat stress. Some authors have described increased firing rate and

enhanced burst activity of dopaminergic neurons after social defeat in susceptible animals

(but not in controls or resilient animals). However, few studies have investigated VTA

neuronal activity in freely behaving animals during a behavioral task of ecological relevance.

In this study, we combined tetrode electrophysiological recordings with ethological

observations to characterize the relationship between the activities of different neuronal

subtypes in the VTA neurons during the social interaction of socially stressed mice. First, we

classified the activity of single neurons using spike waveform and drug-induced modulation

of discharge rate. We assumed as putative DA neurons those units with long spike waveforms

that stopped firing after administration of 1 mg/kg (i.p.) of apomorphine. Second, we

observed that putative DA neurons presented high firing rate in the close vicinity of the

interaction zone during the social interaction test. This activation pattern was stimulus-

dependent since it was not observed during the object condition and it occurred with active

exploration (i.e., social investigation) of the conspecific. Apomorphine-insensitive neurons

(putative non-DA neurons) showed a wider and unspecific firing pattern during the social

interaction test. Third, electrophysiological analysis demonstrates that during social

investigation behavior, putative DA and non-DA neurons in the VTA show opposite firing

rate modulation in resilient and susceptible animals. We propose that after social defeat,

susceptible animals show blunted positive valence assignment to social interactions which

could be associated with the expression of depression-like behavior in these animals.

55

4.2. Introduction

Dopaminergic (DA) neurons in the midbrain ventral tegmental area (VTA) are

essentially involved in the expression of social approach and avoidance responses (Gunaydin

et al., 2014; Tidey and Miczek, 1996), and several studies suggested their important role in

stress-related pathologies (Berton et al., 2006; Cao et al., 2010; Chaudhury et al., 2013;

Friedman et al., 2014; Walsh et al., 2014). Current hypotheses stem from the idea that severe

and prolonged social stress would trigger specific adaptations in the VTA DA neurons and

their projections (Berton et al., 2006; Krishnan et al., 2007; Ortiz et al., 1996; Saal et al.,

2003). Such changes in neural circuits would become persistent in susceptible individuals

promoting social avoidance behavior (Lüthi and Lüscher, 2014; Russo and Nestler, 2013).

Several studies, using both ex vivo brain slice preparations (Krishnan et al., 2007) and with

anesthetized mice (Cao et al., 2010; Razzoli et al., 2011), have reported that midbrain DA

neurons had increased spontaneous and burst firing after social defeat stress. These changes

were observed exclusively in susceptible mice, without significant changes in controls or

resilient ones. These observations suggest that increased DA activity after social defeat stress

may underlie the susceptible phenotype. However, no study has reported the activity of such

neurons in non-anesthetized, freely moving animals submitted to repeated social defeat stress.

Here, we combined tetrode electrophysiological recordings with detailed ethological

observations to explore the relationship between VTA neuronal activity and social interaction

in socially stressed mice. With that approach, we were able to show opposite activity of VTA

putative DA and non-DA neurons during social interaction in susceptible and resilient

animals. This observation corroborates the hypothesis that social stress modify the activity of

VTA neurons during ecologically relevant behavior. We speculate that social stress reduces

the positive valence associated with social interactions, which could contribute to the

expression of depression-related behaviors in these animals.

56

4.3. Material and Methods

4.3.1. Animals

Male 12- to 20-weeks-old C57BL/6J mice, weighting 25–35 g, were used as

experimental subjects. All animals were obtained from the animal facility of the Brain

Institute of the Federal University of Rio Grande do Norte (Natal, Brazil) and were initially

group-housed (maximum of six mice per cage) and, after electrode implantation, remained

single-housed for the rest of the experiment. All animals were maintained at environmental

standard conditions (23±2 C, 12 h light/dark cycle, lights on at 7 am) with food and water

available ad libitum. All procedures were done in accordance to the international guidelines

and the Institutional Animal Care and Use Committee at the Federal University of Rio Grande

do Norte approved all protocols before the beginning of the experiments (Protocol #38/2011).

4.3.2. Manufacture of microelectrode arrays

We used chronically implanted, head-fixed, tetrode arrays to record unit activity from

the VTA. Specifically, four platinum-iridium tetrodes (microwires of 12.5 µm in diameter;

California Fine Wire Inc.) were mounted in a 128 µm guide tubes with a geometrical

configuration of 2x2 and organized to be implanted bilaterally in VTA and spaced by 500 µm

in the anteroposterior orientation, 2 tetrodes in each hemisphere. All tetrodes were mounted in

a single printed circuit board. One day prior the surgery, electrodes impedance measurements

and automated electroplating to impedance matching (Z ≈ 200 kΩ) were performed using

nanoZ TM (Neuralynx, Inc.).

4.3.3. Surgery

Mice were initially given a prophylactic dose of atropine (0.05 mg/kg, i.p.), then

anesthetized and maintained with ~ 1.5 - 2% isoflurane concentration during surgical

procedures. Body temperature was maintained between 35 and 37 °C and aseptic technique

observed. After placing the mouse in a stereotaxic frame, the scalp was shaved, swabbed with

iodine and a central incision was made to expose the skull. Craniotomies were made to allow

57

the placement of the electrodes in the following coordinates (in mm): AP: 3.4 – 3.8 posterior

from bregma, ML: 0.5, DV: 4.2, bilaterally. Reference and ground were short-circuited and

placed in the frontal bone with the assistance of one of the fixation screw. The tetrode arrays

were secured to the cranium with dental cement and four screws served as anchors. All

animals received health care after surgery and a recovery period of at least 7 days before any

experimental manipulation was allowed.

4.3.4. Social defeat stress

We used a modified version of the social defeat model as reported previously

(Koolhaas et al., 1997; Kudryavtseva et al., 1991; Miczek, 1979). Retired breeder Swiss male

mice were used as the resident aggressor. Before entering the experiments, the latency for the

attack was quantified for each male Swiss mouse in four consecutive days. Only animals that

showed consistent attacks (attack by the resident animal for at least two consecutive sessions)

and the latency for the first attack less than 60 seconds in the last screening day were included

in the experiments. After the recovery period (> 7 days), implanted C57BL/6J intruder mice

were subjected to repeated episodes of aggression and social subordination by the resident-

aggressor conspecific followed by sensory contact with the aggressors. Experiments were

carried out with mice submitted to 5 consecutive defeats. For each social defeat session, the

resident’s home cage was first divided into two halves using a perforated plexiglass partition.

The intruder mouse was placed in one side of the cage while the resident occupied the other.

Sensory contact between animals was allowed for 10 minutes before any confrontation. After

that time, the partition was removed and the resident usually initiated attacks against intruder

within the firsts 30 seconds. The attacks lasted for 3 min maximum and were interrupted by

replacing the perforated partition in the cage. After the physical contact phase, resident and

intruder mice were allowed to continue in sensory contact for 24 hours. Control mice

experienced the same conditions without physical contact. All animals were handled daily and

inspected for health conditions. If severe wounds were detected, animals were removed from

the experiment. After the last defeat, animals were single-housed for the rest of the

experiment.

58

4.3.5. Social interaction test

The protocol for the social interaction test was the same described in Chapter 1 and it

used was based on the social preference-avoidance test paradigm previously described by

Berton et al. (2006). All experiments took place during the light period. Initially, mice were

superficially anesthetized with isoflurane (~ 2%) to guarantee a gentle connection of the

headstage cable. Next, animals were transferred to a quiet and dim light illuminated room for

at least 1 h before the beginning of the procedures. After habituation, the mouse was placed in

the center of a 37 x 37 cm square arena with 30 cm high walls made of white plexiglass and

its behavior was monitored by one video camera (50 fps) placed on top of the arena. Animals

were allowed to fully explore the arena twice, for 10 min in each session, under two different

conditions. In the first one, object session, the arena contained an empty perforated plexiglass

cage (the restraining cage, 10 cm length x 6.5 cm wide x 30 cm deep) centered at one of the

walls. In the second one, social session, an unfamiliar Swiss male mouse was introduced into

the retraining cage. Before each session, the arena was wiped with water containing alcohol

(5%) to avoid confounding effects due to the presence of odorant cues. Electrophysiological

recordings were synchronized with the tracking information using commercial software

(Cineplex Studio, Plexon Inc., Dallas, EUA). Between both sessions, the experimental mouse

was removed from the arena (although still connected to the electrophysiological cable), and

returned to its home cage for two minutes.

4.3.6. Electrophysiological recordings and spike sorting

Extracellularly recorded action potentials were isolated from the continuous wideband

signal (digitized at 40 kHz) using Offline Sorter V3 software (Plexon Inc., Dallas, EUA). First,

the wideband signal was high-pass filtered (> 250 Hz) and spike waveforms detected by

threshold (independent thresholds for each electrode; see Figure 9). Tetrode spike data was

then analyzed by concatenating the individual waveforms from each electrode in the tetrode

group to create a single waveform. Then, the number of units were specified manually to

serve as the template for the unit and the waveforms were assigned to clusters using a

template matching algorithm. Because the spike waveforms could varied (i.e., they were not

stable in time) within and between recording sessions, special care was taken regarding

59

waveform stability during spike cutting clustering. Only stable cells (e.g., without drifting in

the amplitude and/or spike waveform) were included in further analysis.

Figure 9 | Tetrode recordings and spike sorting of VTA units. (A) Electrode array tip location. (B) Coronal section showing the location of the recording electrode. (C) Spike waveforms recorded simultaneously from a single tetrode. (D) Principal component analysis for spike waveforms shown in (C). Gray, orange and green clusters represent isolated units 1-3, respectively. PC1 and PC2 represent the first and second principal components, respectively.

4.3.7. Classification of VTA waveforms

It has been proposed that waveform features can be successfully used to classify action

potentials recorded from the extracellular space, mainly the duration of the spike waveform

(Barthó et al., 2004). Thus, we first averaged the waveforms for each isolated unit and

computed the valley-to-peak width and its mean frequency. Putative dopaminergic neurons

show long action potentials and low firing rate (Wang and Tsien, 2011; see Figure 10).

Further validation of putative dopaminergic neurons were done according to its

60

pharmacological response to 1 mg/kg of apomorphine (i.p.). Apomorphine is a D2 receptor

agonist and systemic administration of low doses transiently reduce (or even) block the firing

of the dopaminergic neurons specifically (Fujisawa and Buzsáki, 2011; Wang and Tsien,

2011). The apomorphine test was performed immediately after the social interaction test. For

that, we recorded the neural activity in the VTA of mice in their home cage for at least 30

minutes before injecting apomorphine. We then recorded discharge rate for at least 90

minutes. This method allowed the discrimination of two groups of cells, referred hereafter as

putative DA neurons and putative non-DA neurons.

4.3.8. Analysis of object and social investigation-triggered single unit activity

Peri-event raster plots and histograms were constructed with custom made routines for

Matlab (Mathworks). First, neuronal activity was aligned either on the onset or the offset of

the investigation bouts (object and social). In Chapter 1, we showed that repeated social stress

can significantly modify this behavioral pattern, especially at the onset and offset of the

investigation (see Figure 5). Thus, each investigation bout was considered as an event and it

was represented as a raster plot of spike activity. Second, the combined peri-event time

histograms from each unit were averaged in 50 ms bins, for both the onset and offset of the

investigation. Finally, the grand average of unit discharge rate in 3-sec periods, before and

after the onset and the offset of the investigation bouts, were computed. We used paired

Student t test to compare the before and after moments to test the relation of VTA neurons in

the social investigation behavior. The averaged peri-event time histograms were smoothed by

using a Gaussian filter with the Matlab function smooth (filter width = 5 ms) for clarity

purposes.

4.3.9. Unit firing rate spatial maps

To better visualize the dependency of environmental stimuli (object or social) and unit

activity, heat maps based on spatial discharge rate were created. For that, we first binned the

arena area in 5 mm x 5 mm pixels and calculate the average firing rate of the units when the

mouse was positioned in that region. Then, we normalized this value by animal's occupation.

A Gaussian smoothing operator (spatial size of 3.5 cm) was used to remove spurious firing

61

and reduced the variance in the discharge rate map. The region of the arena with the highest

firing rate was estimated using the smoothed map.

4.3.10. Tissue preparation and histological analysis

At the end of the experiments, animals were disconnected from the recording system

and deeply anaesthetized with pentobarbital (100 mg/kg; i.p.). A 2 s anodal current, 10–15

mA, was applied through one electrode of one selected tetrode per hemisphere. Animals were

then perfused transcardially with a 0.9% saline followed by a 4% buffered paraformaldehyde.

The brains were gently removed, cryoprotected in 30% sucrose solution at 4 °C, cut (50 µm,

coronal sections) using a cryostat, collected serially in glass slides, stained with Nissl’s

method and analyzed in a standard microscope for evaluation of electrode localization. Only

tetrodes restricted to the VTA were used in the following analyses.

4.4. Results

4.4.1. Electrophysiological recording and classification of mice behavioral phenotype in the

social interaction test

We allowed mice to freely explore the object and the social targets in the social

interaction test (Berton et al., 2006) while simultaneously recording the neuronal activity in

the VTA. Overall, a total of 19 units recorded in the VTA of two defeated mice reached our

inclusion criteria. Unfortunately, we didn’t get units from control animals. However, the two

defeated mice we studied here expressed different behavioral phenotypes after our 5-day

protocol of social defeat stress. Mice phenotyping was based on the behavioral parameters

presented in Chapter 1 of this Thesis and included time spent in social interaction zone,

sucrose preference, the approach index (AI), and flight index (FI, see page 31). Mouse #01

showed high scores for social interaction and sucrose preference, as well as a relatively high

AI and no flights. On the other hand, the behavior of mouse #02 was clearly the opposite of

mouse #01, which suggests susceptibility to depression- and anxiety-related phenotype (see

Table 4.1).

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Table 4.1 | Resilient and susceptible mouse display distinct behavioral parameters

Mouse #01

(Resilient)

Mouse #02

(Susceptible)

Controls

(Chapter 2)

Defeated

(Chapter 2)

SI ratio* 0.77 0.45 0.66 ± 0.01 0.57 ± 0.04

Approach index 0.75 0.47 0.81 ± 0.04 0.68 ± 0.06

Number of flights 0 5 2.80 ± 0.70 7.80 ± 0.90

Flight index ‐ ‐0.5 ‐0.66 ± 0.15 ‐0.56 ± 0.25

Relative sucrose consumption 0.82 0.57 0.76 ± 0.02 0.72 ± 0.03

Total time spent in arena center (s) 22.7 7.7 55.1 ± 4.3 36.5 ± 4.0

Total time engaged in social investigation (s) 201.9 100.5 123.1 ± 8.3 117.6 ± 11.3

* Values shown as mean ± SEM for controls and defeated group data.

4.4.2. Classification of putative DA and non-DA neurons

As mentioned in the Methods section, VTA units were classified as putative DA or

non-DA neurons according to i) the spike waveform (Figure 10A) and ii) response to the

administration of the dopamine D2 receptor agonist apomorphine. It has been suggested that

this drug act on dopaminergic autoreceptors decreasing the activity of DA neurons (Freeman

and Bunney, 1987; Wang and Tsien, 2011). Nine putative DA neurons (5 from mouse #01

and 4 from mouse #02) and 10 putative non-DA neurons (5 from mouse #01 and 5 from

mouse #02) were identified (Figure 10B). As shown in the cumulative frequency distribution

of firing rate from two representative neurons (Figure 10C), putative DA neuron (top)

exhibited suppression, while non-DA neurons (bottom) showed no changes in firing rate after

apomorphine injection (neurons from the same animal). Neurons that exhibited markedly

decrease in the firing rate after the drug administration and a valley-to-peak width value

above 0.4 ms (the midpoint between two distribution peaks) were considered putative DA

neurons, otherwise they were considered as non-DA neurons. We verified that the putative

DA neurons typically exhibited broad, triphasic waveforms and low firing rate (Figures 10A,

10B and 10D), which is in agreement with previously established criteria (Grace and Bunney,

1983; Margolis et al., 2008). In contrast, putative non-DA neurons exhibited shorter action

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potentials and a more variable baseline firing rate (1-30 Hz). Additionally, putative DA and

non-DA neurons showed markedly distinct firing patterns, as evidenced by the interspike

interval distribution in Figure 10E.

4.4.3. Putative DA neurons activity during social investigation behavior in resilient and

susceptible mice

Differences in putative DA neuron firing during both object and social sessions of the

social interaction test were compared between resilient and susceptible mice. The susceptible

mouse showed higher global firing rate of putative DA neurons during both object and social

sessions compared to the resilient mouse (Two-way ANOVA, main effect for the phenotype:

F(1,15) = 5.22, P < 0.05; Figure 11). However, we noted that the activity of putative DA

neurons was regular and spatial unspecific in the susceptible mouse during both object and

social sessions (Figure 12A). In contrast, the discharge pattern of putative DA neurons

recorded from the resilient mouse was spatially related to the environmental stimuli, i.e., it

fired when in the close vicinity of the restraining cage during the social session (Figure 12A;

top-right panel). Interestingly, this unit showed increased peak frequency during the social

session relative to the object session as compared with the putative DA neuron from the

susceptible mouse (Figure 12A).

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Figure 10 | Classification of putative DA neurons. (A) Two examples of typically recorded spikes from the VTA showing waveforms for putative DA (blue) and non-DA (black) neurons. The valley-to-peak width was measured as the time interval from the trough to the following peak of the action potential. Blue and black horizontal bars represent the values of valley-to-peak width for the putative DA and non-DA neuron waveforms, respectively. (B) Baseline firing rate and valley-to-peak width of the classified DA (blue) and non-DA (black) neurons. (C) Cumulative spike activity of putative DA (top) and non-DA (bottom) neurons in response to the dopamine receptor agonist apomorphine. In the example both neurons were recorded simultaneously from the same tetrode. (D) Baseline firing rate of classified putative DA and non-DA neurons. (E) Inter-spike intervals of the putative DA and a non-DA neuron shown in A. * P < 0.05; Unpaired Student’s t test.

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Figure 11 | Activity of putative DA neurons of susceptible and resilient mice during free object and social exploration. Chronic social defeat stress increased the global firing rate of putative DA neurons in the susceptible (n = 4), but not in the resilient (n = 5) mouse. Data are shown as mean ± SEM.

The temporal evolution of the discharge pattern of putative DA neurons during the

investigative behavior is shown in Figure 12B. For the resilient mouse, we observed a

predominant increase in the activity of putative DA neurons during the epochs this animal

was engaged in the social investigation (Figure 12B, top-right panel; Figure 13A). This

effect was mainly observed for social investigation, with weak or no effect observed for the

object investigation epochs (Figure 12B, top-left panel; Figure 13A). When we looked for

putative DA neurons activity in the susceptible mouse, we observed both suppression and no

response to the social investigation (Figure 12B, bottom-right panel; Figure 13B).

Interestingly, like the resilient animal, putative DA neurons in the susceptible animal showed

no differences in their activity for the investigation of the object (empty cage) in the object

session (Figure 12B, bottom-left panel; Figure 13B).

As the animals remained immobile when engaged in social investigation, one could

argue that the observed activation pattern reflects reduction in locomotor behavior. Thus, to

rule out (or at least, minimize) the contribution of ambulation we excluded units showing

significantly modulation when the animals stay immobile (exclusion criterion: consistent

change of at least 20 % in the firing rate during immobility in comparison to locomotion

epochs). Here, immobility was defined as the absence of locomotion (less than 1 cm/s for at

least 1 second), although movements of vibrissae and head were allowed to occur.

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Interestingly, most of putative DA neurons did not exhibit significant modulation of their

activity by immobility behavior (data no shown).

Figure 12 | Putative DA neuron activity during social investigation behavior in resilient and susceptible mice. (A) Firing maps showing illustrative examples of putative DA neuron in the resilient (top) and susceptible (bottom) mouse during the object (left) and social (Right) sessions of the social interaction test. The peak firing rate during the session is depicted on the top right corner of each example. The restraining cage is oriented to the top of the figure, in the same configuration as shown in the chapter 2. (B) Illustrative examples of putative DA neuron activity in the resilient (top panels) and susceptible (bottom panels) mouse during both object (left panels) and social (right panels) investigation. The illustrative waveforms are represented in, superimposed individual spikes (gray traces); black ticker line, average spike waveform. Scale bars, 1 mV, 1 s. Raster plots represent 300 s of continuous recording starting in the top left corner and ending in the bottom right for each panel.

At the population level, the averaged firing rate of the resilient mouse increased after

the onset of social investigation in comparison to the preceding epoch (averaged firing rate, in

Hz: 5.3 ± 1.2 and 3.2 ± 0.6, respectively; P < 0.05, paired Student’s t-test; Figure 13C). In

contrast, susceptible mouse showed reduced firing rate after the onset of social investigation

(2.2 ± 0.3 and 3.6 ± 0.3, respectively for the 3-sec period after and before the onset of social

investigation; P < 0.05, paired Student’s t-test; Figure 13C).

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Figure 13 | Putative DA neuron activity is differentially modulated during social investigation behavior in resilient and susceptible mice. (A) Peri-event rasters and smoothed population average peri-event histograms (blue trace) of a putative DA neuron from the resilient mouse. The neuronal activity is referenced to the onset (left panels) and the offset (right panels) for object (top panels) and social investigation bouts. (B) Same description from A for the susceptible mouse. (C) Putative DA neurons showed opposite modulation in the susceptible (n = 4) and resilient (n = 5) mouse for social, but not object investigation.

4.4.4. Putative non-DA neurons activity during social investigation behavior in resilient and

susceptible mice

Putative non-DA neuron firing rate were not different when compared between

resilient and susceptible mice in the object and social sessions in the social interaction test

(Two-way ANOVA, main effect phenotype: F(1,13) = 0.6, P = 0.45; Figure 14). However, we

observed significantly changes in putative non-DA neuron activity during social investigation

behavior. As exemplified in the Figure 15 (same session shown in Figure 12), putative non-

DA neurons in the resilient mouse showed suppressed activity after the onset of social

investigation bouts, while in susceptible mouse it was increased Figure 15A and 15B.

Putative non-DA neurons showed higher firing rate (> 10 Hz) and short spike waveform (<

0.3 ms; see Figure 15B) in comparison to putative DA neuron.

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Figure 14 | Activity of putative non-DA neurons of susceptible and resilient mice during free object and social exploration. The global firing rate of putative non-DA neurons were not different between the susceptible (n = 4) and the resilient (n = 4) mouse. Data is shown as mean ± SEM.

Importantly, some of putative non-DA units showed strong modulation by locomotion.

These units (n = 2) were excluded from further analysis.

Resilient mouse showed decreased activity of putative non-DA neurons after the onset

of social investigation (Figure 15B; top-right panel; Figure 16A). This effect was mainly

observed for social investigation, since no effect was observed in the object investigation

epochs (Figure 15B, top-left panel; Figure 16A). Interestingly, putative non-DA neurons

activity were increased in the susceptible mouse during the social investigation (Figure 15B,

bottom-right panel; Figure 16A). Similar to the putative DA neurons, the activity of non-DA

neurons did not change during the investigation of the object for both resilient and susceptible

mice (Figure 15B, bottom-left panel; Figure 16B).

Analysis of putative non-DA neurons population activity reveal that the averaged

firing rate decreased after the onset of the social investigation in the resilient mouse (in Hz:

12.4 ± 3.2 and 8.5 ± 2.1, for before and after the onset respectively; P < 0.05, paired Student’s

t-test; Figure 16C). In contrast, susceptible mouse showed increased firing rate after the onset

of social investigation (in Hz: 9.5 ± 1.93 3-sec period before onset, 15.4 ± 2.6 3-sec period

after onset; P < 0.05, paired Student’s t-test; Figure 16C).

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Figura 15 | Putative non-DA neuron activity during social investigation behavior in resilient and susceptible mice. (A) Firing maps showing illustrative examples of putative non-DA neuron in the resilient (top) and susceptible (bottom) mouse during the object (left) and social (Right) sessions of the social interaction test. The peak firing rate during the session is depicted on the top right corner of each example. The restraining cage is oriented to the top of the figure, in the same configuration as shown in the chapter 2. (B) Illustrative examples of putative non-DA neuron activity in the resilient (top panels) and susceptible (bottom panels) mouse during both object (left panels) and social (right panels) investigation. The illustrative waveforms are represented as superimposed individual spikes (gray traces); the ticker black line represents the averaged spike waveform. Scale bars, 0.5 mV, 1 s. Raster plots represent 300 s of continuous recording starting in the top left corner and ending in the bottom right for each panel.

In summary, our results present two neuronal classes of VTA population with opposite

patterns during spontaneous and voluntary social investigation in susceptible and resilient

mice. Although preliminary, our results provide insight into the role of VTA activity in

resilience and susceptibility after social defeat stress.

4.5. Discussion

In the present study, we recorded unitary activity from VTA of socially defeated mice

freely behaving while in a social interaction test. We found that putative DA neurons of the

resilient mouse markedly increased their firing rate during bouts of social investigation while

simultaneously recorded putative non-DA neurons showed decreased discharge rate.

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Figure 16 | Putative non-DA neuron activity is differentially modulated during social investigation behavior in resilient and susceptible mice. (A) Peri-event rasters and smoothed population average peri-event histograms (blue trace) of a putative non-DA neuron from the resilient mouse. The neuronal activity is referenced to the onset (left panels) and the offset (right panels) for object (top panels) and social investigation bouts. (B) Same description from A for the susceptible mouse. (C) Putative non-DA neurons showed opposite modulation in the susceptible (n = 4) and resilient (n = 4) mouse for social, but not object investigation.

In markedly contrast, in susceptible mouse, putative DA neurons showed decreased firing rate

while putative non-DA neurons increased it during the bouts of social investigation. Although

preliminary, these results suggest a correlation between social avoidance behavior and

impaired midbrain DA signaling in susceptible animals. As a consequence, we can speculate

that susceptibility to social stress reflects a failure of naturally positive valence signaling

associated to social stimulus. Below, we discuss our results in light of the consequences of

stress-induced plasticity in the mesolimbic dopamine system and the importance of real-time

monitoring midbrain dopaminergic dynamics in order to understand how pathological

behaviors emerge after social stress.

In rodents, dopamine signaling is believed to be important to naturally positive value

associated to social interactions (Gunaydin et al., 2014; Robinson et al., 2002, 2011). Studies

using fast-scan cyclic voltammetry revealed an increased DA release in the striatum during

bouts of social interaction of naïve C57BL/6J mice (Robinson et al., 2002, 2011). In

agreement, Gunaydin et al (2014) found an increase in the activity of VTA DA neurons

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during bouts of social interaction in freely behaving mice, mainly in those which projects to

the NAc. These authors also demonstrated that phasic optogenetic activation of VTA DA

neurons specifically projecting to the NAc elicit robust pro-social behavior, while the

continuous inhibition elicit social avoidance. Our results are in agreement with these data, as

the resilient animal showed social preference and increased activity of putative DA neurons

during social investigation (Figures 12 and 13). On the contrary, susceptible animal showed

opposite neuronal activity, which suggests that VTA plasticity took place during the repeated

social stress leading to valence changes of social stimuli. At the first sight, these results seems

contradictory with results showing that dopaminergic neurons had increased firing rate and

burst activity in ex vivo preparation and anesthetized mice after social defeat stress (Cao et al.,

2010; Krishnan et al., 2007; Razzoli et al., 2011). However, the reduced activity of DA

neurons in susceptible animals reported here occurred specifically during social investigation

behavior. Our hypothesis is that neuronal plasticity process induced by social defeat stress

increases the baseline excitability of VTA DA neurons as a compensatory response to

inhibition of these cells during social interactions. In fact, the global firing rate of DA neurons

in our data was increased in the susceptible mouse in comparison to the resilient one during

free exploration in the object session, corroborating the studies using in vitro and anesthetized

mouse preparations. Our results highlight the importance of studying VTA activity during

ecologically relevant behaviors.

4.5.1. Methodological caveats and study limitations

Conventional criteria used to identify DA neurons are based both on

electrophysiological features of the unit waveforms (e.g. action potential width, valley-to-

peak width) and the pharmacological response to D2 autoreceptors agonists (e.g.

apomorphine, dopamine). However, midbrain DA neurons are quite diverse and show a

substantial heterogeneity in their electrophysiological features (Lammel et al., 2008).

Additionally, at least the mPFC-projecting subpopulation of DA neurons in the VTA do not

express substantial levels of D2 receptors and are irresponsive to D2 receptor agonists induced

inhibition (Lammel et al., 2008; Margolis et al., 2008). Unfortunately, we could not say

anything regarding the projection of these neurons or it effects on target areas.

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4.5.2. Concluding remarks

In summary, we showed that, in resilient animals, putative DA neurons markedly

increased their firing rate during bouts of social investigation while the putative non-DA

neurons decreased it. The opposite changes were observed for the susceptible mouse, with

putative DA neurons showing decreased firing rate while putative non-DA neurons increasing

it during the bouts of social investigation. These results are in agreement with the role of DA

neurons signaling positive valence information to social stimuli, which could be affected in

susceptible animals after social defeat stress. Despite still inconclusive due to insufficient

number of observations, these results provide valuable insight into the dynamics of DA

signaling during social interaction after social defeat stress.

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5. DISCUSSÃO GERAL

Em 1915 Walter Cannon descreveu a resposta de “luta ou fuga” (Cannon, 1915). Duas

décadas depois Hans Selye descobriu o eixo HPA e descreveu sua importância como um

intrincado sistema de regulação fisiológica imprescindível à adaptação dos organismos

(Selye, 1936). Esses trabalhos influenciaram o surgimento de uma série de pesquisas médicas

que ainda hoje continuam crescendo. Hoje podemos dizer que temos uma vasta, porém ainda

incompleta, compreensão de como funcionam as repostas de estresse e das consequências de

sua desregulação para a integridade funcional do organismo (Szabo et al., 2012). Atualmente,

na pesquisa pré-clinica em especial, há grande interesse no aperfeiçoamento dos modelos

animais de estresse social. Esse interesse reside tanto no fato de que o estresse social é crucial

para o desenvolvimento de transtornos psiquiátricos como no reconhecimento de que esses

modelos possuem grande valor na pesquisa translacional. Nesta tese nossa intenção foi a de

contribuir para a compreensão dos efeitos do estresse social no desenvolvimento de

transtornos psiquiátricos utilizando o modelo animal de derrota social. Para isso usamos duas

principais abordagens. Primeiro, apresentamos um estudo com foco sobre o comportamento

animal. O capítulo 1 apresenta um artigo sobre as consequências do estresse de natureza

social para o comportamentos de interação social de camundongos (Mus musculus) da

linhagem isogênica C57BL6/j. Aqui, afirmamos a importância da duração dos ensaios

comportamentais e da quantificação de comportamentos relacionados à ecologia da espécie

estudada para uma caracterização fenotípica adequada.

Segundo, investigamos os padrões de ativação de diferentes populações neuronais da

área tegmentar ventral e sua relação com o comportamento de investigação social de

camundongos nos fenótipos resiliente e susceptível ao estresse social. No capítulo 2,

mostramos que neurônios dopaminérgicos e não-dopaminérgicos da área tegmentar ventral

apresentam padrões de ativação opostos em camundongos resilientes e suscetíveis ao estresse

de derrota social. Apesar de inconclusivos, devido ao número insuficiente de observações,

esses resultados fornecem noções sobre os efeitos do estresse social na dinâmica de

sinalização dopaminérgica durante interações sociais.

Talvez a principal característica associada à exposição crônica ao estresse social seja o

seu efeito complexo sobre a fisiologia e o comportamento de tomada de decisão do indivíduo

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diante de interações sociais (McEwen, 2012). Uma hipótese é que períodos prolongados de

estresse podem induzir formas aberrantes de neuroplasticidade em neurônios dopaminérgicos

da área tegmentar ventral (Hollon et al., 2015; Krishnan et al., 2007). Como é sabido que

esses neurônios desempenham funções importantes na motivação para interagir socialmente

(Gunaydin et al., 2014), alterações no seu funcionamento seriam a causa de sintomas como

aversão social. Entretanto, a literatura muitas vezes apresenta resultados divergentes.

Baseados em estudos com modelos animais de estresse, alguns trabalhos sugerem que o

aumento da atividade de neurônios dopaminérgicos da área tegmentar ventral está associado

ao desenvolvimento dos sintomas psiquiátricos (Cao et al., 2010; Chaudhury et al., 2013;

Friedman et al., 2014; Krishnan et al., 2007; Razzoli et al., 2011). Em contraste, outros

trabalhos propõem que a hipofunção dopaminérgica é causa do desenvolvimento dos sintomas

depressivos (Tye et al., 2013). Os nossos resultados apontam para a segunda hipótese.

É importante ressaltar que os comportamentos específicos analisados nesses trabalhos

podem estar relacionados à diferentes aspectos da sinalização dopaminérgica em processos

motivacionais (Salamone & Correa, 2012). Em um estudo, Chaudhury et al. 2013, usaram

técnicas de optogenética para demonstrar que a estimulação fásica de neurônios DA da VTA é

suficiente para induzir aversão social em camundongos submetidos ao estresse de derrota

sociais. Entretanto, com o protocolo de estimulação utilizado, os autores não controlaram o

aumento da atividade fásica de neurônios DA em relação ao comportamento de investigação

social, que é o principal parâmetro usado para inferir resiliência e susceptibilidade nos

camundongos (Chaudhury et al., 2013; File & Seth, 2003). Uma possibilidade é que o estresse

das derrotas sociais pode ter induzido aversão social inicialmente por ansiedade induzida por

novidade (i.e. neofobia). E assim, a estimulação fásica dos neurônios DA da VTA associada à

permanência inicial desses animais nos cantos da arena tenha induzido preferência pela

ocupação dos cantos da caixa (Tsai et al., 2009). Uma possibilidade é que a preferência pelos

cantos da caixa (devido ao valor de incentivo positivo associado ao aumento da estimulação

fásica nos neurônios DA da VTA) pode ter sido interpretada como susceptibilidade ao

estresse social.

Outro fator importante é a duração das sessões experimentais em testes para

classificação do fenótipo comportamental (Fonio et al., 2012b). Testes com durações

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adequadas são de fundamental importância para evitar fatores de confusão (Hager et al.,

2014). De acordo com Fonio et al. 2012a a ansiedade induzida por novidade é um fator

importante para a caracterização adequada do fenótipo comportamental associado a condições

patológicas crônicas. Nossos resultados comportamentais, apresentados no capítulo dois da

tese, sugerem que sessões de longa duração (>150 segundos) são importantes para a

classificação adequada do fenótipo comportamental após o estresse de derrotas sociais.

Sessões de curta duração (< 150 segundos), como as utilizadas em estudos que se propõem a

investigar a função de neurônios dopaminérgicos sobre comportamentos sociais motivados

(Berton et al., 2006; Chaudhury et al., 2013; Krishnan et al., 2007; Venzala et al., 2012),

podem induzir ao erro na classificação do fenótipo comportamental, já que a aversão social

demonstrada pelos animais pode estar relacionada tanto à ansiedade como à depressão (Toth

and Neumann, 2013).

Para superar essas limitações, o estudo de comportamentos de relevância ecológica

podem ser importantes para a identificação de fenótipos patológicos (Blanchard and

Blanchard, 1988; Chaouloff, 2013; Koolhaas et al., 2006; Sorregotti et al., 2013). Em nossos

resultados, observamos que um subgrupo de camundongos derrotados muda sua estratégia

comportamental de enfrentamento do estímulo estressor durante sessões mais longas de

interação social. Esse resultado foi evidenciado pelo aumento tardio no comportamento de

investigação social e também na redução das posições estendidas durante a investigação

social. Diferenças nas estratégias comportamentais frente ao estímulo estressor também foram

observadas nos comportamentos de fuga. Camundongos que sofreram derrotas sociais

demonstraram mais alta variabilidade interindividual no padrão de fugas exibidas ao longo da

sessão social. Alguns indivíduos demonstraram comportamentos de fuga apenas no início das

sessões de interação social, mudando seu repertório comportamental para investigações

sociais mais. Outro animais exibiram padrão sustentado ou de ocorrência tardia de fugas, não

demonstrando habituação. Esses animais apresentaram baixo consumo de sacarose, sugerindo

que os padrões sustentado e tardio de fugas estão relacionados com o fenótipo depressivo.

Nossos resultados preliminares apresentados no capítulo 2 sugerem a possibilidade de que a

flexibilidade comportamental esteja relacionada à características funcionais em neurônios da

VTA. Assim, a redução na taxa de disparo neuronal, ocorrida especificamente durante os

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comportamentos de investigação social, seria insuficiente para manter o valor de incentivo

associado à interação social. A observação etológica pode ser interessante dado a

possibilidade de analisar a transição comportamental associada a mudanças na atividade dos

neurônios dopaminérgicos da VTA. A observação dessas transições pode se dar em escala

temporal mais curta, por exemplo, durante a redução da distância de investigação (i.e.

transição do comportamento de investigação social realizado em postura estendida para

investigação social relacionada a um contexto afiliativo). Em contrapartida, em escala

temporal mais longa, a atividade neuronal pode ser analisada na transição dos períodos em

que os animais fogem em comparação com os períodos que cessam a ocorrência desse

comportamento.

Por fim, devemos perceber que interações sociais são complexas do ponto de vista dos

processos motivacionais que envolvem. Assim, as abordagens etológicas associadas à

quantificação momento-a-momento do comportamento podem ser de grande valia para a

compreensão das consequências neurobiológicas do estresse de natureza social.

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6. CONCLUSÕES

A partir dos resultados do primeiro estudo (Capítulo 1), concluímos que a

classificação do fenótipo comportamental de camundongos submetidos ao estresse de derrota

social pode ser inapropriada quando baseada esclusivamente na ocupação da zona de

interação e em testes comportamentais de curta duração. Sugerimos que a partir de

abordagens etológicas associadas à sessões de longa duração nos testes de interação social

essas limitações podem ser superadas. Nesse estudo, introduzimos dois índices baseados em

comportamentos de defesa de camundongos. Um índice de aproximação, baseado na distância

de investigação social, foi sugerido como variável informativa para comportamentos

relacionados à ansiedade. Outro índice, o índice de fuga, baseado no padrão de expressão dos

comportamentos de fuga ao longo das sessões de interação social, foi proposto para identificar

o fenótipo relacionado à depressão. Entretanto, estudos de validação farmacológica são

necessários para demonstrar a relevância desses índices para o estudo dos transtornos

psiquiátricos relacionados ao estresse.

No segundo estudo (capítulo 2), foram apresentados resultados sugerindo que

neurônios dopaminérgicos e não-dopaminérgicos da área tegmentar ventral apresentam

padrões de ativação opostos em camundongos resilientes e suscetíveis ao estresse de derrota

social. Apesar de inconclusivos, devido ao número insuficiente de observações, esses

resultados fornecem noções sobre os efeitos do estresse social na dinâmica de sinalização

dopaminérgica durante interações sociais.

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