( PDF ) Estudo dos efeitos comportamentais e neuroquímicos da cetamina em ratos submetidos à modelos animais de depressão

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UNIVERSIDADE DO EXTREMO SUL CATARINENSE – UNESC

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

LÊDA SOARES BRANDÃO GARCIA

ESTUDO DOS EFEITOS COMPORTAMENTAIS E NEUROQUÍMICOS

DA CETAMINA EM RATOS SUBMETIDOS A MODELOS ANIMAIS DE

DEPRESSÃO

CRICIÚMA, JULHO/2008

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LÊDA SOARES BRANDÃO GARCIA

ESTUDO DOS EFEITOS COMPORTAMENTAIS E NEUROQUÍMICOS

DA CETAMINA EM RATOS SUBMETIDOS A MODELOS ANIMAIS DE

DEPRESSÃO

Dissertação de Mestrado apresentada ao

Programa de Pós-Graduação em Ciências da

Saúde para obtenção do

título de Mestre em Ciências da Saúde.

Orientador: Prof. Dr. João Luciano de Quevedo

Co-orientadora: Profa. Dra. Elaine Cristina Gavioli

CRICIÚMA, JULHO DE 2008.

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AGRADECIMENTOS

Para os que fazem o meu entorno, coadjuvantes dos meus resultados:

São eles:

-a família original: mãe, irmãos; pai (in memoriam);

-a família que ajudei a construir: Dario e Fábio;

-os poucos, mas qualificados amigos;

-meus pacientes, permanente foco de atenção;

-meus alunos, ligação mais recente, porém estimulante;

agradecimentos especiais

-ao meu orientador, o jovem e capaz Professor João Quevedo, mentor intelectual

deste feito;

-à minha co-orientadora, Professora Elaine Gaviolli, bela criatura e competente

profissional;

-à Professora Alexandra Zugno, paciente revisora dos meus escritos;

-aos professores do curso, com quem pude interagir:

Álvaro José Back

Carina Rodrigues Boeck

Flávio Merino de Freitas Xavier

Keila Maria Ceresér

Paulo Rômulo de Oliveira Frota

Pedro Roosevelt Torres Romão

Vanessa Morais de Andrade;

-aos colegas do Neurolab, especialmente Clarissa Comim, promissora pesquisadora.

Lêda Soares Brandão Garcia

CRICIÚMA, JULHO/2008.

RESUMO

A depressão, por sua alta prevalência (16,5% ao longo da vida), cursa elevando

taxas de morbidade e de mortalidade, sendo considerada atualmente um problema

de saúde pública. Os tratamentos farmacológicos disponíveis são baseados em

estratégias dependentes do sistema monoaminérgico e apresentam algumas

limitações. Algumas evidências clínicas apontam para o bloqueio dos receptores

NMDA como potencial alvo terapêutico para o tratamento da depressão. Este

trabalho estabeleceu como objetivo avaliar experimentalmente o efeito

antidepressivo da cetamina, um antagonista não competitivo para o glutamato, que

se liga ao sítio da fenciclidina e que, conforme alguns achados clínicos, demonstrou

rápido início de ação para efeitos antidepressivos. Foram realizados três

experimentos: (1) ratos receberam tratamento agudo com cetamina e imipramina e o

comportamento dos animais foi avaliado nos testes do nado forçado e no campo

aberto. Cetamina e imipramina reduziram significativamente o tempo de imobilidade,

não alterando atividade locomotora; (2) utilizando um protocolo semelhante, ratos

receberam tratamento crônico com cetamina e imipramina o que resultou, em todas

as doses testadas, na diminuição do tempo de imobilidade, sem alteração da

atividade locomotora; (3) neste experimento, ratos submetidos a estresse crônico

moderado, apresentaram redução da ingestão de alimentos doces, redução do peso

corporal, aumento do peso da adrenal e dos níveis de cortisol e do hormônio

corticotrófico. A atividade locomotora não foi significativamente afetada. O

tratamento agudo com cetamina foi capaz de reverter a redução do peso corporal, o

aumento do peso da adrenal e o aumento dos níveis do cortisol e do ACTH, mas

não alterou a redução da ingestão de alimentos doces, quando comparado ao grupo

salina; o tratamento crônico com cetamina reverteu todos os parâmetros avaliados.

Conclui-se que, em consonância com dados da literatura, a cetamina apresentou

efeitos do tipo antidepressivo nos testes comportamentais realizados, estimulando

novas e mais esclarecedoras pesquisas sobre seus mecanismos.

Palavras chaves: glutamato; cetamina; receptores N-metil-D-aspartato; teste do

nado forçado; estresse crônico moderado.

ABSTRACT

Depression, a prevalent mood disorder (16,2% lifetime), increases mortality and

morbidity and has been considered a public health problem. Available treatments are

based in monoaminergic system and have several limitations. Growing body of

evidence has pointed to the blochade of N-metyl-D-aspartate (NMDA) receptors

signaling as a potencial therapeutic target for the treatment of major depression. The

present study aimed to evaluate the effects of ketamine, a non-competitive

antagonist to the phencyclidine site of NMDA receptors which some clinical findings

point to a rapid onset of action for antidepressant effects. Three experiments took

place: (1) rats were acutely treated with ketamine and imipramine and animal

behavioral was assessed in the forced swimming and open-field tests. Ketamine and

imipramine reduced time immobility and did not affect locomotor activity; (2) in a

same protocol, rats received chronic treatment and ketamine and imipramine, in all

doses tested, reduced time immobility and did not affect locomotor activity; (3) rats

were exposed to chronic mild stress procedure and decreased sweet intake and

body weight and increased adrenal gland weight and cortisol and

adrenocorticotrophin levels. Locomotor activity was not affected. Acute ketamine

treatment reverted body weight loss and the increase of adrenal weight and the

increase of cortisol and adrenocorticotrophin levels. Acute treatment did not

decrease sweet food intake, compared to saline. Chronic ketamine treatment

reverted all parameters evaluated. In conclusion, and according literature date,

ketamine showed antidepressant-like effects in behavioral tests and further studies

are necessary to investigate its mechanisms.

Keywords: glutamate; ketamine; N-metyl-D-receptors; forced swimming test; chronic

mild stress.

LISTA DE ABREVIATURAS

ACTH - (adrenocorticotropic hormone) - hormônio adrenocorticotrófico

BDNF - (brain derived neurothrophic factor) - fator neurotrófico derivado do

cérebro

CMS - (chronic mild stress) - estresse crônico moderado

CRH - (corticotropin-releasing hormone) - hormônio liberador de corticotrofina

ECT - eletroconvulsoterapia

FST - (forced swimming test) - teste do nado forçado

HPA - (hypothalamic-pituitary-adrenal) - hipotálamo-hipófise-adrenal

IMAO - inibidores da monoamina-oxidase

ISRS - inibidores seletivos da recaptação de serotonina

ISRSN - inibidores seletivos da recaptação de serotonina e de noradrenalina

NGF - (neural growing factor) - fator de crescimento neural

NMDA - (N-methyl-D-aspartate) - N-metil-D-aspartato

PCP - (phenciclidine) - fenciclidina

LISTA DE ILUSTRAÇÕES

Figura 1...........................................................................................................10

SUMÁRIO

1 INTRODUÇÃO................................................................................................1

1.1 Depressão e Estratégias de Tratamento.........................................................2

1.2 Fatores Neurotróficos e Depressão.................................................................5

1.3 Glutamato e Receptores NMDA......................................................................9

1.4 Cetamina........................................................................................................13

1.5 Modelos Animais de Depressão.....................................................................14

1.5.1 Teste do Nado Forçado.................................................................................15

1.5.2 Estresse Crônico Moderado..........................................................................16

2 OBJETIVOS..................................................................................................17

2.1 Objetivo geral.................................................................................................17

2.2 Objetivos Específicos.....................................................................................18

3 ARTIGOS.......................................................................................................19

4 DISCUSSÃO...................................................................................................20

5 REFERÊNCIAS...............................................................................................28

1- INTRODUÇÃO

A necessidade de um melhor entendimento acerca dos mecanismos

fisiopatológicos responsáveis pela depressão tem instigado a pesquisa sobre o tema

uma vez que, até o atual estágio do conhecimento, sequer foi possível definir se as

diferenças observadas nas manifestações clínicas da depressão constituem

diferentes patologias ou variáveis de uma única entidade mórbida (Fleck & Shansis,

2004). Assim, na ausência de evidências, o planejamento de abordagens de

tratamento para a depressão termina por ser estabelecido de modo arbitrário com

enfoque, ora direcionado para os seus aspectos psicossociais, ora, priorizando a

abordagem biológica. Para essa última, houve considerável avanço a partir do

estabelecimento da hipótese monoaminérgica, que associou depressão com

deficiência funcional de catecolaminas (Goodwin & Jamison, 1990). Desde então,

utilizam-se fármacos de ação monoaminérgica como importante ferramenta para o

tratamento da depressão com resultados positivos para grande número de pacientes

portadores do transtorno. Há, entretanto um período de espera, de 2 até 6 semanas

a partir do início do tratamento, para que esses pacientes apresentem sinais clínicos

de melhora (Janicak et al.,1996; Sadock & Sadock, 2005). Além disso, alguns

pacientes não conseguem tolerar os efeitos colaterais desses fármacos enquanto

outros, 10 a 20% em Zarate et al., (2002) e 19 a 34% em Licínio & Wong (2007)

simplesmente não apresentam resposta efetiva às múltiplas tentativas de

tratamento. Tais limitações ao tratamento impõem a busca de novas estratégias

terapêuticas, havendo desde linhas de pesquisas de abordagem não farmacológica,

como a estimulação magnética (Licínio & Wong, 2007), passando por estratégias

neuropeptidérgicas, quais sejam, o bloqueio do hormônio liberador de corticotrofina

(CRH) e o uso de antagonistas da substância P, até as linhas que buscam novos

alvos de ação das drogas, como as pesquisas que envolvem o sistema canabinóide

e o sistema glutamatérgico (Licínio & Wong, 2007). Em relação a esse último,

algumas evidências apontam para o papel dos receptores do tipo N-metil-D-

aspartato (NMDA) na fisiopatologia da depressão e no mecanismo de ação de

tratamentos antidepressivos (Berman et al.,2000; Zarate et al., 2006c). A relevância

do tema motivou o presente trabalho, que estabeleceu como objetivo o estudo dos

efeitos antidepressivos da cetamina em testes comportamentais e a avaliação de

alterações de neurotrofinas em tecido cerebral.

1.1 – Depressão e Estratégias de Tratamento

O Transtorno Depressivo é atualmente considerado um problema de saúde

pública por sua elevada prevalência e curso recorrente, por prejudicar os

desempenhos profissionais e sociais do seu portador, estando ainda associado com

morbidade médica e redução de expectativa de vida (Thase, 2007). Tem sido ainda

responsável por considerável ônus sócio-econômico uma vez que, além dos custos

diretos representados por tratamentos, há que se considerar o prejuízo pela perda

de dias trabalhados (Stahl, 2006). Dados recentes e representativos sobre a

população portadora de transtornos do humor veem do National Comorbidity Survey

Replication (NCS-R) que apresenta estimativas de prevalência para a depressão

maior, na população geral, na ordem de 16,5% ao longo da vida e 6,6% nos últimos

12 meses (Kessler et al., 2007).

A depressão frequentemente se manifesta com ampla variedade de sintomas:

emocionais (vazio e desespero), motivacionais (prejuízo de estímulo e iniciativa),

comportamentais (comprometimento de atividades usuais) e cognitivos (limitações

de concentração e de memória), além de alterações de apetite, sono, atividade

sexual e, não raro, quadros dolorosos e outras somatizações (Kolb, 2002). O

transtorno tem incidência maior no sexo feminino, estimando-se que 64% das

pessoas afetadas se recuperem em seis meses, algumas delas inclusive sem

tratamento (Kolb,2002), havendo, em contrapartida, os indivíduos afetados que,

mesmo sob tratamento adequado, não apresentam melhora do quadro (Zarate et al.,

2002; Licínio & Wong, 2007).

O largo espectro dos sintomas que caracterizam a depressão, afetando

desempenhos pessoais do seu portador, impondo uma infinidade de sintomas e

alterações comportamentais, representa um obstáculo a ser enfrentado pelos

investigadores que trabalham com o paradigma animal, não conseguindo reproduzir

em laboratório o complexo quadro clínico da depressão, ficando assim limitados à

reprodução e observação de aspectos comportamentais mensuráveis (Petit-

Demouliere et al., 2005).

Os fármacos de ação antidepressiva têm representado importantes

ferramentas do arsenal terapêutico para enfrentamento da depressão desde que, na

década de cinqüenta, foi observado que a reserpina, usada no tratamento de

hipertensão arterial, tendia a provocar sintomas depressivos (Iversen, 2007).

Pesquisas, então realizadas, demonstraram que tais sintomas estavam associados à

depleção dos estoques cerebrais de monoaminas, sendo posteriormente observado

a possibilidade de reversão dos sintomas depressivos pela administração de L-

DOPA, um precursor de catecolamina, estabelecendo-se então a associação entre

monoaminas cerebrais e depressão (Iversen, 2007). Seguiu-se a descoberta da

ação inibidora da monoaminaoxidase (MAO), enzima envolvida na degradação de

monoaminas no cérebro, apresentada pela isoniazida, substância usada para

tratamento da tuberculose e que mostrava peculiar efeito na elevação do humor

(Iversen, 2007). Mais adiante, a evidência de que medicamentos tricíclicos

(imipramina e amitriptilina) inibiam a captação de noradrenalina e de serotonina

reforçou o conceito de depressão como estado resultante da deficiência de

monoaminas (Iversen, 2007). Posteriormente foram desenvolvidos fármacos de ação

inibidora seletiva da recaptação de serotonina e os inibidores seletivos da

recaptação de noradrenalina que, possuindo ação mais seletiva e potente de

recaptação de serotonina e de noradrenalina respectivamente, mostraram melhor

perfil de tolerância em relação aos seus antecessores (Stahl, 2006). Persistem,

entretanto, os desafios representados pela demora na obtenção dos efeitos

terapêuticos (Mathew et al., 2008) e pela ausência de eficácia para considerável

número de pacientes Zarate et al., (2002).

Outra linha de pesquisa focada no desenvolvimento de estratégias

terapêuticas para a depressão surgiu a partir de evidências da relação existente

entre esse transtorno e perturbações do eixo hipotálamo-hipófise-adrenal (HPA)

(Licínio & Wong, 2007). Em testes pré-clínicos foi observado que o bloqueio dos

efeitos centrais do CRH, através do uso de substâncias antagonistas, apresentou

efeitos sugestivos de atividade ansiolítica e antidepressiva (Licínio & Wong, 2007).

O papel do estresse na etiopatogenia da depressão também tem sido objeto

de estudos na última década, associando o transtorno aos efeitos deletérios do

estresse na neuroplasticidade (Arantes-Gonçalves & Coelho, 2006). Por

neuroplasticidade entende-se a capacidade apresentada pela célula neuronal de se

transformar, adaptando sua estrutura e função em resposta a exigências internas do

organismo ou a demandas ambientais, podendo ocorrer tais transformações mesmo

em células maduras (Arteni & Alexandre Neto, 2004). Foi observado que os efeitos

deletérios do estresse poderiam ser revertidos pela ação de antidepressivos que, em

uso crônico, influenciam dois aspectos da neuroplasticidade: proliferação e

sobrevivência neuronal (Arantes-Gonçalves & Coelho, 2006).

1.2 - Fatores Neurotróficos e Depressão

Por fatores neurotróficos entendem-se, em última análise, as proteínas

reguladoras da formação de redes neuronais, destacando-se aqui a família das

neurotrofinas composta por quatro elementos: fator de crescimento neural (NGF),

fator de crescimento derivado do cérebro (BDNF), neurotrofina-3 e neurotrofina-4

(Castrén et al., 2007). Tais proteínas agem como mediadores de diferenciação

celular e modulam plasticidade e transmissão sináptica (Shatiel et al., 2007). Têm

origem endógena e lhes tem sido atribuído papel importante na sobrevivência,

manutenção e crescimento de neurônios centrais e periféricos (Kim et al, 2007). O

BDNF é o membro da família com maior expressão cerebral, sendo encontrado não

só no período de desenvolvimento, mas também no cérebro adulto, onde tem função

crítica na plasticidade sináptica e na formação de memórias (Gronli et al., 2006;

Bekinschtein et al., 2008). Estudos experimentais têm demonstrado resultados

consistentes com a hipótese que a menor expressão de BDNF, e possivelmente de

outros fatores de crescimento, seja fator associado à depressão e que, o aumento

dessa neurotrofina desempenha papel na ação de antidepressivos (Duman &

Monteggia, 2006). A diminuição dos níveis de BDNF observada experimentalmente,

na exposição ao estresse, pode ser considerada um indício do envolvimento dessa

neurotrofina na fisiopatologia dos transtornos de humor associados ao estresse

(Shirayama et al. 2002). A noção acerca do efeito deletério do estresse sobre o

sistema nervoso é anterior à descoberta das neurotrofinas e surgiu a partir de

estudos que demonstraram associação entre estresse e atrofia neuronal em

algumas estruturas cerebrais, notadamente no hipocampo (Duman & Monteggia,

2006), estrutura que tem sido implicada no desenvolvimento de transtornos de

humor uma vez que circuitos hipocampais controlam aprendizagem e memória, além

de exercer regulação do eixo HPA e manter conexões com amígdala e córtex pré-

frontal, regiões diretamente envolvidas com emoção e cognição, funções que estão

alteradas na depressão (Duman & Monteggia, 2006). Estudos experimentais têm

demonstrado aumento na concentração do BDNF a partir do uso de antidepressivos

inibidores da monoamina-oxidase (IMAO) e de eletroconvulsoterapia (ECT) apesar

de algumas classes de antidepressivos (os inibidores seletivos da recaptação de

serotonina e os inibidores seletivos da recaptação de serotonina e de noradrenalina)

terem mostrado resultados contraditórios (Duman & Monteggia, 2006). Algumas

evidências do papel das neurotrofinas na depressão, sobretudo naquela associada

ao estresse, podem ser consideradas:

1- O estresse crônico moderado está associado à menor expressão do BDNF no

hipocampo de ratos, comparados com controles não submetidos ao estresse (Gronli

et al., 2006);

2- Em dois modelos animais que avaliam o efeito antidepressivo de fármacos (teste

do nado forçado e paradigma do desamparo aprendido), a administração de BDNF

no hipocampo e no mesencéfalo mostrou resultado comparável ao efeito obtido com

tratamento crônico com antidepressivo. Os camundongos transgênicos que

expressam reduzidos níveis de BDNF no cérebro exibiram reduzida resposta a

antidepressivos em testes comportamentais (Shirayama et al., 2002; Saarelainen et

al., 2003; Sairanen et al.,2005). A infusão crônica de BDNF no mesencéfalo ou a

infusão aguda no giro denteado ou na área CA3 do hipocampo de roedores mostrou

melhorar o desempenho desses animais em testes comportamentais usados para

avaliar efeitos antidepressivos de fármacos (Shaltiel et al, 2007).

3- Tratamentos crônicos, mas não agudos, com antidepressivos elevam níveis do

RNAm que expressa o BDNF no hipocampo e córtex, enquanto

eletroconvulsoterapia (ECT) - o mais eficiente tratamento antidepressivo (UK ECT

Review Group, 2003), mesmo em tratamento agudo aumenta o referido RNAm

(Saaralainen et al., 2003; Mathew et al., 2008).

4- Níveis extra-celulares mais elevados de serotonina e norepinefrina seriam

necessários para ocorrência da proliferação de novas células na neurogênese

hipocampal, enquanto a sobrevivência desses novos neurônios estaria relacionada

com a sinalização do BDNF. Este processo levaria um tempo equivalente àquele

necessário para que tratamentos com antidepressivos iniciem os efeitos clínicos

esperados (Sairanen et al., 2005).

5- Em humanos, estudos de imagem têm demonstrado redução em córtex pré-

frontal, estriado e hipocampo, além de aumento ventricular em portadores de

transtornos do humor comparados a controles sadios (Shaltiel et al., 2007). Pelo

menos parte desse efeito pode ser revertido por fármacos antidepressivos (Fuchs et

al., 2004; Castrén et al., 2007).

6- Os níveis de BDNF se mostraram reduzidos no soro e no plasma de pacientes

deprimidos não tratados, comparados com controles saudáveis (Karege et al., 2003).

Outros estudos encontraram níveis de BDNF mais baixos no plasma de indivíduos

deprimidos que haviam apresentado recente tentativa de suicídio (Lee et al, 2006;

Kim et al., 2006). Também foram encontrados níveis mais baixos de BDNF no

sangue total de mulheres geneticamente predisposta e submetidas a recente evento

estressante (Trajkovska et al., 2008).

O efeito primário das neurotrofinas na sinalização celular seria a ativação de

um receptor tirosina-cinase da família Trk (Arteni & Alexandre Neto, 2004). Assim, o

conjunto de moléculas estabeleceria uma cascata na qual, a partir do momento em

que cada um dos fatores aumenta sua expressão, passaria a fortalecer a expressão

do fator subseqüente. A cascata seria a seguinte: AMPc – MAPcinase – CREB –

BDNF, e teria papel unificador em mecanismos como a reestruturação dendrítica,

aumento da neurogênese hipocampal e aumento da sobrevivência das células do

sistema nervoso central (Arantes-Gonçalves & Coelho, 2005). Entre os elementos da

cascata, o BDNF é considerado o que tem efeito antidepressivo mais potente

(Arantes-Gonçalves & Coelho, 2005), mas, todas essas moléculas já foram

implicadas na fisiopatologia da depressão havendo evidências de que o aumento da

expressão das mesmas seja necessário para a ação terapêutica dos

antidepressivos, o que torna as referidas moléculas alvo terapêutico de potencial

interesse na síntese de novos fármacos antidepressivos (Shaltier et al., 2007).

A pesquisa atual tem também associado a depressão a alterações

estruturais, relacionando o transtorno com deficiências na neuroplasticidade e na

resiliência celular cerebrais, condições que foram observadas em pacientes com

transtorno de humor severo ou recorrente e que, conforme sugerido por recentes

evidências, poderiam estar associadas a alterações na atividade do sistema

glutamatérgico (Maeng & Zarate, 2007).

1.3 – Glutamato e Receptores NMDA

O glutamato é o principal neurotransmissor excitatório do sistema

nervoso de vertebrados e tem ações na excitabilidade e na plasticidade sináptica em

muitos circuitos cerebrais, incluindo vias límbicas envolvidas na depressão (Witkin et

al., 2007). Pode também ter ação neurotóxica, estando assim implicado em

patologias neurológicas de natureza aguda, como isquemia cerebral e epilepsia, ou

processos neuropsiquiátricos degenerativos, de evolução crônica, como esclerose

lateral amiotrófica, coréia de Huntington, mal de Parkinson e doença de Alzheimer

(Meldrum, 2000; Zarate et al., 2002).

A excitotoxicidade é a principal hipótese atual para explicar o mecanismo

neuropatológico que mediaria a via final comum de vários transtornos

neuropsiquiátricos de curso degenerativo (Stahl, 2006). Sob tal circunstância, o

processo normal de neurotransmissão excitatória passaria a ocorrer de modo

desenfreado levando à morte neuronal, o que se daria através da abertura excessiva

dos canais de cálcio e o conseqüente aumento da concentração do cálcio

intracelular, condição que altera a excitabilidade da membrana e promove ativação

excessiva de enzimas intracelulares capazes de disparar uma cascata química

destrutiva (Stahl, 2006).

O interesse pelo papel do sistema glutamatérgico no mecanismo

fisiopatológico dos transtornos de humor cresceu a partir de resultados de estudos

de neuroimagem realizados em indivíduos com transtorno bipolar e com depressão

maior. Tais estudos revelaram alterações morfométricas sugestivas de perda ou de

atrofia celular e assim estimularam pesquisas envolvendo vias implicadas na

neurodegeneração (Zarate et al., 2002).

Para exercer sua ação, em níveis pré e pós-sináptico, o glutamato depende

de receptores específicos que podem ser classificados, por suas características

estruturais, em ionotrópicos e metabotrópicos. Estes últimos, ligados à Proteína-G,

exercem sua ação via segundo mensageiro; os receptores ionotrópicos apresentam

três diferentes subgrupos: NMDA (N-metil-D-aspartato), AMPA (ácido alfa-amino-3-

hidroxi-5-metil-isoxazol-4-propiônico) e KA (cainato) (Maeng & Zarate, 2007). Os

receptores AMPA são responsáveis pela imediata resposta ao glutamato que chega

à fenda sináptica a partir da sua liberação do neurônio pré-sináptico. Tal resposta

consiste na despolarização da membrana neuronal, permitindo a entrada do sódio

na célula, seguida da liberação do magnésio existente no canal do receptor NMDA o

que, em última análise, permite a entrada do cálcio na célula (Pliszka, 2004).

Fig 1: Visão esquemática dos quatro tipos de receptores para o glutamato (Dingledine & McBain,

1999)

Algumas condições (como anoxia e hipoglicemia) perturbam o mecanismo

celular que mantém o glutamato em seu compartimento pré-sináp tico ocorrendo

Algumas condições (como anoxia e hipoglicemia) perturbam o mecanismo celular

que mantém o glutamato em seu compartimento pré-sináptico ocorrendo assim uma

liberação maciça desse neurotransmissor e a seqüência de eventos iniciados nos

receptores AMPA e NMDA e, finalmente, nos receptores metabotrópicos que

exercem sua ação via segundo mensageiro (Zarate et al., 2002). Em estágios

precoces do desenvolvimento, as sinapses demonstram predomínio dos receptores

NMDA, enquanto, em adultos, cerca de 70% das sinapses apresentam tais

receptores em coexistência com receptores AMPA, de cuja ação dependem para

exercer seu funcionamento (Zarate et al, 2002).

Os receptores NMDA são capazes de mediar a neurotransmissão de modo

lento e esta possibilidade de se estender por período prolongado lhes confere

importante papel no desenvolvimento de sinapses e na plasticidade (Sadock &

Sadock, 2005). Na composição do seu canal são conhecidas três subunidades:

NR1, NR2 (NR2A a NR2D) e NR3 (NR3A e NR3B) (Maeng & Zarate, 2007). Os

sítios de ligação para o glutamato foram encontrados na subunidade NR2 enquanto

na subunidade NR1 existem os sítios de ligação para o coagonista glicina. No

interior do canal foram identificados dois sítios: o sítio “s” e o sítio da fenciclidina

(PCP) (Maeng &Zarate, 2007).

Substâncias antagonistas de receptores NMDA têm mostrado ser eficazes

nos modelos que predizem a atividade de antidepressivos (Berman et al, 2000). Por

sua vez, tem sido observado que a administração crônica de antidepressivos é

capaz de afetar esses receptores, que mostraram adaptações em 16, num total de

17 tratamentos com antidepressivos testados (Berman et al, 2000). Tais adaptações

seriam assim importante preditor de atividade antidepressiva (Berman et al., 2000).

O mecanismo da transcrição para esse fenômeno é sugerido pela evidência de que

a administração repetida de antidepressivo regionalmente altera a expressão de

RNAm que codifica múltiplas subunidades de receptores NMDA (Berman et al.,

2000). Estudos preliminares feitos com baixas doses de amantadina, um antagonista

NMDA de baixa potência, sugeriram a redução de sintomas depressivos (Berman et

al., 2000) enquanto observações mais recentes com a memantina, outro antagonista

de receptores NMDA, não constataram eficácia antidepressiva da substância (Zarate

et al., 2006a). A memantina é substância utilizada no tratamento de patologias

neurológicas de natureza degenerativa, como a doença de Alzheimer, onde

considera-se que seus efeitos benéficos na cognição ocorram através da redução na

excessiva atividade dos receptores NMDA, contribuindo assim no reajustamento do

equilíbrio entre inibição e excitação (Johnson & Kotermanski, 2007). Mecanismo

semelhante é encontrado em uma outra substância antagonista de receptores

NMDA, a cetamina, utilizada como anestésico. Apesar da similaridade no

mecanismo de ação, essas duas substâncias apresentam importantes diferenças

clínicas sendo a memantina melhor tolerada. A diversidade talvez decorra de

diferenças farmacocinéticas (a memantina apresenta eliminação mais lenta) ou por

provocarem diferentes alterações químicas nos receptores que inibem, ou ainda, por

sua diferente maneira de se ligarem aos receptores: a memantina liga-se a sítios

superficiais e profundos enquanto a cetamina liga-se ao sítio da fenciclidina

(Johnson & Kotermanski, 2007).

Para Witkin et al., (2007) os dados disponíveis na literatura sugerem que, no

transtorno depressivo, a neurotransmissão excitatória/inibitória se mostre

desregulada o que, por conseqüência, confere às substâncias com ação na

ativação, potenciação ou bloqueio de receptores glutamatérgicos um possível papel

inovador no tratamento dos transtornos do humor.

1.4 – Cetamina

A escolha do cloridrato de cetamina para o presente estudo foi feita em

decorrência de ser a substância um antagonista potente de receptores NMDA

(Berman et al., 2000). A cetamina é usada como anestésico, sendo útil em

procedimentos de pediatria e de pacientes com risco de hipertensão e

broncoespasmo. Tem efeitos colaterais que limitam seu uso rotineiro. Rapidamente

produz estado hipnótico distinto daquele produzido por outros anestésicos (Ferreira,

2004). Pacientes têm profunda analgesia, não respondem a comandos, têm

amnésia, mas podem permanecer de olhos abertos, movimentar os membros

involuntariamente e, frequentemente, ter respiração espontânea. Esse estado

cataléptico tem sido chamado de anestesia dissociativa. A substância é geralmente

administrada por via endovenosa, mas pode ser usada por via intramuscular, oral e

retal. Pode aumentar o fluxo sanguíneo cerebral e a pressão intracraniana. É

possível a ocorrência de delirium com alucinações, ilusões e sonhos vívidos, o que

pode representar uma complicação de difícil manejo num pós-operatório (Ferreira,

2004). Algumas pessoas fazem uso indevido da cetamina, como droga recreativa, às

vezes chamada “special K” (Stahl, 2000).

O mecanismo primário de ação da cetamina dá-se pelo bloqueio dos

receptores NMDA por ligação ao sítio da fenciclidina (PCP), localizado no interior do

canal iônico (Maeng & Zarate, 2007). Isto vale dizer que há inibição da transmissão

excitatória por antagonismo glutamatérgico, resultando em propriedades

farmacológicas responsáveis por sedação, hipnose e analgesia (Ferreira, 2004). A

cetamina tem apresentado efeito antidepressivo mais significativo, quando

comparada com a memantina (Maeng & Zarate, 2007), substância também

antagonista de receptores NMDA e melhor tolerada clinicamente, possivelmente por

apresentar maior afinidade pelo receptor, além de ter ação mais lenta no

bloqueio/desbloqueio do canal (Maeng & Zarate, 2007). A cetamina também interage

com receptores opióides, com receptores monoaminérgicos e muscarínicos e com

canais de cálcio sensíveis à voltagem. De modo diferente em relação a outras

substâncias anestésicas, a cetamina não interage com receptores GABA (Hirota &

Lambert, 1996).

Para a finalidade a que se propôs esse estudo, o cloridrato de cetamina foi

usado em tratamento agudo e crônico, sendo realizados dois testes

comportamentais avaliando-se tempo de imobilidade no nado forçado e atividade

locomotora e, posteriormente, dosado o BDNF hipocampal em ratos.

1.5 – Modelos Animais de Depressão

“Modelo animal de transtorno psiquiátrico pode ser definido operacionalmente

como estados comportamentais incomuns em animais que são especificamente

revertidos pelo mesmo tratamento farmacológico que reverte os sintomas do

transtorno humano“ (Petty & Sherman, 1990).

Os chamados modelos animais de depressão, embora carecendo da

condição humana, são considerados válidos a partir do preenchimento de alguns

critérios que devem se aproximar de condições humanas: (a) semelhança no

sintoma apresentado (validade de face); (b) apresentar melhora com tratamentos

eficazes em humanos e não apresentar resposta sob tratamentos ineficazes em

humanos (validade preditiva); (c) a condição deve ser provocada por eventos

capazes de provocar a condição em humanos (validade etiológica) e (d)

envolvimento de processos neuroquímicos similares (validade de construto)

(Anisman & Matherson, 2005). Assim os modelos animais de depressão terminam

por avaliar apenas alguns sintomas de depressão, como a anedonia que, juntamente

com o humor depressivo, apresenta-se na quase totalidade dos sub-tipos clínicos da

depressão (Anisman & Matherson, 2005).

No presente estudo foram utilizados o teste do nado forçado e o paradigma

do estresse crônico moderado.

1.5.1 – Teste do Nado Forçado (Forced Swiming Test - FST)

Esse teste foi desenvolvido por Porsolt e colaboradores (1978), na American

National Standard, com ratos e, posteriormente, com camundongos. O FST tem

sido, juntamente com o Teste de Suspensão da Cauda e o Teste do Desamparo

Aprendido, a ferramenta mais utilizada para avaliar atividade antidepressiva de

fármacos (Duman & Monteggia, 2006). O modelo é de uso difundido nos laboratórios

por ser de fácil utilização e apresentar confiabilidade no resultado e se baseia na

observação de que ratos colocados num cilindro com água, após tentativas iniciais

de fuga, rapidamente mostram imobilidade, interpretada como prejuízo do

comportamento esperado de tentar fugir ou como desenvolvimento de

comportamento passivo capaz de afastar o animal de formas ativas de

enfrentamento da situação estressora. Se um fármaco antidepressivo for

administrado entre as duas exposições, o animal apresentará comportamento mais

ativo, persistindo na tentativa de escapar por tempo mais prolongado, quando

comparado a animal que recebeu solução inócua (Porsolt et al., 1978; Cryan et al.,

2002). Este teste requer dois dias de procedimentos. O rato é colocado em um

cilindro com água a 23ºC, em nível suficiente para que o animal não consiga apoiar-

se no fundo. No primeiro dia (treinamento) os ratos são forçados a nadar durante 15

minutos. Vinte e quatro horas após o treinamento, cada animal é novamente posto

no cilindro com água e forçado a nadar, agora por 5 minutos, durante os quais se

avaliam parâmetros de imobilidade, aqui se considerando imobilidade total e/ou

movimentos para manter a cabeça fora da água sem intenção de escapar (Porsolt et

al., 1978).

Para avaliar se a redução da imobilidade deveria ser interpretada como um

comportamento relacionado com efeito antidepressivo da substância testada ou a

um efeito estimulante da atividade locomotora, tal como ocorre com uso de

psicoestimulantes (Hunt et al., 2006), foi também realizado o teste do Campo Aberto.

Embora seja ainda de difícil definição, o termo “atividade exploratória” é amplamente

utilizado em pesquisas relacionadas ao comportamento animal. Serão considerados

os números de “crossing” para avaliação da atividade locomotora e os números de

“rearing” para avaliação da atividade exploratória (Vianna et al., 2000).

1.5.2. – Estresse Crônico Moderado (Chronic Mild Stress - CMS)

Modelo desenvolvido por Willner, a partir de estudos iniciais de Katz e

colaboradores (Willner, 1997), no qual ratos são submetidos, por algumas semanas,

a estressores de intensidade moderada e natureza variável, uma vez que a

presença de um mesmo estressor pode levar à adaptação. A medida comumente

usada para avaliar os efeitos é a diminuição no consumo de solução açucarada. Tal

comportamento é considerado significativo de anedonia, ou seja, de incapacidade

para experimentar prazer de qualquer natureza, que constitui achado clínico

primordial nas formas melancólicas da depressão (Willner, 1997). É um modelo

considerado de boa validade de predição, de face e de construto por induzir redução

de resposta frente à recompensa (Willner, 1997). No presente trabalho os ratos

foram submetidos a estressores diversos (descritos no artigo III), por um período de

40 dias, sendo posteriormente avaliados os resultados dos tratamentos agudo e

crônico com cetamina através dos parâmetros: aceitação de alimento açucarado,

peso corporal, níveis de cortisol e de hormônio adrenocorticotrófico, atividade

locomotora e dosagem de BDNF no hipocampo.

2 – OBJETIVOS

2.1 – Objetivo Geral

o Avaliar os efeitos da administração de cetamina nos níveis hipocampais de

BDNF e no comportamento de ratos submetidos ao teste do nado forçado e

ao paradigma do estresse crônico moderado.

2.2 - Objetivos Específicos

o Avaliar os efeitos comportamentais promovidos pela administração aguda e

crônica de cetamina, em doses de 5, 10 e 15 mg/kg, em ratos submetidos ao

teste do nado forçado e ao campo aberto.

o Verificar os níveis de BDNF no hipocampo de ratos tratados, aguda e

cronicamente, com cetamina nas doses de 5, 10 e 15 mg/kg.

o Avaliar os efeitos comportamentais promovidos pela administração aguda e

crônica de cetamina, na dose de 15 mg/kg, em ratos submetidos ao

paradigma do estresse crônico moderado.

o Verificar os níveis de BDNF no hipocampo de ratos submetidos ao estresse

crônico moderado e posteriormente tratados, aguda e cronicamente, com

cetamina na dose de 15 mg/kg.

3. ARTIGOS

Os experimentos realizados tiveram seus respectivos projetos

previamente avaliados e aprovados pelo Comitê de Ética em Pesquisa da UNESC,

onde receberam os seguintes números de protocolo:

(I) Estudo dos efeitos comportamentais da cetamina em ratos submetidos ao teste

do nado forçado. Protocolo: 543/2007.

(II) Avaliação dos efeitos comportamentais do tratamento crônico com cetamina em

ratos submetidos ao teste do nado forçado. Protocolo: 699/2007.

(III) Avaliação do tratamento agudo e crônico com cetamina em ratos submetidos ao

paradigma do estresse crônico moderado. Protocolo: 77/2008.

• Acute administration of ketamine induces antidepressant-like effects in the

forced swimming test and increases BDNF levels in the rat hippocampus (.pdf)

• Chronic Administration of Ketamine Elicits Antidepressant-Like Effects in Rats

without Affecting Hippocampal Brain-Derived Neurotrophic Factor Protein

Levels (.pdf)

• Ketamine Treatment Reverses Behavioral and Physiological Alterations

Induced by Chronic Mild Stress in Rats (.pdf)

Os artigos citados estão disponibilizados no final deste trabalho.

4. DISCUSSÃO

O presente trabalho avaliou inicialmente o efeito do tratamento agudo com

cetamina em ratos, através do teste do nado forçado, demonstrando que uma única

injeção de cetamina (10 e 15 mg/kg) diminuiu o tempo de imobilidade de ratos, no

referido teste comportamental, sem alterar a atividade locomotora espontânea, esta

última avaliada no teste do campo aberto. O resultado está de acordo com estudos

pré-clínicos, como o realizado por Chaturvedi et al. (2001), que demonstrou que a

cetamina conseguiu reverter a imobilidade observada em camundongos submetidos

ao mesmo teste do nado forçado. Em outro estudo, camundongos submetidos ao

teste de suspensão da cauda também apresentaram diminuição do tempo de

imobilidade quando sob efeito da cetamina (Yilmaz et al., 2002; Kos et al., 2006). A

cetamina também foi avaliada clinicamente em estudos como o realizado por

Berman et al. (2000) e replicado por Zarate et al. (2006c), cujos resultados

demonstraram rápida e robusta ação antidepressiva da substância. Esse último

estudo foi ainda seguido de observação dos pacientes que, após duas semanas sem

uso da cetamina, voltaram a receber infusão intravenosa da substância (0,5 mg/kg)

ou salina e, avaliados posteriormente, apresentaram melhores índices de resposta

para a Escala de Avaliação de Hamilton, a partir de 110 minutos após injeção, por

até uma semana depois (Maeng & Zarate, 2007).

Substâncias de ação antagonista de receptores NMDA, como a cetamina,

têm demonstrado em estudos clínicos proporcionar rápida ação antidepressiva

(Berman et al., 2000; Zarate et al., 2006c; Liebrenz et al., 2007). Também no estudo

experimental realizado por Yilmaz et al. (2002) foi observado que uma única injeção

da cetamina, em dose anestésica (160 mg/kg), induziu efeito do tipo antidepressivo

em ratos submetidos ao teste do nado forçado, no 3º, 7º ou no 10º dia após o

tratamento.

Os dados resultantes do tratamento agudo aqui realizado e referido

demonstraram ainda que o uso da cetamina promoveu aumento significativo dos

níveis de BDNF no hipocampo dos ratos que receberam a substância, sendo que tal

efeito não foi observado nos animais que receberam salina ou imipramina. O

aumento dos níveis desse fator neurotrófico tem sido associado a efeito

antidepressivo e alguns estudos sugerem que maior sinalização de BDNF e de seu

receptor TrkB seria condição necessária para que ocorresse ação antidepressiva

(Castrén et al., 2007). O aumento dos níveis do RNAm do BDNF tem sido observado

no uso crônico dos fármacos antidepressivos conhecidos mas ECT, de reconhecida

ação antidepressiva, em uso agudo, apresenta o mesmo resultado (Saarelainen et

al., (2003). Assim, deve ser considerada a possibilidade de a cetamina apresentar

rápida ação antidepressiva por produzir, de forma mais direta, alguns efeitos

resultantes de adaptações, como: redução funcional dos receptores NMDA para o

glutamato, favorecimento de ação rápida dos receptores AMPA e rápida indução do

fator de transcrição CREB, com reflexo nos níveis de BDNF (Krystal, 2007) e os

conseqüentes efeitos comportamentais característicos da ação de antidepressivos.

Os resultados obtidos no presente trabalho, em consonância com dados da

literatura, mostram que o tratamento agudo com a cetamina apresentou efeitos do

tipo antidepressivo, caracterizados por diminuição da imobilidade dos animais

submetidos ao FST e aumento dos níveis de BDNF no hipocampo de ratos.

Este trabalho também avaliou o efeito do tratamento crônico (14 dias) com

cetamina, em ratos igualmente submetidos aos testes comportamentais realizados

no tratamento agudo, e os resultados obtidos demonstraram que cetamina e

imipramina diminuíram o tempo de imobilidade dos ratos no teste do nado forçado,

em todas as doses testadas. A atividade locomotora foi também avaliada pelo teste

do campo aberto sendo demonstrado que a substância em estudo, tanto quanto a

imipramina, não alterou a atividade locomotora espontânea. Para esse experimento

foi ainda realizada dosagem dos níveis de BDNF no hipocampo dos animais

tratados, não se observando alterações significativas dos níveis da proteína referida,

resultado válido para cetamina e imipramina, em todas as doses testadas.

Neste mesmo experimento, o tratamento com imipramina (14 dias) resultou

em diminuição do tempo de imobilidade observada no FST, em todas as doses

testadas, inclusive na menor dose utilizada, comprovadamente ineficaz no

tratamento agudo. Também a cetamina, em uso crônico, apresentou redução do

tempo de imobilidade observada no FST, em todas as doses testadas, inclusive

naquelas ineficazes no tratamento agudo (5mg/kg), sugerindo eficácia em doses

baixas.

Outro aspecto importante observado refere-se à ausência de sinais de

tolerância observada na utilização das dosagens mais altas utilizadas (15m/kg) uma

vez que há registros de utilização da substância como droga de abuso (ver

introdução).

Detke et al., (1995), utilizando o teste do nado forçado, observaram que

drogas de efeito noradrenérgico (desipramina, maprotilina) aumentaram o

comportamento de elevação dos animais testados, enquanto antidepressivos de

ação serotonérgica (fluoxetina, sertralina, paroxetina) aumentaram o tempo de nado.

O presente experimento demonstrou que a cetamina, assim como a imipramina,

aumentou o tempo de elevação (climbing) e o tempo de natação dos animais

sugerindo a existência de um diferente mecanismo de ação ou a combinação de

diferentes mecanismos, o que certamente requer novos estudos.

Em relação ao BDNF, o efeito antidepressivo crônico tem sido largamente

associado com a elevação dos seus níveis. O presente trabalho, que registrou

elevação significativa dos níveis dessa proteína no hipocampo dos animais tratados

com cetamina, de forma aguda, observou ausência de alterações significativas em

seus valores no tratamento crônico. O resultado obtido permite pensar se tal

aumento não poderia ocorrer no início do uso da cetamina e não ser detectado em

tempo mais tardio, em função de possível mecanismo adaptativo.

Dúvidas, portanto, permanecem sobre o mecanismo de ação da substância

testada. Tem sido proposto que a ação neuroprotetora seja decorrência da redução

à transmissão excessiva de cálcio, assim como de um aumento na expressão de

fatores neurotróficos (Sanacora et al., 2003). Também há que se cogitar sobre

participação dos receptores AMPA, que apresentam sugestiva coexistência com

receptores NMDA em sinapses maduras. Estudos experimentais têm sugerido que

antagonistas de receptores glutamatérgicos do tipo NMDA mostram efeito

antidepressivo em modelo animal e eficácia semelhante aos antidepressivos

tricíclicos em ratos e em camundongos (Zarate et al., 2002), assim, tais substâncias

antagonistas poderiam ter efeito sinérgico com antidepressivos tradicionais o que as

tornaria possíveis agentes para terapêutica combinada em casos de depressão

resistente (Zarate et al., 2002)

Finalizando os experimentos do presente trabalho, a cetamina foi utilizada em

tratamento agudo e crônico, em ratos submetidos a estresse crônico variado.

Vários estudos têm demonstrado que exposição a situações estressoras pode

influenciar o comportamento alimentar em ratos e que a exposição a estresse

crônico pode alterar inclusive o peso corporal desses animais (Gamaro et al., 2003).

A condição de ameaça instalada e a necessidade de retomada do equilíbrio resultam

em maior estimulação do eixo hipotálamo-pituitária-adrenal (HPA) e a conseqüente

secreção de glicocorticóides que funciona, a um só tempo, como resposta ao

estresse e como mediador das mudanças fisiopatológicas doravante passíveis de

ocorrer (Mello et al., 2003).

Neste estudo, após 40 dias de exposição a estressores variados, evitando-se

assim possível adaptação dos animais, foi observada ocorrência de redução da

ingestão de alimentos açucarados e do peso corporal e aumento do peso da

glândula adrenal e dos níveis de cortisol e do hormônio corticotrópico (ACTH).

O tratamento agudo com cetamina conseguiu reverter a redução do peso

corporal, mas não alterou a ingestão de alimentos doces que havia sido reduzida

pelo estresse. Por sua vez, a administração crônica de cetamina foi capaz de

aumentar a ingestão de alimentos doces também no grupo de animais controle.

Dados da literatura dão conta que perda de peso e menor ingestão de alimentos

doces podem ser fenômenos dissociados, havendo inclusive evidências de

experimentos em que a redução na ingestão de alimentos foi menor em animais que

perderam mais peso (Wilner,1997). Assim, o aumento da ingestão de alimentos

doces observado nos animais não submetidos ao CMS, e tratados com repetidas

doses de cetamina, poderia estar relacionado a um aumento indiscriminado da

ingestão de alimentos, que a literatura refere como uma possível combinação de

ações centrais e viscerais exercidas por antagonistas de receptores NMDA (Burns &

Ritter, 1998; Treece et al., 2000; Jahng & Houpt, 2001). Burns & Ritter (1998)

mostraram que a inibição de neurônio aferente visceral, responsável pela condução

da saciedade ao sistema nervoso central, em ratos tratados com MK801, resultou

em bloqueio no aumento da ingestão de alimentos. Posteriormente, Treece et al.

(2000) demonstraram que lesão vagal, em ratos tratados com MK801, resultou em

consumo de alimentos doces em quantidade muito próxima àquela consumida pelos

animais que receberam salina. Assim, não se pode afastar a hipótese que a

reversão do comportamento de anedonia em animais submetidos ao CMS,

observada com o tratamento por cetamina, seja devida a uma ação direta da droga

sobre a ingestão de alimentos. O modelo, entretanto, está focado na observação da

redução do comportamento hedônico manifestada pelos animais submetidos ao

estresse e a redução no consumo do alimento doce observada no modelo, que pode

ser duradoura e revertida pela ação de fármacos antidepressivos, é interpretada

como sinal de anedonia (Wilner, 1997).

O estudo mostrou ainda que os animais submetidos ao estresse crônico

apresentaram aumento do peso da adrenal, além de elevação dos níveis de cortisol

e do ACTH, resultado compatível com dados da literatura sobre exposição de

animais ao estresse crônico moderado e sinais correlatos de maior atividade do eixo

HPA, incluindo hipertrofia adrenal e hipersecreção de hormônios corticosteróides

(Wilner, 1997; Harro et al., 2001). Conforme a validade preditiva do modelo, tais

alterações devem ser revertidas pelo uso de antidepressivos, resultado compatível

com aquele aqui observado nos tratamentos agudo e crônico com a cetamina.

Não foram observadas alterações na atividade locomotora, avaliada pelo teste

do campo aberto, resultado válido tanto para o tratamento agudo quanto para o

crônico. Os níveis de BDNF no hipocampo também não mostraram alterações

dependentes do estresse. Tampouco foram observadas alterações nos níveis da

proteína, após tratamentos agudo e crônico com a cetamina. Tais dados estão de

acordo com a literatura: Gronli et al. (2006) demonstraram que ratos submetidos ao

CMS por 5 semanas apresentaram redução da expressão do BDNF no giro

denteado, entretanto, tal alteração não foi observada no hipocampo.

Os efeitos clinico-comportamentais de outros antagonistas de receptores

NMDA também despertam interesse como possíveis agentes terapêuticos: a

memantina – de menor especificidade, mostra-se melhor tolerada clinicamente mas,

seu efeito antidepressivo não foi comprovado em ensaio de 8 semanas levado a

efeito por Zarate et al. (2006a), entretanto, os próprios autores, posteriomente,

questionaram se as doses utilizadas não teriam sido baixas (Zarate et al., 2006b).

Outro antagonista de receptores NMDA, a fenciclidina (PCP) – este de grande

especificidade, apresenta forte potencial de ação psicotomimética (Stahl, 2006),

dificultando a avaliação de outros possíveis efeitos.

O presente trabalho, utilizando dois modelos animais de depressão, avaliou a

cetamina, em tratamento agudo e crônico, inicialmente através do teste do nado

forçado e, posteriormente, através do paradigma do estresse crônico moderado,

obtendo resultados compatíveis com a literatura acerca dos efeitos antidepressivos

da substância testada. Como antagonista não competitivo de receptores NMDA para

o glutamato, a cetamina, também aqui, apresentou promissores efeitos do tipo

antidepressivos, a saber:

i) diminuiu o tempo de imobilidade no FST, nas doses de 10 e 15 mg/kg no

tratamento agudo e, em todas as doses testadas, no tratamento crônico;

ii) não induziu aumento da atividade locomotora, avaliada no teste do campo

aberto, nas doses testadas, nos 3 diferentes experimentos;

iii) o tratamento agudo com cetamina (15 mg/kg) mostrou significativo

aumento dos níveis de BDNF no hipocampo dos animais tratados. Não houve

diferença no tratamento crônico. Os animais submetidos ao paradigma do estresse

crônico moderado também não apresentaram alterações dos níveis de BDNF;

iv) no paradigma do CMS, o tratamento com cetamina (agudo e crônico)

mostrou-se capaz de reverter: o aumento do peso da adrenal; o aumento dos níveis

de corticosterona e de ACTH, e a diminuição do peso corporal, induzidos pelo

estresse. O tratamento crônico com a cetamina (mas não o agudo) foi capaz de

reverter a diminuição da ingestão de alimentos doces, observada no modelo.

O conjunto dos resultados obtidos no presente trabalho mostra-se consistente

com relatos prévios sobre a hipótese da disfunção de receptores glutamatérgicos

NMDA na depressão e os efeitos de seus antagonistas como possível via alternativa

de abordagem terapêutica devendo entretanto ser considerado que, embora a

cetamina seja relativamente seletiva para receptores NMDA, a possibilidade de os

presentes resultados serem mediados por interações com outros receptores não

pode ser totalmente descartada, requerendo assim novos estudos.

Quanto ao possível uso clínico da cetamina como agente antidepressivo,

alguns desafios deverão ser previamente suplantados. Alguns, já elencados por

Mathew et al., (2008), referem-se à necessidade de estudos acerca de dosagens e

dos resultados de seu uso em associações com outros fármacos. Também se faz

necessária uma melhor compreensão de possíveis efeitos colaterais sobre a

cognição e a senso-percepção, assim como das eventuais conseqüências do seu

uso repetido, já que os dados disponíveis sugerem efeito rápido.

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Acute administration of ketamine induces antidepressant-like effects in the

forced swimming test and increases BDNF levels in the rat hippocampus

Lêda S.B. Garcia

, Clarissa M. Comim

, Samira S. Valvassori

, Gislaine Z. Réus

Luciana M. Barbosa

, Ana Cristina Andreazza

, Laura Stertz

, Gabriel R. Fries

Elaine Cristina Gavioli

, Flavio Kapczinski

, João Quevedo

⁎

Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde,

Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil

Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Bipolar Disorders Program,

Centro de Pesquisas, Hospital de Clínicas. Rua Ramiro Barcelos, 2350, 90035-003 Porto Alegre, RS, Brazil

Received 24 May 2007; received in revised form 31 July 2007; accepted 31 July 2007

Abstract

Ketamine is a non-competitive antagonist to the phencyclidine site of N-methyl-

D-aspartate (NMDA) receptor. Clinical findings point to a

rapid onset of action for ketamine on the treatment of major depression. Considering that classic antidepressants may take long-lasting time to

exhibit their main therapeutic effects, the present study aims to compare the behavioral effects and the BDNF hippocampus levels of

acute administration of ketamine and imipramine in rats. To this aim, rats were acutely treated with ketamine (5, 10 and 15 mg/kg) and imipramine

(10, 20 and 30 mg/kg) and animal behavioral was assessed in the forced swimming and open-field tests. Afterwards, BDNF protein

hippocampal levels were assessed in imipramine- and ketamine-treated rats by ELISA-sandwich assay. We observed that ketamine at the doses of

10 and 15 mg/kg, and imipramine at 20 and 30 mg/kg reduced immobility time compared to saline group, without affecting locomotor activity.

Interesting enough, acute administration of ketamine at the higher dose, but not imipramine, increased BDNF protein levels in the rat

hippocampus. In conclusion, our findings suggest that the increase of hippocampal BDNF protein levels induced by ketamine might be necessary

to produce a rapid onset of antidepressant action.

Keywords: Antidepressants; BDNF; Forced swimming test; Imipramine; Ketamine; NMDA receptor

1. Introduction

Depression is one of the most prevalent and costly psy-

chopathologies and a leading cause of morbidity and mortality in

the world. It is worthy of note that the pharmacotherapy of

depression is costly and widely prescribed by physicians, although

less than half of treated patients attain complete remission after

therapy with a single antidepressant. Others exhibit partial,

refractory or intolerant responses to the pharmacological treat-

ment, emphasizing the need to discover novel antidepressants

(Pacher et al., 2001). The challenges for the design of new agents

to treat depression are threefold: rapid onset of antidepressant

response, broader efficacy, and fewer adverse effects. While

progress has been made to reduce side-effects, currently available

antidepressants do not show convincing evidence for a shorter

delay of onset of therapeutic actions neither for improved efficacy

on the treatment of major depression (Nutt, 2002). Thus, there is

clearly a need to develop rapidly acting and potent treatments for

major depression.

Glutamate is the primary excitatory neurotransmitter in the

mammalian brain. Glutamatergic neurotransmission may be mo-

dulated in the brain by different receptor types, including

Progress in Neuro-Psychopharmacology & Biological Psychiatry xx (2007) xxx– xxx

+ MOD EL

PNP-06789; No of Pages 5

www.elsevier.com/locate/pnpbp

Abbreviations: BDNF, brain-derived-neurotrophic factor; IP, intraperitoneal;

EGTA, ethylene glycol tetraacetic acid; IMI, imipramine; KET, ketamine;

NMDA, N-methyl-D-aspartate; OD, optical density; PMSF, phenylmethylsulfo-

nyl fluoride; PBS, phosphate buffer solution.

⁎

Corresponding author. Fax: +55 48 3443 4817.

E-mail address: [email protected] (J. Quevedo).

doi:10.1016/j.pnpbp.2007.07.027

ARTICLE IN PRESS

Please cite this article as: Garcia LSB et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF

levels in the rat hippocampus. Prog Neuro-Psychopharmacol Biol Psychiatry (2007), doi:10.1016/j.pnpbp.2007.07.027

ionotropic and metabotropic receptors. Studies have pointed to the

ionotropic glutamate N-methyl-

D-aspartate receptor (NMDA) as

an important player in the etiology of psychopathologies, such as

anxiety and major depression (Javitt, 2004; Krystal et al., 1999).

Several preclinical studies have demonstrated that NMDA

antagonists, such as MK-801, AP7, CPP, neramexane and others,

display anxiolytic- and antidepressant-like effects in rats injected

into distinct brain areas and subjected to various animal models of

anxiety and depression (Kos et al., 2006a; Molchanov and

Guimarães, 2002; Menard and Treit, 2000; Adamec et al., 1999;

Matheus and Guimarães, 1997; Przegalinski et al., 1997; Skolnick

et al., 1996; Maj et al., 1992; Trullas and Skolnick, 1990).

Ketamine is a non-competitive antagonist to the phencyclidine

site of N-methyl-

D-aspartate (NMDA) receptor for glutamate, but

it also interacts with voltage sensitive Ca

channels, and opioid,

monoaminergic, and muscarinic receptors (for a review see: Hirota

and Lambert, 1996). Recently, clinical studies suggested that acute

administration of ketamine ameliorate depressive symptoms in

patients suffering from major depression (Zarate et al., 2006;

Berman et al., 2000). In agreement with these clinical findings,

some evidence from the literature suggests that ketamine induces

anxiolytic- and antidepressant-like effects in rodents subjected to

animal models of anxiety and depression (Kos et al., 2006b;

Yilmaz et al., 2002; Chaturvedi et al., 2001; Silvestre et al., 1997).

Brain-derived-neurotrophic factor (BDNF) is one of several

endogenous proteins that play critical roles in the survival,

maintenance, and growth of the brain and peripheral neurons

(Lewin and Barde, 1997). A growing body of evidence suggests

that BDNF could be mediating the pathophysiology of mood

disorders. In fact, reduced brain BDNF levels have been found in

postmortem samples from depressed patients (Karege et al.,

2002), whereas brain infusion of BDNF produces antidepressant-

like action in rats (Siuciak et al., 1997). In addition, exposure to

stress decreases levels of BDNF in brain regions associated with

depression, while antidepressant treatment produces opposite

actions and blocks the effects of stress on BDNF (for a review see:

Duman and Monteggia, 2006). Interestingly, chronic, but not

acute, antidepressant treatment induces increasing of BDNF

expression and BDNF immunoreactive fibers in the hippocampus

of rodents (Nibuya et al., 1996; De Foubert et al., 2004). Thus,

agents capable of rapidly enhancing BDNF levels may lead aid

the development of innovative antidepressant drugs.

The main aim of the present study was to compare behavioral

and molecular effects induced by acute administration of ketamine

and imipramine in rats. The behavioral effects of both drugs were

evaluated in the forced swimming test, which is a behavioral

despair assay widely used for screening antidepressant drugs

(McArthur and Borsini, 2006). The BDNF protein levels were

measured using an ELISA kit in the hippocampus of rats acutely

treated with ketamine and imipramine.

2. Materials and methods

2.1. Animals

Male Adult Wistar rats (60 days old) were obtained from

UNESC (Universidade do Extremo Sul Catarinense, Criciúma,

Brazil) breeding colony. They were housed five per cage with

food and water available ad libitum and were maintained on a

12-h light/dark cycle (lights on at 7:00 AM). All experi mental

procedures involving animals were performed in accordance

with the NIH Guide for the Care and Use of Laboratory Animals

and the Brazilian Society for Neuroscience and Behavior

(SBNeC) recommendations for animal care.

2.2. Drugs and treatments

Ketamine was obtained from Fort Dodge (Brazil) and imip-

ramine, the standard antidepressant, from Novartis Pharmaceuti-

cal Industry (Brazil). Different groups of rats (n=15 each) were

administered intraperitoneally (IP) with saline or different doses

of ketamine (5, 10 and 15 mg/kg) or imipramine (10, 20 and

30 mg/kg) 60 minutes before the test sessions, i.e. forced swim-

ming or open-field tests. All treatments were administered in a

volume of 1 ml/kg. The range of doses of ketamine employed in

this work was chosen based on a previous study, which reported an

increase of spontaneous locomotion at 25 mg/kg, while no

changes were observed at 10 mg/kg (Hunt et al., 2006).

2.3. App aratus

The forced swimming test was conducted according to pre-

vious reports (Porsolt et al., 1977; Detke et al., 1995). The test

involves two individual exposures to a cylindrical tank with water

in which rats cannot touch the bottom of the tank or escape. The

tank is made of transparent Plexiglas, 80 cm tall, 30 cm in

diameter, and filled with water (22–23 °C) to a depth of 40 cm.

Water in the tank was changed after each rat. For the first

exposure, rats without drug treatment were placed in the water for

15 min (pre-test session). Twenty-four hours later, rats were

placed in the water again for a 5 min session (test session), and the

immobility time of rats were recorded in seconds. Rats were

treated with ketamine, imipramine or saline only 60 min before

the second exposure to the cylindrical tank of water (test session).

In a separate series of experiments, naïve rats were treated with

ketamine (5–15 mg/kg), imipramine (10–30 mg/kg) and saline

60 min before the exposure to the open-field apparatus, in order to

assess possible effects of drug treatment on spontaneous loco-

motor activity. Analysis of rat spontaneous activity was carried

out in an open field apparatus, which is an arena 45× 60 cm

surrounded by 50 cm high walls made of brown plywood with a

frontal glass wall. The floor of the open field was divided into 9

rectangles (15×20 cm each) by black lines. Animals were gently

placed on the left rear quadrant, and left to explore the arena for

5 min. The number of horizontal (crossings) and vertical

(rearings) activity performed by each rat during the 5-min

observation period was counted by an expert observer.

2.4. Exp erimental procedure

Immediately after the forced swimming test, acutely saline,

imipramine and ketamine-treated rats were sacrificed and the

skulls were removed and hippocampus was dissected and stored at

− 80 °C for biochemical analyses. BDNF levels in hippocampus

2 L.S.B. Garcia et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xx (2007) xxx–xxx

ARTICLE IN PRESS

Please cite this article as: Garcia LSB et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF

levels in the rat hippocampus. Prog Neuro-Psychopharmacol Biol Psychiatry (2007), doi:10.1016/j.pnpbp.2007.07.027

were measured by anti-BDNF sandwich-ELISA, according to the

manufacturer instructions (Chemicon, USA). Briefly, rat hippo-

campus was homogenized in phosphate buffer solution (PBS)

with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM

(EGTA). Microtiter plates (96-well flat-bottom) were coated for

24 h with the samples diluted 1:2 in sample diluent and standard

curve ranged from 7.8 to 500 pg/ml of BNDF. The plates were

then washed four times with sample diluent and a monoclonal

anti-BNDF rabbit antibody diluted 1:1000 in sample diluent was

added to each well and incubated for 3 h at room temperature.

After washing, a peroxidase conjugated anti-rabbit antibody

(diluted 1:1000) was added to each well and incubated at room

temperature for 1 h. After addition of streptavidin-enzyme,

substrate and stop solution, the amount of BDNF was determined

by absorbance in 450 nm. The standard curve demonstrates a

direct relationship between Optical Density (OD) and BDNF

concentration. Total protein was measured by Lowry's method

using bovine serum albumin as a standard, as previously described

by Frey et al. (2006).

2.5. Statistical analysis

All data are presented as mean± S.E.M. Differences among

experimental groups in the forced swimming, open field test and

in the assessment of BDNF levels were determined by one-way

ANOVA, followed by Tukey post-hoc test when ANOVA was

significant; p values less than 0.05 were considered to be statistical

significant.

3. Results

As depicted in Fig. 1, the acute administration of the standard

antidepressant imipramine reduced, in a significant manner, the

immobility time of rats at 20 and 30 mg/kg compared to saline

(6–97)

=5.45; pb 0.05; Fig. 1). The intraperitoneal treatment

with ketamine at the doses of 10 and 15 mg/kg decreased

significantly the immobility time of rats compared to saline group

(6–97)

=5.45; p b 0.05; Fig. 1). In the open-field test, the

treatment with ketamine and imiprimine at all doses tested did

not modify the number of crossing and rearing compared to saline

treated-rats (Fig. 2A and B).

Fig. 3 illustrated the effects of the acute treatment with imip-

ramine (10, 20 and 30 mg/kg), ketamine (5, 10 and 15 mg/kg) and

saline in BDNF protein hippocampus levels of rats. A statistical

significant increase in BDNF protein levels in the hippocampus

was observed in rats treated with ketamine only at the higher dose

(15 mg/kg; F

(3–36)

=5.73; p b 0.05), but not with imipramine,

compared to saline group.

4. Discussion

The present study demonstrated that: (1) the acute treat-

ment with ketamine (10 and 15 mg/kg) and imipramine (20

and 30 mg/kg) decreased the immobility time of rats in the

forced swimming test; (2) ketamine and imipramine did not

affect spontaneous locomotor activity in the open-field test;

and (3) the acute treatment with ketamine at the higher dose,

Fig. 1. Effects of the acute administration of ketamine (5, 10 and 15 mg/kg, i.p.)

and imipramine (10, 20 and 30 mg/kg, i.p.) on the immobility time of rats

subjected to the forced swimming test. Bars represent means ± S.E.M. of 15 rats.

⁎

p b 0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.

Fig. 2. Effects of the acute administration of ketamine (5, 10 and 15 mg/kg, i.p.)

and imipramine (10, 20 and 30 mg/kg, i.p.) on the number of crossings (A) and

rearings (B) of rats subjected to the open field test. Bars represent means ±S.E.M. of

15 rats.

⁎

pb 0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.

Fig. 3. Effects of the acute administration of ketamine (5, 10 and 15 mg/kg, i.p.)

and imipramine (10, 20 and 30 mg/kg, i.p.) on the BDNF levels in the rat

hippocampus. Bars represent means ± S.E.M. of 15 rats.

⁎

p b 0.05 vs. saline

according to ANOVA followed by Tukey post-hoc test.

3L.S.B. Garcia et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xx (2007) xxx–xxx

ARTICLE IN PRESS

Please cite this article as: Garcia LSB et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF

levels in the rat hippocampus. Prog Neuro-Psychopharmacol Biol Psychiatry (2007), doi:10.1016/j.pnpbp.2007.07.027

but not imipramine, increased BDNF protein levels in the rat

hippocampus.

The behavioral effects induced by ketamine in rats reported in

the present study are in agreement with literature data, which

support an antidepressant action for ketamine in basic and clinical

studies. In fact, the treatment with ketamine reversed the shock-

induced increase of immobility time in the mouse forced

swimming test (Chaturvedi et al ., 2001). Another study

demonstrated that a single injection of an anesthetic dose of

ketamine (160 mg/kg) induced antidepressant-like effects in rats

tested in 3, 7, or 10 days after the forced swimming test (Yilmaz

et al., 2002). Additionally, in the mouse tail suspension test

ketamine produced anti-immobility effects, suggesting an

antidepressant-like action in mice (Kos et al., 2006b). Taken

together, our findings are in agreement with these previous

observations, and are strengthening the view that ketamine

induces antidepressant-like effects in rodents.

In 2000, a pilot study showed that a single dose of ketam ine

produced antidepressant effects in patients suffering from major

depression (Berman et al., 2000). Recently, Zarate et al. (2006)

extended this study to a higher number of patients, and they

found that the acute administration of ketamine rapidly

improved depressive symptoms in patients with major depres-

sion. In this study, ketamine ameliorates the symptoms of

depression within 110 minutes after injection, and these effects

remained significant until 7 days after ketamine injection

(Zarate et al., 2006). Therefore, these data strongly suggest that

ketamine can induce robust and rapid antidepressant effects in

depressed patients after a single intravenous injection.

Our finding s also showed that acute administration of keta-

mine, but not imipramine, significantly increased BDNF

protein levels in the rat hippocampus compared with saline

group. Several studies have suggested that normal BDNF-TrkB

receptor signaling is both necessary and sufficient for anti-

depressant drug action (for a review see: Castrén et a l., 2007).

Some authors have found that antidepressants acting through

different mechanisms rapidly increase TrkB receptor activation

and signaling within an hour after drug administration

(Saarel aine n et al., 20 03; Ra ntam ak i et al., 200 6). Despite

that, studies have shown that rats treated with fluoxetine for 4,

7, 14 and 21 days displayed unaltered hippocampal BDNF

proteinlevelswhenassessedbyELISAassay(De Foubert et

al., 2004),andthesameholdstrueto3weeksoftreatmentwith

desipramine (Jacobs en and Mork, 2004). Th us, taken together

our data are showing that acute admi nistration of ketamine

increase hippocampal BDNF protein leve ls, whist acute

(present data) and chronic treatment with classic antidepressant

did not affect it.

A growing body of evidence support an important role of

neurotrophic factors in mood disorders. In fact, reduced brain

BDNF levels predispose to depression, whereas increases in

brain BDNF levels produce an antidepressant action (for a

review see: Castrén et al., 2007). Our present findings revealed

that acute administration of ketamine causes an increase of

BDNF hippocampal levels detect ed immediately after the

forced swimming test. Importantly, our data did not evaluate

the durat ion of ketamine effects on BNDF levels in rats. Further

studies aiming to establis h a time–response curve to ketamine in

behavioral and molecular assays are worthy of doing.

Altogether, our findings support a quite unique effect induced

by ketamine in the hippocampal BDNF protein levels, which

suggests that the rapid onset of action of ketamine in the clinic

might be due to the increase of hippocampal BDNF protein

levels. Additionally, our findings contribute in explaining the

slow onset of antidepressant activity observed with classic

antidepressants.

Finally, it should be noted that although ketamine is a high-

affinity NMDA receptor antagonist, it has less, but potentially

relevant, affinity for μ opiate, monoaminergic, and muscarinic

receptors and also interacts with voltage sensitive Ca

channels

(Lindefors et al., 1997; Elliott et al., 1995; Wong et al., 1996;

Kapur a nd Seeman, 2001; Eide e t al., 1997). Thus, the

antidepressant-like effects of ketamine observed in the present

study could be due to interactions of ketamine with several

receptor systems, not only with NMDA receptors, which could

produce synergic effects on the brain pathways involved in the

modulation of behavioral and molecular actions of antidepres-

sants. Lastly, basic and clinical findings suggest that brain

pathways modulated by ketamine could play an important role in

reducing the onset of action of antidepressants.

5. Conclusion

The antidepressant-like effects of ketamine are in agreement

with literature data, which support an important role played by the

NMDA receptor signaling in major depression. However, it

should be kept in mind that, besides NMDA receptors, ketamine

interacts with distinct receptor systems, such opioid, monoamin-

ergic, muscarinic receptors and voltage sensitive Ca

channels,

which could produce synergic effects on the brain pathways

involved in the modulation of behavioral and molecular actions of

antidepressants.

Interestingly enough, our study demonstrated that ketamine,

but not imipramine, increased BDNF levels in rat hippocampus

after one single injection. Altogether, basic and clinical findings

might suggest that acute increase of BDNF protein levels in

hippocampus might be critical to antidepressant drugs with rapid

onset of action. Future studies need to be carried out in an

attempt to further investigate pharmacological and molecular

mechanism by whi ch ketamine, and other NMDA antagonists,

induce antidepressant-like effects.

Acknowledgements

This study was supported in part by CNPq (Brazil), UNESC

(Brazil), FAPESC (Brazil).

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Doi: 10.1111/j.1742-7843.2008.00210.x

Journal compilation

. Basic & Clinical Pharmacology & Toxicology

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Blackwell Publishing Ltd

Chronic Administration of Ketamine Elicits Antidepressant-Like Effects

in Rats without Affecting Hippocampal Brain-Derived Neurotrophic

Factor Protein Levels

Lêda S. Garcia

, Clarissa M. Comim

, Samira S. Valvassori

, Gislaine Z. Réus

, Ana Cristina Andreazza

, Laura Stertz

, Gabriel R. Fries

Elaine Cristina Gavioli

, Flavio Kapczinski

and João Quevedo

Laboratory of Neurosciences, Postgraduate Program in Health Sciences, Unidade Acadêmica de Ciências da Saúde, Universidade do

Extremo Sul Catarinense, SC, Brazil, and

Bipolar Disorders Program, Centro de Pesquisas, Hospital de Clínicas de Porto Alegre, RS, Brazil

(Received September 23, 2007; Accepted October 29, 2007)

Abstract:

growing body of evidence has pointed to the blockade of the N-methyl-



-aspartate receptor (NMDA) receptor

signaling as a potential therapeutic target for the treatment of major depression. The present study was aimed to evaluate

the behavioural and molecular effects of the chronic treatment with ketamine and imipramine in rats. To this aim, rats were

14 days treated once a day with ketamine (5, 10 and 15 mg/kg) and imipramine (10, 20 and 30 mg/kg) and then subjected

to the forced swimming and open-ﬁeld tests. Ketamine and imipramine, at the all doses tested, reduced immobility time,

and increased both climbing and swimming time of rats compared to the saline group, without affecting spontaneous

locomotor activity. Brain-derived neurotrophic factor

(

BDNF) hippocampal levels were assessed in imipramine- and

ketamine-treated rats by ELISA sandwich assay. Chronic administration of both drugs, ketamine and imipramine, did not

modify BDNF protein levels in the rat hippocampus. In conclusion, our ﬁndings demonstrate for the ﬁrst time that chronic

administration of acute inactive doses of ketamine (5 mg/kg) becomes active after chronic treatment, while no signs of

tolerance to the behavioural effects of ketamine were observed after chronic administration of acute active doses (10 and

15 mg/kg). Finally, these ﬁndings further support the hypothesis that NMDA receptor could be a new pharmacological

target for the treatment of mood disorders.

Lately, several studies have been investigating into the optimal

treatment for major depression, despite that the adequate

management of this psychopathology remains a challenge.

The primary reasons for these pitfalls include (i) the incidence

of undesired side effects produced by currently available

antidepressants, (ii) the apparent delay in achieving measurable

therapeutic beneﬁt and (iii) a high percentage of treatment-

resistant patients [1,2]. Thus, there is a compelling need for

the development of novel antidepressant therapies with an

improved pharmacological proﬁle of action [3].

A growing body of evidence has pointed to the ionotropic

glutamate N-methyl-



-aspartate receptor (NMDA) as an

important player in the aetiology of psychopathologies,

including anxiety and major depression [4,5]. In fact, drugs

that block the NMDA receptor signaling have shown anti-

depressant properties in both clinical and preclinical studies

[6]. In this way, chemical compounds acting on NMDA

receptors could be interesting as pharmacological targets for

the treatment of mood disorders.

Ketamine, as well as phencyclidine, MK-801 and memantine,

are a non-competitive antagonist of NMDA receptor for

glutamate, which works in a use- and voltage-dependent

manner. Actually, ketamine binds the ionic channel of NMDA

receptor, thus blocking the entrance of Ca

to the neuron

[7,8]. However, distinct from other NMDA receptor

antagonists, ketamine also interacts with voltage-sensitive

channels, and opioid, monoaminergic and muscarinic

receptors [9].

Clinical studies suggest that acute administration of ketamine

ameliorates depressive symptoms in patients suffering

from major depression [10–12]. Preclinical ﬁndings, which

evaluated the behavioural effects of rodents acutely treated

with ketamine, also support this view. In fact, very recently,

we demonstrated that acute administration of ketamine

decreased immobility time in the forced swimming test [13].

In addition, some authors also suggest that ketamine induces

rapid and robust behavioural effects similar to classical

antidepressant in rodents subjected to animal models of

depression [14–17].

Neurotrophins, and particularly brain-derived neurotrophic

factor (BDNF), have been shown to function as a key

regulator of neurite outgrowth, synaptic plasticity and the

selection of functional neuronal connections in the central

nervous system, which make neurotrophins potential mediators

of the plastic changes induced by antidepressants [18]. In

fact, some authors have shown that chronic administration

of classic antidepressants increases mRNA encoding BDNF

and BDNF-immunoreactive ﬁbres in the hippocampus of

rodents [18]. Interestingly, few studies reporting hippocampal

BDNF protein levels in rats treated with antidepressants are

Author for correspondence: João Quevedo, Laboratory of

Neurosciences, Programa de Pós-Graduação em Ciências da Saúde,

Unidade Acadêmica de Ciências da Saúde, Universidade do

Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil (fax +55

48 3443 4817, e-mail [email protected]).

LÊDA S. GARCIA

ET AL.

UNCORRECTED PROOF

Journal compilation

Basic & Clinical Pharmacology & Toxicology

still available, and mostly these studies indicate that chronic

antidepressant treatment did not affect the BDNF levels

[19–21]. Very recently, we demonstrated that acute ketamine,

but not imipramine, increased BDNF protein levels in the

hippocampus of rats [13]. Thus, we suggest that ketamine

could be an innovative antidepressant drug by inducing

distinct and molecular effects on the rat hippocampus

compared to classic antidepressant.

The present study was aimed to assess the behavioural

effects of chronic treatment with ketamine in rats, in order to

provide evidence that the repeated administration of ketamine

does not induce tolerance. The behavioural effect of ketamine

was assessed in the forced swimming test, which is a

behavioural assay widely used for screening antidepressant

drugs [22]. The hippocampal BDNF protein levels were

also measured in rats 14 days treated with ketamine at 5, 10

and 15 mg/kg. Behavioural and molecular effects elicited by

chronic administration of ketamine were compared to

imipramine, a standard antidepressant drug.

Material and Methods

Animals.

Male adult Wistar rats (60 days old; weighing 300–350 g)

were obtained from UNESC (Universidade do Extremo Sul

Catarinense, Criciúma, Brazil) breeding colony. They were housed

ﬁve per cage (40

20 cm) with food and water available

ad libitum

and were maintained on a 12-hr light:dark cycle (lights on at

7:00 a.m.). All experimental procedures involving animals were

performed following the recommendations of the Brazilian Society

for Neuroscience and Behavior, which was in accordance with the

National Institutes of Health guidelines for the Animal Care and

Use of Laboratory Animals.

Drugs and treatments.

Ketamine employed in the present study was

purchased from Fort Dodge Animal Health (Fort Dodge, IA, USA)

as injectable solution (concentration 0.1 g/ml), and imipramine

from Novartis Pharmaceutical Industry (Basel, Switzerland).

Different groups of rats (n = 15 each) were administered intraperi-

toneally once a day with saline or different doses of ketamine

(5, 10 and 15 mg/kg) or imipramine (10, 20 and 30 mg/kg) during

14 days. Imipramine and ketamine were dissolved in saline imme-

diately before the intraperitoneal injections. All treatments were

administered in a volume of 1 ml/kg. The range of doses of ketamine

employed in this work was chosen based on our previous study

[13].

Rats were tested in the open ﬁeld and forced swim test following

chronic imipramine and ketamine treatments. Beginning on day 12

of chronic treatment, rats were tested in the open ﬁeld in order to

assess the spontaneous locomotor activity. On day 13 and 14 of

chronic treatment, rats were then tested in the forced swimming

test. From day 12 to 14 of chronic treatment, drug administration

was done 60 min. before the assessment of animal behaviour in the

open ﬁeld (day 12) and forced swimming test (days 13 and 14).

Forced swimming test.

The forced swimming test was conducted

according to previous reports [23,24]. The test involves two individual

exposures to a cylindrical tank with water in which rats cannot touch

the bottom of the tank or escape. The tank is made of transparent

plexiglas, 80 cm tall, 30 cm in diameter, and ﬁlled with water (22–

C) to a depth of 40 cm. Water in the tank was changed after

each rat. On day 13 of chronic treatment, 1 hr after drug treatment

rats were individually placed in the cylinder containing water for

15 min. (pre-test session). On the 14th day, rats received the last

intraperitoneal drug treatment, and after 1 hr, they were subjected

again to the forced swimming test for a 5-min. session (test session).

During the test session, were recorded in seconds some behavioural

parameters, such as immobility time (i.e. no additional activity is

observed other than that required to keep the rat’s head above the

water), climbing time, which is deﬁned as upward-directed move-

ments of the forepaws along the side of the swim chamber, and

swimming time (i.e. movement usually horizontal throughout the

swim chamber).

Open-ﬁeld test.

This apparatus consists of a brown plywood arena

60 cm surrounded by wood 50 cm high walls and containing a

frontal glass wall. The ﬂoor of the open ﬁeld was divided into nine

rectangles (15

20 cm each) by black lines. Animals were gently

placed on the left rear quadrant, and left to explore the arena for

5 min. After 12 days of treatment, rats were exposed to the open-ﬁeld

apparatus, and the number of horizontal (crossings) and vertical

(rearings) activity performed by each rat during the 5-min. observation

period was counted by an expert observer.

Rat hippocampal BDNF level measurement.

The measurement of

BDNF hippocampal levels was performed as previously described

[25]. Immediately after the forced swimming test, the 14 days

chronically treated rat were killed and the skulls were removed and

hippocampus was dissected and stored at –80

C for biochemical

analyses. BDNF levels in the hippocampus were measured by anti-

BDNF sandwich-ELISA, according to the manufacturer instructions

(Chemicon International Inc., Temecula, CA, USA). Brieﬂy, rat

hippocampus was homogenized in phosphate-buffered solution

with 1 mM phenylmethylsulfonyl ﬂuoride and 1 mM ethylenegly-

coltetraacetic acid. Microtiter plates (96-well ﬂat-bottom) were

coated for 24 hr with the samples diluted 1:2 in sample diluent and

standard curve ranged from 7.8 to 500 pg/ml of BNDF. The plates

were then washed four times with sample diluent and a monoclonal

anti-BNDF rabbit antibody diluted 1:1000 in sample diluent was

added to each well and incubated for 3 hr at room temperature.

After washing, a peroxidase-conjugated anti-rabbit antibody (horse-

radish peroxidase enzyme; diluted 1:1000) was added to each well

and incubated at room temperature for 1 hr. After addition of

streptavidin enzyme, substrate (3,3

′

,5,5

′

-tetramethylbenzidine) and

stop solution, the amount of BDNF was determined by absorbance

in 450 nm. The standard curve demonstrates a direct relationship

between optical density and BDNF concentration. BDNF was

expressed as pg of BDNF per ml of serum obtained from hippocampal

homogenate. Total protein was measured by Lowry’s method using

bovine serum albumin as a standard.

Statistical analysis.

All data are presented as mean ± S.E.M. Dif-

ferences among experimental groups in the forced swimming, and

open-ﬁeld test in the assessment of BDNF levels were determined

by one-way

,

followed by Turkey’s

post hoc

test when



was signiﬁcant; P-values of <0.05 were considered to be statistical

signiﬁcant.

Results

As depicted in ﬁg. 1, chronic administration of the standard

antidepressant imipramine reduced in a signiﬁcant manner

the immobility time of rats at 10, 20 and 30 mg/kg compared

to saline (F

(3.54)

= 6.66; P < 0.001). Interestingly, the intra-

peritoneal treatment with ketamine at the doses of 5, 10 and

15 mg/kg signiﬁcantly decreased the immobility time of rats

compared to saline group (ﬁg. 2; F

(3.55)

= 6.26; P = 0.001). In

the open-ﬁeld test, the treatment with ketamine and imiprimine

at all doses tested did not modify the number of crossing

and rearing compared to saline-treated rats (ﬁg. 3A and B;

P > 0.05).

XXXXXX

UNCORRECTED PROOF

Journal compilation

Basic & Clinical Pharmacology & Toxicology

Figure 4 illustrated the effects of the chronic treatment

with imipramine (10, 20 and 30 mg/kg), ketamine (5, 10 and

15 mg/kg) and saline in BDNF levels of rat hippocampus.

The treatment with ketamine and imiprimine at all doses

tested did not modify the BDNF levels in the rat hippocampus

compared to saline group (P > 0.05).

Discussion

The present study demonstrates that (i) the chronic treatment

with all doses of ketamine and imipramine decreased the

immobility time of rats in the forced swimming test; (ii)

ketamine and imipramine did not affect spontaneous

locomotor activity in the open-ﬁeld test; and (iii) chronic

treatment with ketamine and imipramine did not modify

BDNF protein levels in the rat hippocampus. Thus, consider-

ing the abuse potential and the risk of tolerance to the

effects of ketamine, our ﬁndings are quite relevant, because

preclinical and clinical available data report ‘antidepressant

effects’ just for acute administration of ketamine.

In fact, very recently, we have demonstrated that a single

injection of ketamine (10 and 15 mg/kg) and imipramine (20

and 30 mg/kg) decreased the immobility time of rats in the

forced swimming test, without modifying the locomotor

activity [13]. In the tail suspension test, ketamine also

produced anti-immobility effects in mice [15]. Yilmaz et al.

Fig. 1. Effects of chronic administration of imipramine (10, 20 and

30 mg/kg, intraperitoneally) on the immobility, swimming and

climbing time of rats subjected to the forced swimming test. Bars

represent means ± S.E.M. of 15 rats. *P < 0.05 versus saline

according to  followed by Turkey’s post hoc test.

Fig. 2. Effects of chronic administration of ketamine (5, 10 and

15 mg/kg, intraperitoneally) on the immobility, swimming and

climbing time of rats subjected to the forced swimming test. Bars

represent means ± S.E.M. of 15 rats. *P < 0.05 versus saline

according to  followed by Turkey’s post hoc test.

Fig. 3. Effects of chronic administration of ketamine (5, 10 and

15 mg/kg, intraperitoneally) and imipramine (10, 20 and 30 mg/kg,

intraperitoneally) on the number of crossings (A) and rearings (B)

of rats subjected to the open-ﬁeld test. Bars represent means ±

S.E.M. of 15 rats.

Fig. 4. Effects of chronic administration of ketamine (5, 10 and

15 mg/kg, intraperitoneally) and imipramine (10, 20 and 30 mg/kg,

intraperitoneally) on the BDNF hippocampal levels in rats. Bars

represent means ± S.E.M. of 10 rats.

LÊDA S. GARCIA

ET AL.

UNCORRECTED PROOF

Journal compilation

Basic & Clinical Pharmacology & Toxicology

[16] demonstrated that a single injection of an anaesthetic

dose of ketamine (160 mg/kg) reduced the immobility time

in the rat forced swimming test assessed 3, 7 or 10 days after

the injection. Other literature ﬁndings show that the treatment

with ketamine reversed the shock-induced increase of

immobility time in the mouse forced swimming test [17].

Maeng et al. [14] recently demonstrated that a single injection

ketamine reversed learned helplessness behaviour in mice,

and reduced immobility time in the mouse forced swimming

test assessed 30 min. and 2 weeks after the drug administration.

Taken together, these ﬁndings support robust and sustained

effects to ketamine on behavioural tests used for screening

antidepressant drugs.

The present study demonstrates that 14 days of imipramine

treatment, at doses of 10, 20 and 30 mg/kg reduced immobility

time of rats subjected to the forced swimming test. These

ﬁndings are in agreement with other authors that support

reduction of immobility time in the forced swimming test

after repeated administration of imipramine, especially at low

doses (such as 10 mg/kg), which were acutely inactive [26,27].

Interestingly enough, ketamine also reduced immobility

time in the forced swimming test at all doses tested, including

doses that were inactive after acute administration, such as

5 mg/kg, as we previously reported [13]. These ﬁndings

show that chronic administration of ketamine at low doses

(i.e. 5 mg/kg) induces behavioural responses that were not

elicited acutely, while at higher doses (i.e. 10 and 15 mg/kg)

no signs of tolerance were observed after the chronic exposure.

In 1995, Detke et al. [24] reported that despite the

anti-immobility effects, antidepressant drugs that enhance

noradrenergic neurotransmission increase climbing behaviour,

whereas the enhancement of serotonergic neurotransmission

increases swimming time in the rat forced swimming test.

Our ﬁndings indicate that ketamine, like imipramine, con-

sistently reduced immobility time and signiﬁcantly increased

climbing and swimming behaviour in rats. Literature data

show that imipramine dose-dependently reduces immobility

time, and equally increases both climbing and swimming

behaviour in the rat forced swimming test [28]. It should be

mentioned that minalcipram, a dual NE/5-HT reuptake

inhibitor [29], or BW373U86, a

-opioid agonist [30], produced

a pattern of response similar to imipramine in the rat forced

swimming test. Thus, these unique behavioural effects appear

to be a combination of differently acting antidepressants, or

they could be due to a totally new mechanism of antidepressant

action. Further studies aiming to investigate the mechanism(s)

by which ketamine produces antidepressant-like effects are

required.

Several ﬁndings support the important role played by

neurotrophic factors in mood disorders. Actually, reduced

brain BDNF levels have been found in post-mortem samples

from depressed patients, and antidepressant therapy restores

BDNF brain levels to the normal range in human beings

[18]. Interestingly, preclinical ﬁndings suggest that chronic

treatment with classic antidepressants most commonly

increases mRNA encoding BDNF [18], than BDNF protein

levels, in the rat hippocampus [19,20]. Possible explanations

of the lack of increasing BDNF levels in rats chronically

treated with imipramine could be that (i) the half-life of

BDNF is too short for detecting changes, while the concentra-

tion of the BDNF transcript is stronger and more stable;

and (ii) the level of stress experienced by animals (acute and

chronic) probably inﬂuences the turnover and expression of

BDNF, and consequently its availability in the brain. In

fact, studies have shown that naïve rats chronically

treated with classic antidepressants such as ﬂuoxetine and

desipramine, display unaltered hippocampal BDNF protein

levels [19,20]. In contrast, learned helplessness rats display

reduced BDNF levels in the hippocampus compared to

naïve animals, and repeated treatment with imipramine

restores BDNF levels in learned helplessness rats [21]. These

observations suggest that BDNF levels could be reduced in

the hippocampus under chronic stressful situations, and

repeated antidepressant treatment could normalize it.

Recently, we reported that one single injection of ketamine,

but not imipramine, increased BDNF levels in the rat hippo-

campus, suggesting that ketamine can alter acutely, but not

chronically (present ﬁndings) BDNF hippocampal levels in

rats. Thus, an increase of BDNF hippocampal levels could

possibly occur early during chronic administration (such as

observed after acute injection of ketamine), but it may not

be more detectable at a later time because of adaptive

mechanisms or due to the development of tolerance to

ketamine effects on hippocampal BDNF levels.

Taken together, the present data demonstrate that the

repeated administration of NMDA receptor antagonist

ketamine reduces immobility time at all doses tested in the

rat forced swimming test. These ﬁndings are absolutely relevant

to the clinical use of ketamine, due to (i) acute inactive

doses of ketamine (i.e. 5 mg/kg) become active after chronic

treatment, and (ii) acute active doses of ketamine (i.e. 10

and 15 mg/kg) do not produce tolerance to the behavioural

effects when injected chronically in rats assessed in the

forced swimming test.

Our data also show that ketamine, similar to imipramine,

did not alter BDNF protein levels in the hippocampus of

rats subjected to the forced swimming test. It could be due

to the fact that repeated administration of antidepressants

increases BDNF hippocampal levels under chronic, stressful

stimulus, but not under acute, stressful situations such as

forced swim. Another fact is that acute (previous ﬁndings),

but not chronic (present data) administration of ketamine

increases BDNF levels in the hippocampus. These contrasting

effects could be due to adaptive mechanisms or induction of

tolerance to the effects of ketamine on BDNF protein levels.

Finally, further studies aiming to investigate the mechanisms

by which ketamine induces antidepressant-like effects is

mandatory to the development of novel antidepressant

drugs with rapid and robust onset of action.

Acknowledgements

This study was supported in part by CNPq (Brazil),

UNESC (Brazil), FAPESC (Brazil) and Instituto Cérebro e

Mente (Brazil).

XXXXXX

UNCORRECTED PROOF

Journal compilation

Basic & Clinical Pharmacology & Toxicology

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uery

orm

Journal: Basic & Clinical Pharmacology & Toxicology

Article: pto_210.fm

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KETAMINE TREATMENT REVERSES BEHAVIORAL AND PHYSIOLOGICAL

ALTERATIONS INDUCED BY CHRONIC MILD STRESS IN RATS

Lêda S.B. Garcia

, Clarissa M. Comim

, Samira S. Valvassori

, Gislaine Z.

Réus

, Laura Stertz

, Flávio Kapczinski

, Elaine C. Gavioli

, João Quevedo

Laboratory of Neurosciences, Postgraduate Programme in Health Sciences,

University of Extremo Sul Catarinense, Criciúma, SC, Brazil

Bipolar Disorders Program and Molecular Psychiatry Laboratory, 90035-003

Porto Alegre, RS, Brazil.

*Corresponding author:

Prof. João Quevedo, MD, PhD

Laboratório de Neurociências,

Programa de Pós-Graduação em Ciências da Saúde,

Unidade Acadêmica de Ciências da Saúde,

Universidade do Extremo Sul Catarinense,

88806-000 Criciúma, SC, Brazil

Fax: +55 48 3443 4817.

E-mail: [email protected]

ABSTRACT

Several studies have supported the idea that ionotropic glutamate N-methyl-D-

aspartate receptor (NMDA) is an important player in the etiology of

psychopathologies, such as anxiety disorders and major depression. A growing

body of evidence has pointed to the blockade of the NMDA receptor signaling

as a potential therapeutic target for the treatment of major depression. Available

antidepressants may take long-lasting time to exhibit their main therapeutic

effects nevertheless. The purpose of the present study was to evaluate

behavioral and physiological effects of acute and chronic administration of

ketamine, a NMDA receptor antagonist, in rats exposed to Chronic Mild Stress

procedure. To this aim, after 40 days of exposure to CMS procedure, rats were

treated with ketamine (15 mg/kg) and assessed sweet food consumption, body

and adrenal gland weight, corticosterone and adrenocorticotropic (ACTH)

hormone levels, and hippocampal BDNF protein levels. Our findings

demonstrated that chronic stressful situations induced anhedonia, evoked

hypertrophy of adrenal gland weight, impaired gain of body weight and

increased cortisol and ACTH circulating levels in rats. Acute and chronic

treatment with ketamine reversed the increase of adrenal gland weight,

promoted regain of body weight, and normalized cortisol and ACTH circulating

levels. Repeated, but not acute, administration of ketamine reversed anhedonia.

Finally, acute and chronic ketamine treatment did not alter hippocampal BDNF

protein levels in CMS rats. In conclusion, these findings support to the idea of a

potencial role of NMDA receptors in depression-like symptoms and the rapid

and robust effects of ketamine in the reversion of anhedonic behavior and

physiological alterations induced by chronic mild stressful situations in rats.

KEY WORDS: chronic mild stress; rat; ketamine; NMDA receptor; anhedonia.

ABBREVIATIONS:

ACTH (adrenocorticotropic hormone);

BDNF (brain-derived neurotrofic factor);

CMS (chronic mild stress);

(HPA) hypothalamic-pituitary-adrenal.

NMDA (N-methyl-D-aspartate)

1. INTRODUCTION

Major depression is a serious and recurrent disorder often manifested

with psychological, behavioral and physiological symptoms. It affects 17-20% of

the population of the world resulting in premature death, major social deficits

and economic consequences (Kessler et al., 1994). Among people with major

depression, 75-85% have recurrent episodes (Keller et al., 1986; Mueller, 1999)

and 10-30% recover incompletely and have persistent and residual depressive

symptoms (Mann, 2005).

The pharmacotherapy of depression is widely prescribed by physicians,

although less than half of treated patients attain complete remission after

therapy with a single antidepressant. Others exhibit partial, refractory or

intolerant responses to the pharmacological treatment, emphasizing the need to

discover novel antidepressants (Pacher et al., 2001). The challenges for the

design of new antidepressant agents are threefold: rapid onset of

antidepressant response, broader efficacy, and fewer adverse effects. While

progress has been made to reduce side-effects, currently available

antidepressants do not show convincing evidence for a shorter delay of onset of

therapeutic actions neither for improved efficacy on the treatment of major

depression (Nutt, 2002).

Several studies have supported the idea that ionotropic glutamate N-

methyl-D-aspartate receptor (NMDA) is an important player in the etiology of

psychopathologies, such as anxiety disorders and major depression (Javitt,

2004; Krystal et al., 1999). A growing body of evidence has pointed to the

blockade of the NMDA receptor signaling as a potential therapeutic target for

the treatment of major depression. Several preclinical studies have

demonstrated that NMDA antagonists, such as MK-801, AP7, CPP,

neramexane and others, display anxiolytic- and antidepressant-like effects in

rats injected into distinct brain areas and subjected to various animal models of

anxiety and depression (Kos et al., 2006; Molchanov and Guimarães, 2002;

Menard and Treit, 2000; Matheus and Guimarães, 1997; Skolnick et al., 1996;

Maj et al., 1992).

Ketamine is a NMDA receptor antagonist which displays high affinity to

the phencyclidine binding site, within the ionotropic channel. The quite unique

ability of ketamine to block the NMDA receptor is based on: 1) higher affinity for

the NMDA receptor; 2) much slower open-channel blocking/unblocking kinetics;

3) a different type of channel closure (ie, “trapping block” as opposed to “partial

trapping” properties); 4) a non selective blockage of voltage-sensitive Ca2+

channels, and opioid, monoaminergic and muscarinic receptors, than other

NMDA receptor antagonist (Bolshakov et al., 2003; for a review: Hirota and

Lambert, 1996). Some of these properties may explain why ketamine was found

to have significant antidepressant actions. In fact, administration of ketamine

has been shown to induce antidepressant effects in humans as well as in

rodents subjected to animal models of depression (for a review see: Maeng and

Zarate, 2007). In a pilot study developed by Berman et al. (2000), seven human

patients with treatment resistant depression showed significant improvement in

depressive symptoms within 72 hours of ketamine treatment. Other studies

replicated these findings and the results confirmed a rapid onset of action for

ketamine on the treatment of major depression (Zarate et al., 2006a; Liebrenz

et al., 2007a; Liebrenz et al., 2007b). Studies with animals models showed that

acute and chronic administration of ketamine induces robust and sustained

antidepressant-like effects in distinct animal models of depression, such as

learned helplessness and forced swimming test (Chatuverdi et al., 2001;

Yalmaz et al., 2002; Garcia et al., 2008a, 2008b; Maeng et al., 2008).

The role played by neurotrophic factors, particularly the brain-derived

neurotrophic factor (BDNF), in neurogenesis has also been considered

important in the mechanism of action of antidepressant drugs (for a review see:

Castren et al., 2007). BDNF has important functions in the adult human brain as

a regulator of neuronal survival, fast synaptic transmission, and activity-

dependent synaptic plasticity (Lewin and Barde, 1996; Hashimoto et al., 2004;

Blum and Konnert, 2005). However, in rodents, a limited number of studies has

investigated the hippocampal BDNF protein levels in antidepressant-treated

animals and available data indicate that antidepressant treatment did not affect

the total amount of that hippocampal protein (Jacobsen and Mork, 2004; Garcia

et al., 2008a, 2008b). Very recently, we demonstrated that distinct from

imipramine – a tricyclic antidepressant drug, acute injection of ketamine

increased BDNF protein levels in the hippocampus of rats (Garcia et al.,

2008a)., suggesting therefore that ketamine could be an innovative

antidepressant drug by inducing distinct molecular effects on the rat

hippocampus compared to classic antidepressant.

In this context, the chronic mild stress (CMS) model has been shown to

induce lower consumption of sucrose (sweet food), postulated to reflect

anhedonia (the loss of interest or pleasure) in animals, one of the two core

symptoms required for diagnosis of a major depressive episode in humans

(Katz, 1981; Willner, 1987; Willner, 1997). The exposure of rats to CMS also

induces changes in hypothalamic-pituitary-adrenal axis, loss of body weight,

and adrenal hypertrophy, which leads to corticosterone hypersecretion

(Vollmayr and Henn, 2003). The CMS paradigm described by Willner and

collaborators (1987) is a model of depression obtained by using chronic

unpredictable mild stressors (Wilner, 2005). The CMS model is based on

intensive labor, space demanding and long stress procedure which consists of

exposing animals sequentially to a variety of mild and unpredictable “stressors”

(e.g. isolation, water and food deprivation, restraint, forced swimming, flashing

light exposure) for a period of 4-6 weeks. The protocol is regarded as being

close to model the human situation, consisting more of daily hassles than

traumatic events (Wilner, 2005).

The present study aimed to investigate behavioral and physiological effects of

acute and repeated administration of ketamine in rats submitted to the chronic

mild stress procedure. Rats were subjected to 40 days of chronic unpredictable

stressful stimuli and, afterwards, sucrose consumption was assessed through

repeated sessions in saline- and ketamine-treated rats. Additionally, as

physiological parameters, body and adrenal gland were weighed, corticosterone

and ACTH circulating levels were analyzed in the serum. Finally, the amount of

hippocampal BDNF protein levels was assessed in all experimental groups.

The present study aimed to investigate behavioral and physiological

effects of acute and chronic administration of ketamine in rats submitted to the

chronic mild stress procedure. Rats were subjected to 40 days of chronic

unpredictable stressful stimuli, and afterwards sucrose consumption was

assessed through repeated sessions in saline- and ketamine-treated rats.

Additionally, as physiological parameters, body and adrenal gland were

weighed, corticosterone and ACTH circulating levels were analyzed in the

serum. Finally, the amount of hippocampal BDNF protein levels was assessed

in all experimental groups.

2. MATERIAL AND METHODS

2.1 Animals

Male Wistar rats (3-4 months, 220-310 g) were obtained from our

breeding colony (UNESC). The animals were housed 5 to a cage with food and

water available ad libitum (except for the stressed group during the period when

the stressor applied required no food or no water) and were maintained on a 12-

h light/dark cycle (lights on at 7:00 am). All experimental procedures involving

animals were performed in accordance with the NIH Guide for the Care and

Use of Laboratory Animals and the Brazilian Society for Neuroscience and

Behavior (SBNeC) recommendations for animal care.

2.2 Drugs and Treatment

Ketamine (Fort Dodge Animal Health - Fort Dodge, IA, USA) in dose of

15 mg/kg (as previously used by Garcia et al., 2008a, 2008b) was injected

intraperitoneally, one day (acute treatment) or once a day, across 7 days

(chronic treatment) after CMS procedure. All treatments were administered in a

volume of 1 ml/kg.

To develop this study we employed 90 animals (n=15) separated in six groups,

as follows: 1) non stressed – saline; 2) non stressed - acute ketamine; 3) non

stressed - chronic ketamine; 4) stressed – saline; 5) stressed - acute ketamine;

6) stressed - chronic ketamine. The chronic treatment with ketamine was

performed during the anhedonia test, once a day, across 7 days, 60 min prior to

behavioral assessment. In the acute treatment, the unique dose of ketamine

was administered in the first day of the anhedonia test, also 60 min before the

behavioral test.

2.3 Experimental Procedure

Chronic mild stress (CMS) protocol was adapted from the procedure

described by Gamaro and collaborators (2003). The animals were divided in

two groups: control and stressed. During the 40 days of treatment, control rats

were kept undisturbed in their home cages according conditions previously

refered (see above: 2.1 Animals). The 40-days CMS paradigm was used for the

stressed group rats. Individual stressors and length of time applied each day

are listed on Table 1. The following stressors were used: (i) 24h of food

deprivation; (ii) 24h of water deprivation; (iii) 1-3 h of restraint as described later,

(iv) 1,5-2 h of restraint at 4°C; (v) forced swimming during 10 or 15 min as

described later; (vi) flashing light for 120-210 min; (vii) isolation (2-3 days).

Stressors stimuli were applied at different time everyday, in order to minimize its

predictability.

Restraint was carried out by placing the animal in a 25 x 7cm plastic tube

and adjusting it with plaster tape on the outside, so that the animal was unable

to move. There was a 1cm hole at the far end for breathing. Forced swimming

was carried out by placing the animal in a glass tank measuring 50 x 47cm with

30 cm of water at 23±2°C. Exposure to flashing light was made by placing the

animal in a 60 x 60 x 25 cm plywood made box divided in 16 cells of 15 x 15 x

25 cm with a frontal glass wall. It was used a 40 w lamp flashing at frequency of

60 flashes/minute.

2.4 Sweet food consumption (anhedonia test)

After 40 days of treatment, consumption of sweet food was measured to

verify anhedonia. The animals were placed in a rectangular box (40 × 15 × 20

cm) with a glass ceiling, floor and side walls made of wood and divided into 9

equal rectangles by black lines. This test was performed between 8:00 a.m. and

12:00 a.m. Ten Froot Loops (Kellogg’s® pellets of wheat and corn starch and

sucrose) were placed on an extremity of the box. Animals were submitted to 5

sessions of 3 min each, once a day, to familiarize with this food (training

sessions) and, afterwards, the animals were exposed to two test sessions, 3

min each, and the number of ingested pellets and the spontaneous locomotor

activity (number of crossings over the black lines and rearings) were measured

(Gamaro et al., 2003; Frey et al., 2006). After each rat be tested, it took 40

seconds to clean the apparatus in order to prevent the smell of the previous rat

from affecting the behavior of subsequent animals.

It was established that when the animal ate part of the Froot (e.g. 1/3 or

1/4), this fraction would be considered as one unit according previous studies

(Katz et al., 1981; Gamaro et al., 2003; Wilner, 2005; Grønli et al., 2006). These

two evaluations were made with the animals fed ad libitum. It was done since

food deprivation, which is used in many behavior tasks as a motivating stimulus,

may also be an acute stressor (Katz et al., 1981; Gamaro et al., 2003). Body

weight was measured at the beginning (1

day) of the chronic mild stress

protocol and after 7 days of finished protocol.

On the seventh day of consumption of sweet food, immediately after the

last Froot Loop session, under ether anesthesia, blood was collected by cardiac

puncture, for subsequent analyses of corticosterone and adrenocorticotropic

(ACTH) serum levels. After euthanizing the rats by decapitation, the brains were

dissected and hippocampi were isolated immediately and then stored at -80°C

for later analysis of BDNF protein levels. The adrenal gland (only the right one)

was removed, through laparotomy, and weighed in an analytical balance.

Adrenal gland weight was used in this study as an indirect parameter of

hypothalamic-pituitary-adrenal axis activation (Gamaro et al., 2003).

2.5 Corticosterone and ACTH circulating levels

Corticosterone levels were determined using enzyme immunoassay kits.

Serum concentrations of ACTH were determined using commercially available

radioimmunoassay kits for animals. Both analyses were performed by a

commercial laboratory blind to the experiments.

2.6 Rat hippocampal BDNF level measurement.

The measurement of BDNF hippocampal levels was performed, as

previously described (Frey et al., 2006), by anti-BDNF sandwich-ELISA,

according to the manufacturer instructions (Chemicon International Inc.,

Temecula, CA, USA). Briefly, rat hippocampus was homogenized in phosphate-

buffered solution with 1 mM phenylmethylsulfonyl fluoride and 1 mM

ethyleneglycoltetraacetic acid. Microtiter plates (96-well flat-bottom) were

coated for 24 hr with the samples diluted 1:2 in sample diluent and standard

curve ranged from 7.8 to 500 pg/ml of BNDF. The plates were then washed four

times with sample diluent and a monoclonal anti-BDNF rabbit antibody diluted

1:1000 in sample diluent was added to each well and incubated for 3 hr at room

temperature. After washing, a peroxidase-conjugated anti-BDNF rabbit antibody

(horseradish peroxidase enzyme; diluted 1:1000) was added to each well and

incubated at room temperature for 1 hr. After addition of streptavidin enzyme,

substrate (3,3′,5,5′-tetramethylbenzidine) and stop solution, the amount of

BDNF was determined by absorbance in 450 nm. The standard curve

demonstrates a direct relationship between optical density and BDNF

concentration. BDNF was expressed as pg of BDNF per ml of serum obtained

from hippocampal homogenate. Total protein was measured by Lowry’s method

using bovine serum albumin as standard.

2.7 Statistical analysis

The Statistical Package for the Social Sciences (SPSS) 15.0 was utilized

for statistical analyses. All data are presented as mean ± S.E.M (the data was

normally distributed). Differences among experimental groups were determined

by one-way ANOVA followed by Tukey’s post hoc test for sweet food intake,

locomotor activity, adrenal gland weight, corticosterone and ACTH circulating

levels and BDNF hippocampal levels. By contrast, body weight was analyzed by

employing Student’s t-test for paired data. P values less than 0.05 were

considered statistically significant.

RESULTS

As depicted in Figure 1, stressed rats treated with saline displayed

decreased sweet food intake compared with control rats treated with saline (F

(5-

77)

=24,109; p=0,0001). Statistical analyses revealed that chronic treatment with

ketamine increased sweet food intake when compared with stressed rats

treated with saline (F

(5-77)

=24,109; p=0,0001). Acute treatment with ketamine

(stressed rats) did not alter sweet food intake when compared with stressed

rats treated with saline (F

(5-77)

=24,109; p=0,218). Additionally, it should be noted

that non-stressed rats chronically treated with ketamine increased sweet food

intake when compared with control rats treated with saline (F

(5-77)

=24,109;

p=0,012).

The open-field test evaluate locomotor activity in rats after 40-days of

chronic stressful stimuli and it was not observed any changes in the number of

crossings (F

(5-77)

=0,088 p=0,994) and rearings (F

(5-77)

=1,400; p=0,234)

displayed by all groups (date not shown).

The effects of the CMS protocol in body weight is illustrated in Figure 2.

Stressed rats treated with saline failed to gain weight (t= 1.065; df=13;

p=0,306). In contrast, stressed animals gained weight after the treatment with

ketamine (acute and chronic) compared with the body weight assessed at the

beginning of the experiment. Before experimental procedure it was not

observed body weight difference between groups (F

(5-75)

=0,941; p=0,459).

Corticosterone, ACTH hormone levels and adrenal gland weight are

shown in Figure 3A, 3B and 3C respectively. Rats subjected to the CMS

procedure, and treated with saline, displayed increased corticosterone (F

(5-

23)

=5.149; p=0,023) and ACTH (F

(5-21)

=4.276; p=0,033) hormone levels

compared with non-stressed rats injected with saline. The treatment of stressed

rats with ketamine compared with those treated with saline reverted the

increase of circulating cortisol in acute (F

(5-23)

=5.149; p=0,004) and chronic

treatment (F

(5-23)

=5.149; p=0,002) and ACTH in acute (F

(5-21)

=4.276; p=0,017)

and chronic treatment (F

(5-21)

=4.276; p=0,008) hormone levels compared with

stressed rats treated with saline. CMS procedure induced an increase of rat

adrenal gland weight in stressed rats treated with saline compared with non-

stressed rats injected with saline (F

(5-67)

=14,717; p=0,001). Interesting enough,

the treatment with ketamine (acute and chronic) reestablished a normal range

of adrenal gland weight in stressed rats. In the figure 2, non-stressed rats

treated with saline or ketamine (acute and chronic) gained weight compared

with the beginning of the experiment.

The analyses of hippocampus BDNF levels in stressed rats, as well as in

non stressed animals, injected with saline did not display any alteration (F

(5-

32)

=0.926; p=0,409). In the same way, ketamine (acute and chronic) treatment

did not alter BDNF protein levels in the hippocampus independent of stress

condition (date not shown).

DISCUSSION

The present study demonstrated that: (1) CMS rats displayed reduced sweet

food intake, without significant changes in locomotor activity; (2) CMS rats failed

to gain weight, and displayed hypertrophy of adrenal gland compared to non-

stressed rats; (3) CMS rats displayed increase of corticosterone and ACTH

circulating levels compared to non-stressed rats; (4) hippocampus BDNF

protein levels were not altered in the CMS rats; (5) chronic (but not acute)

administration of ketamine reversed the anhedonic behavior induced by chronic

stressful stimuli, and (6) acute/chronic ketamine treatment reversed the

increase of adrenal gland weight and corticosterone and ACTH circulating

levels, and prompted the gain of body weight in CMS rats and finally (7)

acute/chronic ketamine treatment did not alter hippocampal BDNF protein levels

in CMS rats.

The CMS paradigm is a model of depression characterized for chronic

unpredictable mild stressors (Willner, 2005). In the CMS model, both

consumption of and preference for sucrose intake, as well as decreased

intracranial self stimulation behavior, have served as markers of generalized

decrease in sensitivity to reward and it is quite related to anhedonia (Willner et

al., 1987; Gamaro et al., 2003; Berkris et al., 2005). In accordance with the

literature, present data confirm that rats exposed to CMS procedure, and

treated with saline, consumed less sweet food compared to non-stressed rats

treated with saline. These findings suggest that, under our experimental

conditions, the CMS procedure induced anhedonic behavior in our rats.

The present findings revealed that repeated administration of ketamine

reversed the anhedonic behavior in CMS rats and, indeed, increased sweet

food consumption in non-stressed rats. In fact, literature findings show that

distinct NMDA receptor antagonists, such as AP-1 and MK801, increase food

intake in rodents (Burns and Ritter, 1998; Treece et al., 2000; Jahng and Houpt,

2001). These behavioral effects seem to be a mixture of central and visceral

actions of NMDA receptor antagonists. Burns and Ritter (1998) have shown that

the destruction of small unmyelinated visceral afferent neurons, which carry the

satiety signals from viscera to satiety centre in the CNS, blocked the increase of

food intake in MK801 treated rats. Additionally, Treece and colleagues (2000)

have shown that the lesion of caudomedial subnucleus of the solitary tract

and/or dorsal motor nucleus of the vagus in MK801-treated rats resulted in

nearly the same amount of consumption of sucrose as compared with saline.

Based on these findings, we cannot rule out the hypothesis that ketamine

reversed the anhedonic behavior in rats subjected to CMS due to a direct effect

of this drug on food consumption. However, failed to gain weight in stressed

rats should not be ignored. Emotional changes, such as exposure to stress

situations, can influence feeding behavior, and studies have demonstrated that

chronic exposure to stressors may alter body weight of rats (Dess et al., 1988;

Gamaro et al., 2003; Berkris et al., 2005). In accordance with literature data, our

findings also demonstrated loss of body weight in CMS rats compared to non-

stressed rats. Interesting enough, CMS rats acutely or chronically treated with

ketamine regain body weight compared with stressed rats injected with saline.

Thus, the rapid recovery of body weight in stressed rats could suggest that

NMDA receptors could be modulating feeding behavior during stressful

situations.

Our findings also demonstrated an increase in adrenal gland weight in

CMS rats compared with non-stressed rats. Interestingly, acute and repeated

administration of ketamine reversed the hypertrophy of adrenal gland in

stressed rats. Distinct authors have already suggested an increase of rat

adrenal weight after 14 (Harro et al., 2001) or 28 days (Konarska et al., 1990) of

CMS paradigm. The hypertrophy of adrenal gland could be mediated by the

increase of ACTH circulating hormone, which is released in high concentrations

during stressful situations by anterior pituitary gland (O'Connor et al., 2000).

According to this view, our findings showed that ACTH and corticosterone

circulating levels were increased in stressed rats compared with non-stressed

animals. Importantly, acute and repeated administration of ketamine reversed

these hormonal alterations in CMS rats, thus, reinforcing the idea of a relevant

role played by glutamatergic signaling, via NMDA receptors, in the mediation of

physiological aspects of stress.

In the context, several studies support the hypothesis of BDNF

involvement in depression and suggest that depressive disorders induce a

marked decrease in plasma BDNF levels (for a review see: Castren et al., 2007;

Lee et al., 2007). Actually, reduced brain BDNF levels have been found in

postmortem samples from depressed patients, and antidepressant therapy

restores BDNF brain levels to the normal range in human beings (Castren et al.,

2007). Importantly, present study showed that CMS rats treated with saline did

not display alteration in BDNF protein levels in the rat hippocampus compared

with non-stressed group. In accordance with our findings, Grønli and colleagues

(2006) demonstrated that rats exposed for 5 weeks to repeated, unpredictable,

mild stressors showed reduced BDNF expression and inhibited in the dentate

gyrus, whereas no significant effects were observed in the hippocampus proper.

Thus, suggesting that BDNF protein alterations could occur after stressful

situations in some specific hippocampal areas, but not in the entirely structure.

Additionally, we should not rule out the fact that hippocampal BDNF protein

levels could be normalized while the BDNF measurement was being performed,

since these analyses were done on the 7th day after CMS protocol. In acute

administration of ketamine (15 mg/kg) it was demonstrated an increase in

BDNF protein levels in the rat hippocampus (Garcia et al., 2008a), while

repeated (14 days) administration of ketamine (at the same dose) did not alter

this hippocampal protein levels (Garcia et al., 2008b).

The hippocampus has been implicated in many cognitive aspects of

depression and the heterogeneity of clinical findings in major depression

suggests that multiple neurocircuits and neurochemicals aspects are involved in

its pathogenesis. Anhedonia is a particularly characteristic feature of major

depression and may provide insights into its underlying neurobiology.

Importantly, this symptom appears to be mediated by dopaminergic mesolimbic

and mesostriatal projections that are, in turn, influenced by key gene variants

and environment stressors (for a review: Stein, 2008). It is known that stress

decreases the expression of BDNF in limbic structures and that chronic

antidepressant treatment reverses the effects of stress. These decreased levels

of BDNF could contribute to the atrophy of certain limbic structures, including

the hippocampus and prefrontal cortex, that has been observed in depressed

subjects (for reviews: Duman and Monteggia, 2006; Castren et al., 2007).

In this study, we did not evaluated available BDNF levels in mesolimbic

system, but we cannot rule out the idea that this protein can be found in this

area. We evaluated hippocampus, one of the several limbic structures that have

been implicated in mood disorders. Hippocampal circuitry is also involved in

learning and memory functions and in regulation of the hypothalamic-pituitary-

adrenal axis that are altered in depression. In addition, the hippocampus has

connections with amygdala and prefrontal cortex, regions that show straight

involvement in emotion and cognition and, thereby contribute to other major

symptoms of depression (for review: Duman and Monteggia, 2006). Our data

demonstrate that repeated administration of ketamine (15 mg/kg) did not modify

hippocampal BDNF protein levels neither in CMS rats nor in control animals

however, it should be considered that the failure in reduction of hippocampal

BDNF protein levels in CMS rats could influence a possible effect of ketamine

on the amount of BDNF protein available in the hippocampus or, in other words,

ketamine would not be able to increase BDNF levels if stress did not reduce the

amount of hippocampal protein. Thus, ketamine action could have suffered

consequences of changes in HPA axis or even received interference of other

signalizations pathways.

CONCLUSION

The present findings suggest that chronic treatment with ketamine reversed the

effect of stress in rats subjected to CMS model and, thus reversed the decrease

on sweet food intake and this behavior is related to anhedonia. Additionally, our

findings revealed that acute as well as repeated administration of ketamine

reversed loss of body weight, the hypertrophy of adrenal glands, and the

increase of ACTH and corticosterone circulating levels. These findings could

suggest a participation of glutamatergic activation, via NMDA receptors, in the

mediation of physiological changes in rats subjected to stressful situations.

Finally, further studies aiming to investigate the mechanisms by which ketamine

induces antidepressant-like effects are mandatory for the development of

innovative antidepressant drugs with rapid onset of action.

Acknowledgements: This research was supported by grants from CNPq

(ECG, FK and JQ), FAPESC (ECG and JQ), Instituto Cérebro e Mente (FK,

ECG and JQ) and UNESC (ECG and JQ). FK and JQ are CNPq Research

Fellows. SSV is holder of a CAPES studentship, CMC is holder of a CNPq

Studentship, and GZR is holder of a FAPESC/CAPES studentship.

Statement of Interest: none.

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antagonist in treatment-resistant major depression. Arch Gen Psychiatry 2006;

63:856–864.

TABLES

Table 1 - Schedule of stressor agent used during the chronic treatment.

Day of

treatment

Stressor

used

Duration Time of

day

Day 1 Water deprivation 24 h 8:00 a.m.

Day 2 Food deprivation 24 h 1:00 p.m.

Day 3 Isolation 24 h 6:00 p.m.

Day 4 Isolation 24 h -

Day 5 Isolation 24 h -

Day 6 Flashing light 3 h 3:00 p.m.

Day 7 Food deprivation 24 h 7:00 a.m.

Day 8 Forced swimming 10 min 8:00 p.m.

Day 9 Restraint 1 h 12:00 p.m.

Day 10 Water deprivation 24 h 10:00 a.m.

Day 11 No stressor

applied

- -

Day 12 No stressor

applied

- -

Day 13 Restraint + cold 2 h 9:00 a.m.

Day 14 Flashing light 2.5 h 1 p.m.

Day 15 Food deprivation 24 h 4 p.m.

Day 16 Forced swimming 15 min 11 a.m.

Day 17 Isolation 24 h 8:00 p.m.

Day 18 Isolation 24 h -

Day 19 Isolation 24 h -

Day 20 Water deprivation 24 h 10:00 a.m.

Day 21 Food deprivation 24 h 2:00 p.m.

Day 22 Flashing light 3 h 9:00 p.m.

Day 23 Restraint 2 h 7:00 a.m.

Day 24 Isolation 24 h 4 p.m.

Day 25 Isolation 24 h -

Day 26 Restraint + cold 1.5 h 11:00 a.m.

Day 27 Forced swimming 10 min 3:00 p.m.

Day 28 Flashing light 3.5 h 5:00 p.m.

Day 29 No stressor

applied

- -

Day 30 Food deprivation 24 h 10:00 a.m.

Day 31 Restraint 3 h 10:00 p.m.

Day 32 Flashing light 2 h 11:00 a.m.

Day 33 Water deprivation 24 h 1:00 p.m.

Day 34 Restraint + cold 2 h 7:00 a.m.

Day 35 Forced swimming 15 min 10:00 p.m.

Day 36 Isolation 24 h 9:00 a.m.

Day 37 Isolation 24 h 3:00 a.m.

Day 38 No stressor

applied

- -

Day 39 Flashing light 3 h 6:00 p.m.

Day 40 Forced swimming 10 min 8:00 a.m.

FIGURES

FIGURE 1 - Garcia et al.

FIGURE 2 - Garcia et al.

Control +

Saline

Control +

Acute

Ketamine

Control +

Chronic

Ketamine

CMS +

Saline

CMS + Acute

Ketamine

CMS +

Chronic

Ketamine

Sucrose Intake

(mean of pellets consume

in test sessions)

***

100

150

200

250

300

350

400

450

500

Control +

Saline

Control +

Acute

Ketamine

Control +

Chronic

Ketamine

CMS +

Saline

CMS +

Acute

Ketamine

CMS +

Chronic

Ketamine

body weight (g)

Before

After

FIGURE 3 - Garcia et al.

0,5

1,5

2,5

3,5

Control +

Saline

Control +

Acute

Ketamine

Control +

Chronic

Ketamine

CMS +

Saline

CMS +

Acute

Ketamine

CMS +

Chronic

Ketamine

Corticosterone levels ug/dL

***

100

200

300

400

500

Control +

Saline

Control +

Acute

Ketamine

Control +

Chronic

Ketamine

CMS + Saline CMS + Acute

Ketamine

CMS +

Chronic

Ketamine

ACTH levels pg/ml

***

0,05

0,1

0,15

0,2

0,25

Control +

Saline

Control +

Acute

Ketamine

Control +

Chronic

Ketamine

CMS +

Saline

CMS +

Acute

Ketamine

CMS +

Chronic

Ketamine

adrenal weight (g)

***

LEGENDS OF FIGURES

Figure 1 – Effects of CMS procedure on sweet food consumption (pellets

consumed in 2 days of tests) in rats acute and repeatedly treated with ketamine.

Bars represent means ± S.E.M. of 15 rats. *p<0.05 vs. non-stressed + saline,

**p<0,05 vs. CMS + saline, # p<0,05 vs. non-stressed + saline, according to

ANOVA post-hoc Tukey test.

Figure 2 - Effects of CMS procedure on body weight of rats acute and

repeatedly treated with ketamine. Bars represent means ± S.E.M. of 15 rats.

*p<0,05 vs. before CMS protocol, according to Student t test for paired date.

Figure 3 - Effects of CMS procedure on corticosterone (3A), adrenocorticotropic

hormone (3B) circulating levels and adrenal gland weight (3C) in rats acute and

repeatedly treated with ketamine. Bars represent means ± S.E.M. of 15 rats.

*p<0.05 vs. control + saline, **p<0.05 vs. CMS + acute ketamine and ***p<0.05

vs. CMS + chronic ketamine, according to ANOVA post-hoc Tukey test.

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