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SUSANA MARIA SOUSA SILVA FERREIRA

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DISSERTAÇÃO DE CANDIDATURA AO GRAU DE DOUTOR APRESENTADA À FACULDADE DE

MEDICINA DA UNIVERSIDADE DO PORTO

PORTO 2012

Artigo 48º, parágrafo 3 – A Faculdade não responde pelas doutrinas expendidas na

dissertação. (Regulamento da Faculdade de Medicina do Porto – Decreto 19 337, de 29 de

Janeiro de 1931).

Esta investigação foi realizada no Centro de Morfologia Experimental – Unidade 121/94 da

Fundação para a Ciência e a Tecnologia (FCT), sediado no Departamento de Anatomia da

Faculdade de Medicina da Universidade do Porto.

Orientador: Professora Doutora Maria Dulce Cordeiro Madeira (Professora Catedrática da

Faculdade de Medicina da Universidade do Porto).

A candidata declara que teve uma contribuição determinante na realização do trabalho

experimental (execução de protocolos e elaboração técnica), na interpretação dos resultados e

discussão dos mesmos. Além disso, contribuiu activamente para a redacção dos trabalhos

apresentados.

Corpo Catedrático da Faculdade de Medicina da Universidade do Porto

Professores Efectivos

Doutor Manuel Alberto Coimbra Sobrinho Simões

Doutor Jorge Manuel Mergulhão Castro Tavares

Doutora Maria Amelia Duarte Ferreira

Doutor José Agostinho Marques Lopes

Doutor Patrício Manuel Vieira Araújo Soares Silva

Doutor Daniel Filipe Lima Moura

Doutor Alberto Manuel Barros da Silva

Doutor José Manuel Lopes Teixeira Amarante

Doutor José Henrique Dias Pinto de Barros

Doutora Maria Fátima Machado Henriques Carneiro

Doutora Isabel Maria Amorim Pereira Ramos

Doutora Deolinda Maria Valente Alves Lima Teixeira

Doutora Maria Dulce Cordeiro Madeira

Doutor Altamiro Manuel Rodrigues Costa Pereira

Doutor Rui Manuel Almeida Mota Cardoso

Doutor António Carlos Freitas Ribeiro Saraiva

Doutor Álvaro Jerónimo Leal Machado de Aguiar

Doutor José Carlos Neves da Cunha Areias

Doutor Manuel Jesus Falcão Pestana Vasconcelos

Doutor João Francisco Montenegro Andrade Lima Bernardes

Doutora Maria Leonor Martins Soares David

Doutor Rui Manuel Lopes Nunes

Doutor José Eduardo Torres Eckenroth Guimarães

Doutor Francisco Fernando Rocha Gonçalves

Doutor José Manuel Pereira Dias de Castro Lopes

Doutor Manuel António Caldeira Pais Clemente

Doutor António Albino Coelho Marques Abrantes Teixeira

Doutor Joaquim Adelino Correia Ferreira Leite Moreira

Professores Jubilados ou Aposentados

Doutor Abel José Sampaio da Costa Tavares

Doutor Abel Vitorino Trigo Cabral

Doutor Alexandre Alberto Guerra Sousa Pinto

Doutor Álvaro Jerónimo Leal Machado de Aguiar

Doutor Amândio Gomes Sampaio Tavares

Doutor António Augusto Lopes Vaz

Doutor António Carvalho Almeida Coimbra

Doutor António Fernandes da Fonseca

Doutor António Fernandes Oliveira Barbosa Ribeiro Braga

Doutor António Germano Pina Silva Leal

Doutor António José Pacheco Palha

Doutor António Luís Tomé da Rocha Ribeiro

Doutor António Manuel Sampaio de Araújo Teixeira

Doutor Belmiro dos Santos Patrício

Doutor Cândido Alves Hipólito Reis

Doutor Carlos Rodrigo Magalhães Ramalhão

Doutor Cassiano Pena de Abreu e Lima

Doutor Daniel Santos Pinto Serrão

Doutor Eduardo Jorge Cunha Rodrigues Pereira

Doutor Fernando de Carvalho Cerqueira Magro Ferreira

Doutor Fernando Tavarela Veloso

Doutor Francisco de Sousa Lé

Doutor Henrique José Ferreira Gonçalves Lecour de Menezes

Doutor José Augusto Fleming Torrinha

Doutor José Carvalho de Oliveira

Doutor José Fernando Barros Castro Correia

Doutor José Luís Medina Vieira

Doutor José Manuel Costa Mesquita Guimarães

Doutor Levi Eugénio Ribeiro Guerra

Doutor Luís Alberto Martins Gomes de Almeida

Doutor Manuel Augusto Cardoso de Oliveira

Doutor Manuel Machado Rodrigues Gomes

Doutor Manuel Maria Paula Barbosa

Doutora Maria da Conceição Fernandes Marques Magalhães

Doutora Maria Isabel Amorim de Azevedo

Doutor Mário José Cerqueira Gomes Braga

Doutor Serafim Correia Pinto Guimarães

Doutor Valdemar Miguel Botelho dos Santos Cardoso

Doutor Walter Friedrich Alfred Osswald

Aos meus Pais,

à Sofia

Ao Vítor,

ao Miguel

À Professora Doutora Maria Dulce Cordeiro Madeira

AAGGRRAADDEECCIIMMEENNTTOOSS

Um trabalho desta natureza é resultado de um longo percurso, comportando uma série de anseios,

preocupações e desafios que, normalmente, se enfrentam sozinho. Este, certamente, não foi o caso.

Assim, este trabalho é fruto de contributos múltiplos, sem os quais a sua realização se tornaria quase

impossível. Foi graças ao apoio incondicional de algumas pessoas, às quais não posso deixar de

manifestar o meu profundo agradecimento, que hoje esta tese constitui uma realidade.

À Professora Doutora Maria Dulce Cordeiro Madeira, a quem quero expressar o meu mais sentido

reconhecimento pelo seu apoio e ensinamentos, não apenas como Orientadora mas, acima de tudo,

como uma verdadeira “MÃE” do mundo profissional. São alguns apanágios da sua personalidade, o

esforço, a dedicação, o entusiasmo, o rigor, o empenho, o dinamismo, a determinação, que

igualmente transmite com todo o carinho e detalhe a cada um dos seus discípulos. Foi pelas suas

mãos que dei os primeiros passos no mundo da morfologia e, mais tarde, na docência. Aprendi com o

seu exemplo e orientação as características que definem um verdadeiro universitário. Cada linha

desta dissertação é obra sua. Estou-lhe profundamente agradecida, mesmo nos momentos mais

difíceis as suas palavras foram sempre motivadoras, muitas vezes resumidas numa única palavra,

ATITUDE.

Ao Professor Doutor Manuel Maria Paula Barbosa, pela mão de quem cheguei ao então Instituto de

Anatomia, o meu profundo agradecimento pelo empenho constante na minha formação científica e

pedagógica. Desde o primeiro dia a sua disponibilidade, rigor e dedicação, a par das críticas e

sugestões orientadoras em momentos oportunos, marcaram e serão sempre perpectuados na minha

caminhada profissional. Foi pela sua mão que aprendi a ver o núcleo paraventricular, cerne desta

dissertação, com o detalhe e exactidão que hoje o vejo. Agradecer tão preciosa ajuda parece-me

muito pouco, quando ao meu lado tive sempre o exemplo de um verdadeiro MESTRE e AMIGO.

Ao Professor Doutor José Paulo Andrade agradeço a amizade e todo o apoio que sempre me

dispensou desde a primeira hora. Aos Professores Doutores António Cadete Leite e Carlos Ruela

agradeço a amizade e o apoio que deles também recebi.

Entre os colegas do Departamento gostaria particularmente de agradecer àqueles com quem de perto

laborei nas estradas da morfologia. Ao Professor Doutor Pedro Pereira, com quem tanto discuti na

alegria e na tristeza, desejo realçar a amizade que nos une já desde longa data e salientar as suas

exímias qualidades de docente e investigador dedicado e perseverante. À Professora Doutora Susana

Sá, a sua confiança nos momentos mais difíceis foi crucial. A nossa amizade teve início ainda na

Faculdade de Ciências do Porto, espero que perdure por muitos e longos anos. Ao Professor Doutor

Armando Cardoso, meu também companheiro, a certeza de que dificilmente se encontra hoje um

grupo tão coeso e unido pelos laços do trabalho. Tenho imenso orgulho de integrar este

extraordinário grupo dedicado ao ensino e à ciência.

Reconheço igualmente o grande valor de todo o corpo docente e técnico do Departamento de

Anatomia e agradeço toda a amizade, colaboração e preciosos ensinamentos com que sempre contei.

Recordo e agradeço a todos os elementos que, algum dia fizeram também parte desta enorme equipa,

através da sua generosidade e deferência enriqueceram ao longo destes anos cada dia de labuta neste

Departamento.

Aos meus Pais, meu orgulho, minha referência, meu esteio, minha inspiração, meus professores,

agradeço-vos pelas oportunidades que me ofereceram, por acreditarem incondicionalmente em mim

mesmo quando eu duvidava. As vossas palavras, que nunca esquecerei, fizeram certamente a

diferença. Tudo o que hoje sou a vocês o devo e sem o vosso apoio aqui nunca teria chegado.

À Sofia, que para além de ser a melhor irmã do mundo, sempre me demonstrou confiança, força,

incentivo e determinação, fortalecendo-me nos momentos em que me sentia a fraquejar. Tornaste

sempre claro aquilo que para mim muitas vezes era uma incerteza.

Da mesma forma não podiam também ser esquecidos... todos os meus familiares e amigos pelo apoio

e compreensão de sempre…

Ao meu príncipe, Miguel, a maior riqueza que possuo. O meu pesar pelas poucas horas que

brincamos juntos e as muitas em que não estive presente para te ver crescer, mas a certeza de que,

como sempre, será tudo para ti…

A ti Vítor, amor da minha vida, o meu muito obrigado pela compreensão e inesgotável paciência com

que suportaste as tantas horas de ausência a que este trabalho obrigou. Aqui testemunho a admiração

que sinto por ti e a felicidade de te ter como fiel companheiro e apaixonado pai do nosso “piolhito”.

LLIISSTTAA DDEE AABBRREEVVIIAATTUURRAASS

A1 Grupo celular A1 da formação reticular noradrenérgica do tronco cerebral

A2 Grupo celular A2 da formação reticular noradrenérgica do tronco cerebral

ACTH Hormona adrenocorticotrófica

C1 Grupo celular C1 da formação reticular catecolaminérgica do tronco cerebral

CRH Corticoliberina

ER-α Receptores de estrogénios do tipo α

ER-β Receptores de estrogénios do tipo β

GABAA Receptor ionotrópico do ácido gama-aminobutírico

HPA Eixo hipotálamo-hipófise-suprarrenal

IL-1 β Interleucina-1 β

IL-6 Interleucina-6

LPS Lipopolissacarídeo

mRNA Ácido ribonucleico mensageiro

NMDA N-metil D-Aspartato

PVN Núcleo paraventricular do hipotálamo

PVNmp Divisão parvocelular medial do núcleo paraventricular do hipotálamo

SNC Sistema nervoso central

TNF-α Factor de necrose tumoral-α

TRH Hormona libertadora de tireotrofina

ÍÍNNDDIICCEE

INTRODUÇÃO 1

TRABALHOS 15

Prolonged alcohol intake leads to reversible depression of

corticotropin-releasing hormone and vasopressin immunoreactivity

and mRNA levels in the parvocellular neurons of the paraventricular

nucleus 17

Sexually dimorphic response of the hypothalamo-pituitary-adrenal

axis to chronic alcohol consumption and withdrawal 31

Effects of chronic alcohol consumption and withdrawal on the

response of the male and female hypothalamic-pituitary-adrenal

axis to acute immune stress 47

Prolonged alcohol intake leads to irreversible loss of vasopressin and

oxytocin neurons in the paraventricular nucleus of the hypothalamus 61

DISCUSSÃO GERAL 77

CONCLUSÕES 89

REFERÊNCIAS 93

RESUMO 113

ABSTRACT 117

1

IINNTTRROODDUUÇÇÃÃOO

3

O consumo de álcool, e o exagero de que frequentemente é alvo, acompanha a história da

humanidade desde os seus primórdios, sendo o alcoolismo hoje amplamente reconhecido

como uma doença crónica, em cuja etiologia e desenvolvimento são relevantes tanto a

vulnerabilidade genética como factores sociais e ambientais (McLellan et al., 2000). De entre

as substâncias de abuso comum, o álcool é a mais frequentemente consumida em todo o

mundo, tendo ocupado desde sempre lugar privilegiado a nível sociocultural, como ícone de

rituais religiosos e de celebrações de natureza secular. Sendo uma molécula muito solúvel na

água, difunde-se facilmente por todos os tecidos (Mukherjee et al., 2007) e para o interior das

suas células (Hipólito-Reis, 2008). É, por isso, responsável pelo aparecimento de uma

panóplia de patologias que afectam potencialmente todos os órgãos do corpo (Lieber, 1995;

Rehm et al., 1996; NIAAA, 2000b; Mukherjee et al., 2007), de que se destacam as do foro

gastrenterológico, cardiovascular, ósseo, imunológico, muscular e neurológico (NIAAA,

2000a; Mukherjee et al., 2007). Apesar dos efeitos benéficos que têm sido atribuídos ao

consumo moderado de álcool, especialmente na redução do risco de doença coronária,

acidentes vasculares cerebrais e diabetes mellitus (Sacco et al., 1999; Rehm et al., 2003;

Collins et al., 2009), o álcool é apontado como factor causal de aproximadamente 2,5 milhões

de mortes por ano (WHO, 2011). Como factor de risco associado a diferentes patologias, o

álcool ocupa o terceiro lugar nos países desenvolvidos, e o primeiro nos países com reduzida

taxa de mortalidade infantil (Li, 2008).

Cerca de 2 biliões de pessoas em todo o mundo consomem álcool, estimando-se que,

destas, 76 milhões sofram de patologias decorrentes da sua ingestão. Dados constantes do

Global Status Report on Alcohol and Health (WHO, 2011) indicam que o consumo crónico

de álcool contribui, por si só, para 4,5% do peso global da doença. De acordo com os

resultados publicados pelo World Drink Trends (2005), referentes a estimativas do consumo

de álcool por pessoas com idade superior a 15 anos, Portugal ocupava, em 2003, a oitava

posição no ranking mundial, com um consumo de 9,6 litros per capita. Este consumo parece

vir a aumentar progressivamente já que, segundo os últimos dados estatísticos disponíveis e

que constam do Global Status Report on Alcohol and Health (WHO, 2011), o consumo de

4

álcool em Portugal foi, em 2005, de 12,4 litros por pessoa. Curiosamente, o consumo médio

de álcool em todo o mundo era de 6,1 litros por pessoa e por ano, valor este inferior às

estimativas referentes ao consumo per capita em Portugal no mesmo ano (WHO, 2011). Este

valor é, como seria de esperar, superior ao estimado para o consumo de vinho pela mesma

população, tendo Portugal ocupado, entre 2003-2005, a terceira posição no ranking mundial,

logo atrás do Luxemburgo e da França, com um consumo de 6,7 litros per capita (WHO,

2011).

Trabalhos desenvolvidos nas últimas décadas têm demonstrado que as consequências

do consumo crónico de álcool são diferentes nos dois sexos, sendo mais nefastas na mulher

(Angove e Fothergill, 2003; Dettling et al., 2007). Este facto poderá estar relacionado com os

níveis de alcoolémia. Com efeito, embora o consumo médio de álcool seja, na maioria das

populações estudadas, cerca de quatro vezes superior no homem (WHO, 2011), as mulheres

atingem alcoolémias mais elevadas após ingestão de idêntica quantidade de álcool (Scott,

2000). Pensa-se que para este facto contribuem diferenças sexuais na massa muscular, no

volume total de água e no teor de lipídeos (Dettling et al., 2008) e, ainda, na taxa de

metabolização de álcool, dadas as diferenças existentes entre os sexos tanto na actividade da

desidrogenase alcoólica gástrica como na taxa de oxidação hepática do álcool (Baraona et al.,

2001; Chrostek et al., 2003). Compreende-se, pois, que as mulheres apresentem, quando

comparadas com os homens, risco acrescido de cirrose hepática e pancreatite, mesmo após

ingestão de menores quantidades de álcool e mais curtas histórias de alcoolismo (Thakker,

1998; Mann et al., 2003), e que sejam mais susceptíveis ao desenvolvimento de doenças

cardiovasculares e miopatias (Urbano-Márquez et al., 1995; Murray et al., 2002). É hoje

também claro que a probabilidade do consumo crónico de álcool causar danos no sistema

nervoso central (SNC) é maior na mulher que no homem (revisto em Hommer, 2003; Ceylan-

Isik et al., 2010). Com efeito, a prevalência de alterações neurológicas resultantes do

consumo de álcool, de que são exemplo algumas demências, disfunções cognitivas e o

clássico síndrome de Wernicke-Korsakoff, é mais elevada nas mulheres que nos homens

(Hommer et al., 2001; Sohrabji, 2002). Sabe-se, também, que a ingestão prolongada de álcool

nas mulheres se associa a redução do volume da formação do hipocampo (Agartz et al., 1999)

e da área do corpo caloso (Hommer et al., 1996), alterações estas não identificadas até hoje

nos homens, e a atrofia cerebral de grau semelhante ao documentado em homens com história

de consumo de álcool mais prolongada (Hommer et al., 2001; Mann et al., 2005).

Embora as técnicas neuroimagiológicas actualmente disponíveis tenham vindo a

contribuir, de modo significativo, para desvendar as alterações neurológicas decorrentes da

5

ingestão prolongada de álcool e para o desenvolvimento de terapêuticas que auxiliem no

combate a esta doença (Garbutt et al., 2005), a compreensão do modus operandi desta noxa

no SNC continua, ainda hoje, a representar um desafio para os investigadores. No decurso das

últimas décadas, muito se progrediu na identificação e caracterização das alterações

morfológicas e perturbações funcionais resultantes do consumo crónico de álcool através da

utilização de modelos experimentais de alcoolização. Assim, foi possível demonstrar que, ao

invés das exposições agudas, o consumo crónico de álcool provoca alterações degenerativas,

incluindo morte neuronal (Walker et al., 1980; Phillips e Cragg, 1983; Cadete-Leite et al.,

1988b; Madeira et al., 1993; Paula-Barbosa et al., 1993; Lukoyanov et al., 1999), redução do

comprimento das arborizações dendríticas (Tavares et al., 1983; Cadete-Leite et al., 1988b,

1989a), perda de sinapses (Tavares e Paula-Barbosa, 1984; Cadete-Leite et al., 1986; Cadete-

Leite et al., 1989b) e de poros nucleares (Andrade et al., 1988), alterações dos organelos

celulares (Paula-Barbosa et al., 1986; Cadete-Leite et al., 1988a; Ruela et al., 1994) e

modificação do padrão neuroquímico de populações neuronais (Cadete-Leite et al., 1995,

1997, 2003; Falco et al., 2009) em áreas tão diversas como os córtices cerebral e cerebeloso, a

formação do hipocampo, a amígdala, o hipotálamo, as áreas septais e os núcleos da base. Para

além disso, desencadeia fenómenos de reorganização neuronal em diversas regiões do SNC

(Tavares et al., 1986; Cadete-Leite et al., 1988b) e altera muitos dos sistemas de

neurotransmissores cerebrais (revisto em De Witte, 1996; Fadda e Rossetti, 1998) e seus

receptores, sendo de salientar o seu efeito potenciador da acção dos receptores NMDA e

depressor da função dos receptores GABAA (revisto em Dodd et al., 2000; Kumar et al.,

2009).

O núcleo paraventricular (PVN) é de todos os agrupamentos celulares do hipotálamo o que

melhor reflecte a extrema complexidade estrutural e funcional desta área diencefálica.

Fundamental para a integração das respostas somatomotoras, endócrinas e autónomas

necessárias à manutenção da homeostasia, encontra-se localizado na zona periventricular da

região anterior (quiasmática ou supra-óptica) do hipotálamo (Simerly, 2004). De forma alar e

com cerca de 0,5 mm3 de volume, é um agregado celular caracterizado pela diversidade

morfológica e neuroquímica dos seus neurónios e pela multiplicidade das conexões que

estabelece com outras regiões do SNC (para revisão, ver Pacák, 2000; Herman et al., 2003).

Embora não exista uniformidade na nomenclatura relativa ao parcelamento do PVN

6

(Armstrong et al., 1980; Swanson et al., 1986; Kiss et al., 1991), a mais utilizada identifica

oito divisões, todas elas heterogéneas quanto às características citológicas, neuroquímicas e

hodológicas dos seus neurónios. Nesta classificação, da responsabilidade de Swanson e

Kuypers (1980), são reconhecidas as divisões magnocelulares anterior, medial e posterior, e

as parvocelulares anterior, periventricular, medial, dorsal e lateral. Algumas das eferências

destas divisões estão relativamente bem caracterizadas, o que permitiu a sua agregação em

dois grandes grupos que se distinguem sobretudo pelos seus atributos funcionais (Swanson e

Sawchenko, 1983; Simmons e Swanson, 2008): os neurónios visceromotores, que projectam

para núcleos do tronco cerebral e da medula espinhal e são responsáveis pelo controlo de

respostas autónomas e de comportamentos somatomotores, e os neurónios neurosecretores,

que estão envolvidos na regulação da produção hormonal. Destes, uns são magnocelulares e

projectam para a neuro-hipófise e, os restantes, parvocelulares e enviam as suas projecções

para a lâmina externa da eminência mediana (revisto em Swanson e Sawchenko, 1983).

Os neurónios visceromotores, ou pré-autónomos, do PVN são maioritariamente

mediocelulares (Stern, 2001) e ocupam as divisões parvocelulares dorsal e lateral (Swanson e

Sawchenko, 1983). Classificados como multipolares, mas com corpos celulares alongados e

de maior eixo dirigido mediolateralmente (van den Pol, 1982; Rho e Swanson, 1989),

produzem principalmente vasopressina e oxitocina (Tóth et al., 1999; Hallbeck et al., 2001).

No seu conjunto, contribuem para cerca de um terço do número total de axónios que integram

a projecção hipotalâmica descendente associada ao sistema nervoso autónomo (Hosoya,

1980). Desta forma, o PVN tem a capacidade de regular a actividade do sistema nervoso

simpático de forma quer directa, pelas projecções que envia para os núcleos das colunas

intermediomedial e intermediolateral da medula espinhal, quer indirecta, através das suas

eferências para a porção rostral do bolbo ventrolateral e de colaterais que aqui têm origem

(Pyner e Coote, 2000; Moon et al., 2002). Para além das áreas visceromotoras, são-lhe

também reconhecidos outros alvos, tais como a substância cinzenta periaquedutal e os núcleos

do feixe solitário, motor dorsal do vago, ambíguo, oculomotor acessório, tegmentar

pedunculopontino, cerúleo e parabraquial (Geerling et al., 2010). A projecção com origem na

divisão parvocelular dorsal atinge sobretudo os alvos mais distais e é predominantemente

oxitocinérgica, enquanto que a proveniente da divisão parvocelular lateral enerva tanto as

áreas-alvo mais rostrais, sobretudo através de fibras vasopressinérgicas, como a medula

espinhal, esta principalmente através de fibras oxitocinérgicas (Sawchenko e Swanson, 1982;

Shafton et al., 1998). A estimulação dos neurónios destas divisões induz alterações profundas

das funções cardiovasculares (Duan et al., 1997; Coote, 2005), da homeostasia energética

7

(Jansen et al., 1997; Blevins et al., 2004), da modulação somatossensorial (Horn et al., 1994;

Pan et al., 1999) e das funções sexuais, nomeadamente das masculinas (Véronneau-

Longueville et al., 1999; Argiolas e Melis, 2005).

Os neurónios neurosecretores magnocelulares, responsáveis pela regulação da

actividade neuro-hipofisária, concentram-se nas regiões laterais do PVN e formam as divisões

magnocelulares anterior, medial e posterior (Swanson e Sawchenko, 1983). Sintetizam os

neuropeptídeos vasopressina e oxitocina que, após transporte axonal para o lobo posterior da

hipófise, aí são armazenados em grandes vesículas de secreção até que, em resposta a

estímulos apropriados, são lançados na circulação sistémica (Scharrer and Scharrer, 1940;

Swanson e Sawchenko, 1983; Morris et al., 2000; Landgraf e Neumann, 2004). A

vasopressina é produzida pelos neurónios das divisões magnocelulares medial e posterior

(Rhodes et al., 1981; Swanson et al., 1981). É sintetizada e libertada em resposta a variações

do volume e osmolaridade sanguíneas e tem como principal função a manutenção do

equilíbrio hidro-electrolítico. Já a oxitocina, produzida por neurónios de todas as divisões

magnocelulares (Rhodes et al., 1981; Swanson et al., 1981), está sobretudo envolvida em

funções de carácter reprodutivo, como a contracção uterina no decurso do parto e a ejecção de

leite, e energético, tais como a secreção de insulina e de glucagão, podendo ainda, e em

resposta a alterações osmóticas e volémicas, potenciar a acção da vasopressina (Telleria-Diaz

et al., 2001). É de salientar que alguns neurónios neurosecretores magnocelulares dão origem

a colaterais que se dirigem para a eminência mediana onde libertam vasopressina, e que

outros, sobretudo os localizados na divisão magnocelular posterior, constituem ponto de

partida de projecções descentes para neurónios pré-ganglionares simpáticos localizados nos

segmentos torácicos superiores (revisto em Benarroch, 2005; ver também Hosoya et al.,

1995).

Os neurónios neurosecretores parvocelulares do PVN são responsáveis pela síntese de

numerosas hormonas com função estimuladora ou inibidora, as quais, após transporte para a

eminência mediana, são libertadas e veiculadas pela circulação porta-hipofisária para a adeno-

hipófise, cuja actividade condicionam. Estes neurónios são de pequenas ou médias dimensões

e ocupam as divisões parvocelulares anterior, periventricular e medial do PVN (Swanson e

Sawchenko, 1983). Ao contrário da divisão anterior, onde os neurónios peptidérgicos são

esparsos e produzem principalmente somatostatina e hormona libertadora da tireotrofina

(TRH; para revisão, ver Swanson e Sawchenko, 1983; Simmons e Swanson, 2009), nas

divisões localizadas mais posteriormente, a periventricular e a medial, os neurónios

hipofisiotróficos são numerosos e agrupam-se em três colunas verticais: uma, adjacente ao

8

epêndima ventricular, onde predominam neurónios somatostatinérgicos (revisto em Swanson

e Sawchenko, 1983; Simmons e Swanson, 2009), outra, em posição intermédia, rica em

neurónios produtores de TRH (para revisão, ver Swanson e Sawchenko, 1983; Simmons e

Swanson, 2009) e, a mais lateral, onde se concentram, formando um denso agregado celular,

os neurónios que produzem corticoliberina (CRH; revisto em Swanson e Sawchenko, 1983;

Simmons e Swanson, 2009; ver também Cummings et al., 1983). Assim, é a divisão

parvocelular medial (PVNmp) a principal responsável pela produção de CRH e, nalgumas

circunstâncias, também de vasopressina, encefalina e neurotensina, entre outros (Sawchenko

et al., 1984a,b). Compreende-se, pois, que o PVNmp desempenhe papel de relevo na

regulação da actividade do eixo neuroendócrino responsável pela produção de

glucocorticóides, o eixo hipotálamo-hipófise-suprarrenal (HPA; Antoni, 1986). De facto, a

CRH é o principal secretagogo da hormona adrenocorticotrófica (ACTH) produzida pela

adeno-hipófise, a qual, actuando sobre o córtex da glândula suprarrenal, estimula a síntese e a

libertação de glucocorticóides (Whitnall, 1993). Embora este neuropeptídeo seja o principal

modulador da actividade do eixo HPA (Vale et al., 1981), não é o único, sendo a sua acção

fortemente potenciada pela vasopressina que, embora seja em condições basais um fraco

secretagogo da ACTH, é co-produzida em quantidades aumentadas em situações de activação

crónica do eixo HPA pelos neurónios produtores de CRH (para revisão, ver Aguilera e Liu,

2012). Para além da sua acção na regulação da homeostasia, tanto em condições basais como

em resposta ao stresse, os glucocorticóides exercem acção inibitória directa sobre a adeno-

hipófise, o hipotálamo e centros extra-hipotalâmicos, designadamente o hipocampo,

contribuindo deste modo para regular, através de mecanismos de feedback negativo, a

actividade do eixo HPA (Ziegler e Herman, 2002; Dallman, 2005).

Estudos realizados tanto no Homem como em animais de experiência têm vindo a demonstrar

que a actividade do eixo HPA é modulada pelos esteróides sexuais (revisto em Rhodes e

Rubin, 1999; Kudielka e Kirschbaum, 2005). Quando comparadas com os machos, as fêmeas

possuem níveis basais mais elevados de corticosterona (Lesniewska et al., 1990; Carey et al.,

1995; Atkinson e Wanddell, 1997) e respondem ao stresse com aumentos mais exuberantes

das concentrações plasmáticas destas hormonas (Viau e Meaney, 1991; Ogilvie e Rivier,

1997; Figueiredo et al., 2007). Embora alguns estudos sugiram que estas diferenças são

determinadas pelo ambiente hormonal perinatal típico de cada sexo (Patchev et al., 1995;

9

McCormick et al., 1998), é hoje amplamente aceite que os efeitos “activacionais” dos

esteróides sexuais são fundamentais para a sua expressão. Com efeito, os dados obtidos em

trabalhos realizados em ratos machos e fêmeas gonadectomizados e ulteriormente

suplementados, ou não, com doses fisiológicas das respectivas hormonas sexuais mostram

que a testosterona inibe e, pelo contrário, os estrogénios estimulam a actividade do eixo HPA

(revisto em Handa et al., 2009; ver também Lunga e Herbert, 2004; Seale et al., 2004a,b;

Figueiredo et al., 2007).

Dos componentes do eixo HPA, são sensíveis aos esteróides sexuais tanto o córtex

suprarrenal (Kitay, 1961; Critchlow et al., 1963; Bornstein et al., 2008) como os neurónios

parvocelulares do PVN. Nos machos, a redução dos níveis circulantes de testosterona

induzida por orquidectomia provoca aumento da expressão de Fos e da síntese de CRH e

vasopressina no PVN (Seale et al., 2004a; Viau et al., 2003), sendo estes efeitos revertidos

pela administração de testosterona (Seale et al., 2004b). Porém, nas fêmeas, a acção dos

esteróides sexuais sobre a actividade dos neurónios do PVNmp parece ser mais complexa.

Embora seja consensual que a ovariectomia reduz a síntese de CRH e de vasopressina (Seale

et al., 2004a,b), o mesmo não se verifica em relação aos efeitos dos estrogénios sobre a

síntese destes neuropetídeos. Com efeito, há estudos que mostram que os níveis de mRNA da

CRH se encontram aumentados em ratos que, após ovariectomia, foram tratados com doses

fisiológicas de estradiol (Patchev et al., 1995; Seale et al., 2004b), enquanto outros

demonstram que, nas mesmas condições experimentais, esses níveis estão diminuidos

(Paulmyer-Lacroix et al., 1996; Lunga e Herbert, 2004) ou, até, inalterados (Figueiredo et al.,

2007). Embora de modo menos assertivo, o mesmo se passa em relação à vasopressina já que

a par de autores que observaram aumento dos níveis do mRNA deste neuropetídeo (Patchev et

al., 1995; Seale et al., 2004b), outros há que não detectaram qualquer alteração (Lunga e

Herbert, 2004) em ratos que, após ovariectomia, foram tratados com doses fisiológicas de

estradiol.

Nas fêmeas, os estrogénios parecem modular a actividade do eixo HPA através de

mecanismos directos e indirectos. Com efeito, os receptores de estrogénios do tipo β (ER-β)

são expressos abundantemente apenas pelos neurónios pré-autónomos do PVN produtores de

CRH (Shughrue and Merchenthaler, 2001; Stern e Zhang, 2003), sendo os receptores de

estrogénios do tipo α (ER-α) abundantemente expressos pelos neurónios de áreas envolventes

do PVN e que se sabe estarem implicadas na inibição do eixo HPA (Suzuki e Handa, 2005).

Os neurónios do PVNmp também não expressam receptores para a testosterona, admitindo-se

que a modulação da sua actividade, nos machos, se faz por via trans-sináptica a partir de áreas

10

que expressam receptores para os androgénios e para os estrogénios (Simerly et al., 1990;

Handa et al., 1996) e que possuem níveis elevados de actividade da enzima aromatase

(Roselli et al., 1997), como é o caso do bed nucleus of the stria terminalis e da área pré-óptica

medial (Viau e Meaney, 1996). Alguns dados sugerem que a modulação da actividade do eixo

HPA pelos esteróides sexuais poderá ser também mediada pela interacção dos estrogénios e

da testosterona com a expressão e a taxa de ocupação dos receptores para os glucocorticóides

(Viau e Meaney, 1996) e mineralocorticóides (Carey et al., 1995).

É actualmente irrefragável que a ingestão esporádica de álcool provoca intensa e abrupta

activação do eixo HPA, conduzindo ao aumento das concentrações plasmáticas de cortisol no

Homem (Jenkins e Connolly, 1968; Schuckit et al., 1987; Lex et al., 1991; Ekman et al.,

1994) e de corticosterona nos roedores (Ellis, 1966; Rivier et al., 1984; Spencer e McEwen,

1990). Porém, já o mesmo se não pode dizer quanto às consequências da ingestão prolongada,

ou crónica, de álcool. De facto, a par de estudos que apontam para a presença de elevadas

concentrações plasmáticas de cortisol no Homem (Schuckit et al., 1987; Gianoulakis et al.,

2003) e de corticosterona em animais de experiência (Schuckit et al., 1987; Rasmussen et al.,

2000), outros demonstram que tanto as concentrações plasmáticas de cortisol (Lex et al.,

1991; del Arbol et al., 1995) como as de corticosterona (Rachamin et al., 1989; Little et al.,

2008) se encontram inalteradas. Ao contrário das alterações hormonais, os dados disponíveis

na literatura sobre os efeitos do álcool na actividade dos neurónios hipofisiotróficos do PVN

resultam exclusivamente de trabalhos realizados em animais de experiência, na sua quase

totalidade em modelos de exposição aguda ou de curta duração ao álcool. Estes estudos

revelaram que a exposição aguda ao álcool aumenta, de forma rápida e intensa, a síntese de

CRH e de vasopressina pelos neurónios parvocelulares do PVN (Rivier e Lee, 1996; Ogilvie

et al., 1997), e que estes efeitos são neutralizados, na ausência de exposição adicional ao

álcool, algumas horas mais tarde (Zoeller e Rudeen, 1992). Nas exposições repetidas, mas de

curta duração, os níveis de corticosterona continuam elevados, mas a síntese de CRH pelo

PVNmp está aumentada (Rivier et al., 1990) ou inalterada (Lee e Rivier, 1993; Zhou et al.,

2000), a de vasopressina diminuída (Gulya et al., 1991; Sanna et al., 1993), e o conteúdo de

CRH na eminência mediana reduzido (Rivier et al., 1984).

Apesar destes dados revelarem serem distintas as consequências da exposição aguda

ou de curta duração ao álcool das decorrentes da sua ingestão prolongada, e de sugerirem que

11

o eixo HPA sofre um processo de adaptação quando exposto ao álcool por longos períodos,

nenhuma investigação de que se tenha conhecimento se centrou no estudo das repercussões

desta condição experimental sobre a organização estrutural do PVNmp e a actividade dos seus

neurónios produtores dos neuropeptídeos que regulam a actividade do eixo HPA.

Assim, a presente dissertação teve como primeiro objectivo avaliar se a ingestão prolongada

de álcool provoca alterações degenerativas e/ou da actividade dos neurónios do PVNmp que

produzem CRH e vasopressina. Com efeito, em investigações anteriores que utilizaram o

mesmo modelo experimental de alcoolização demonstrou-se que o consumo crónico de álcool

provoca morte neuronal no núcleo supra-óptico e, simultaneamente, hipertrofia dos seus

neurónios sobreviventes (Madeira et al., 1993). Estes efeitos são distintos dos observados em

núcleos hipotalâmicos formados por agregados de neurónios parvocelulares, designadamente

no núcleo supraquiasmático, onde a alcoolização crónica não induz morte celular, mas

deprime a síntese e a expressão de neuropeptídeos tais como a vasopressina, o polipeptídeo

intestinal vasoactivo, o peptídeo libertador da gastrina e a somatostatina (Madeira et al.,

1997).

Sabe-se também que a abstinência, após longos períodos de alcoolização, está

associada a fenómenos de reorganização neuronal e de recuperação funcional nalgumas

regiões do SNC (Tavares et al., 1986; Cadete-Leite et al., 1988b; Madeira et al., 1993; Ruela

et al., 1994). Porém, esta resposta não é uniforme e, noutras regiões, a abstinência alcoólica

funciona como noxa adicional, levando ao agravamento das alterações provocadas pela

ingestão crónica de álcool. Observaram-se respostas desta natureza, por exemplo, na

formação do hipocampo (Paula-Barbosa et al., 1993; Lukoyanov et al., 1999), no córtex pré-

frontal (Cadete-Leite et al., 1990), no prosencéfalo basal (Cadete-Leite et al., 2003), e

também no hipotálamo, designadamente no núcleo supraquiasmático, onde a abstinência

provoca depressão mais acentuada da capacidade de síntese dos neuropeptídeos mais

abundantemente expressos pelos seus neurónios (Madeira et al., 1997). Por este motivo, e

face aos resultados obtidos nos trabalhos em que se estudaram os efeitos da alcoolização

crónica, foi também objectivo da presente dissertação analisar se a abstinência alcoólica

permite, ou não, recuperação da actividade tanto dos neurónios do PVNmp como do eixo

HPA.

12

Para responder a estas questões, determinaram-se as concentrações plasmáticas de

corticosterona e estimaram-se, através da aplicação de técnicas estereológicas, o volume e o

número total de neurónios do PVNmp em material corado pelo Giemsa, e o número total de

neurónios produtores de CRH e de vasopressina em material corado imunocitoquimicamente.

Com o objectivo de analisar se as alterações da expressão de CRH e vasopressina pelos

neurónios do PVNmp reflectiam alterações da sua síntese, aplicaram-se técnicas semi-

quantitativas a material tratado por hibridização in situ para estimar os níveis de mRNA de

cada um destes neuropeptídeos.

São cada vez mais numerosos os trabalhos indiciadores de que o stresse crónico pode

condicionar a resposta do eixo HPA à ulterior exposição a estímulos indutores de stresse tanto

de natureza homóloga como heteróloga (revisto em Aguilera et al., 2008; Jankord e Herman,

2008). Estudos realizados em modelos experimentais de alcoolização mostraram que a

administração intragástrica de álcool durante três dias consecutivos atenua o aumento

esperado, face à exposição aguda a esta noxa, da secreção de CRH e ACTH (Lee e Rivier,

1997, 2003; Rivier e Lee, 2001). Porém, foi também demonstrado que, nas mesmas condições

experimentais, a resposta do eixo HPA a um estímulo heterólogo, tal como a administração de

electric foot shocks (Lee e Rivier, 1993, 1997; Rivier e Lee, 2001) ou de citocinas (Lee e

Rivier, 1993; Rivier, 1997), era, pelo contrário, efectiva (Lee e Rivier, 1997; Rivier, 1997;

Lee et al., 2000). Assim, e com o objectivo de avaliar se, após ingestão prolongada de álcool e

subsequente abstinência, o eixo HPA possui, ou não, capacidade de elaborar uma resposta

adequada face à exposição aguda a um estímulo heterólogo indutor de stresse, quantificaram

-se os níveis de mRNA da CRH e da vasopressina no PVNmp e determinaram-se as

concentrações plasmáticas de corticosterona em ratos que, no último dia do período de

alcoolização e de abstinência, receberam uma injecção, por via intraperitoneal, de

lipopolissacarídeo (LPS).

Por último, e com o propósito de avaliar se os esteróides sexuais típicos de cada sexo,

e os níveis plasmáticos de estrogénios nas fêmeas, influenciam a actividade basal e a resposta

ao stresse do eixo HPA durante a ingestão crónica de álcool e a abstinência alcoólica, os

trabalhos incluídos na presente disertação foram realizados em machos e em fêmeas, algumas

em fases aleatórias ciclo éstrico e, outras, ovariectomizadas e depois injectadas com benzoato

de estradiol ou com veículo.

13

*************

O conjunto de trabalhos incluídos na presente dissertação relatam o esforço feito para

clarificar aspectos relativos ao eixo HPA no decurso da resposta ao stresse. Assim, pretendeu-

se avaliar se

a) A ingestão crónica de álcool modifica a organização citoarquitectónica do PVNmp

e a actividade dos seus neurónios envolvidos na regulação da actividade do eixo

HPA, i.e., os produtores de CRH e vasopressina.

b) Os efeitos da ingestão crónica de álcool sobre o PVNmp e a actividade do eixo

HPA são permanentes ou, pelo contrário, reversíveis após abstinência alcoólica.

c) A ingestão crónica de álcool e a abstinência alcoólica alteram a resposta do eixo

HPA à estimulação aguda por um factor indutor de stresse sistémico.

d) As repercussões da ingestão crónica de álcool e da abstinência alcoólica na

organização estrutural e neuroquímica do PVNmp e na resposta do eixo HPA ao

stresse diferem entre machos e fêmeas, e se são, nas fêmeas, influenciadas pelos

níveis circulantes de estrogénios.

15

TTRRAABBAALLHHOOSS

17

II..

Prolonged alcohol intake leads to reversible depression of corticotropin-

releasing hormone and vasopressin immunoreactivity and mRNA levels in

the parvocellular neurons of the paraventricular nucleus

19

Brain Research 954 (2002) 82–93www.elsevier.com/ locate/bres

Research report

P rolonged alcohol intake leads to reversible depression ofcorticotropin-releasing hormone and vasopressin immunoreactivity and

mRNA levels in the parvocellular neurons of the paraventricularnucleus

Susana M. Silva, Manuel M. Paula-Barbosa, M. Dulce Madeira*

Department of Anatomy, Porto Medical School, Alameda Hernaniˆ Monteiro, 4200-319 Porto, Portugal

Accepted 25 July 2002

Abstract

The ability of alcohol to activate the hypothalamic–pituitary–adrenal (HPA) axis is well documented in investigations based in acuteand short-term experimental paradigms. Herein, we have addressed the possibility that the prolonged exposure to ethanol concentrationsthat are initially effective in stimulating corticosteroid secretion might induce alterations in the response of the HPA axis that cannot beevinced by shorter exposures. Using conventional histological techniques, immunohistochemistry and in situ hybridization, we haveexamined the medial parvocellular division of the paraventricular nucleus (PVNmp), and the synthesis and expression of corticotropin-releasing hormone (CRH) and vasopressin (VP) by its constituent neurons, in rats submitted to 6 months of ethanol treatment and towithdrawal (2 months after 6 months of alcohol intake). Ethanol treatment and withdrawal did not produce neuronal loss in the PVNmp.However, the total number of CRH- and VP-immunoreactive neurons and the CRH mRNA levels were significantly decreased by ethanoltreatment. In withdrawn rats, the number of CRH- and VP-immunostained neurons and the gene expression of CRH were increasedrelative to ethanol-treated rats and did not differ from those of controls. No significant variations were detected in VP mRNA levels as aresult of ethanol treatment or withdrawal. These results show that prolonged alcohol intake blunts the expression of CRH and VP in theparvocellular neurons of the PVN, and that this effect is, partially at least, reversible by withdrawal. They also suggest that thedevelopment of tolerance to the effects of ethanol involve changes that take place at the hypothalamic level. 2002 Elsevier Science B.V. All rights reserved.

Theme: Endocrine and autonomic regulation

Topic: Hypothalamic–pituitary–adrenal regulation

Keywords: Paraventricular nucleus; Alcohol; Withdrawal; Corticotropin-releasing hormone; Vasopressin

1 . Introduction exposure to alcohol. On that account, there is convincingevidence that acute ethanol exposure leads to a transitory

The effects of alcohol on the brain have been studied for and dose-related increase in the plasma levels of ACTHmany years in the hypothalamus, the regulator of the and corticosterone [34,44], and that this response dependsactivity of the peripheral endocrine glands (reviewed in on the delivery to the pituitary of the hypothalamicRef. [32]). In particular, an enormous progress has been peptides corticotropin-releasing hormone (CRH) and vas-achieved during the last decade on the characterization of opressin (VP), which are synthesized by specific subpopu-the activity of the hypothalamic–pituitary–adrenal (HPA) lations of parvocellular neurons located in the paraven-axis following either acute administration or short-term tricular nucleus (PVN). A single exposure to alcohol,

achieved either through intraperitoneal or intragastricinjections or by means of inhalation, was shown to induce*Corresponding author. Tel.:1351-22-509-6808; fax:1351-22-550-

5640. transient expression of immediate-early gene markers ofE-mail address: [email protected](M.D. Madeira). cellular activation, such asc-fos and NGFI-B, and to

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03346-2

20

83S.M. Silva et al. / Brain Research 954 (2002) 82–93

increase the heteronuclear and the mRNA levels of CRH neurons. We have focused on the parvocellular neuronsand VP in the PVN [34,35,42]. There is also considerable producing CRH and VP, primarily because they are potentdata showing that corticosterone secretion is increased in modulators of the activity of the HPA axis [5,41] and theyrats exposed to high concentrations of alcohol for periods exhibit changes in their activity after acute and short-termranging between 3 days and 3 weeks [9,44,45,52,55]. exposure to alcohol. Finally, because there are conflictingHowever, while VP mRNA levels have been consistently data about the effects of abstinence on the activity of thereported as decreased, both in mice fed an ethanol liquid HPA axis, with different studies showing either a completediet during 7 days [12,13,18] and in rats exposed to normalization [56] or persistence [1,8,29,38] of HPAalcohol vapors for 2 weeks [47], data about the effects of functional abnormalities under basal conditions or aftershort-term alcohol exposure on the activity of CRH- experimental manipulations, we have examined whether orproducing neurons are controversial. In fact, there are not withdrawal modifies the structural and/or the neuro-studies showing that the CRH mRNA levels in the chemical changes induced by long-term ethanol treatmentparvocellular division of the PVN are increased in rats in the PVNmp.exposed to alcohol vapors during 7 days [45] and un-changed in rats fed with an alcohol diet for 10 days [22].

In contrast with the wealthy information about theeffects, and the underlying mechanisms, of acute and 2 . Materials and methodsshort-term exposure to alcohol, few studies have addressedthe repercussions of prolonged alcohol exposure (i.e., for 2 .1. Animals and treatmentsperiods greater than 3 weeks) on the activity of the HPAaxis. In addition, the available investigations have been The studies were carried out in male Wistar rats derivedconfined to hormonal determinations, with different studies from the colony of the Gulbenkian Institute of Scienceshowing either no effect [8,26] or an increase [49] in (Oeiras, Portugal). The animals were maintained through-plasma cortisol levels in individuals consuming alcohol out the experiment under standard laboratory conditions:over several years, and unchanged morning basal plasma 12-h light /dark cycle (lights on at 07:00 h) and tempera-corticosterone concentrations in rats fed with an ethanol ture of 228C. Solid diet and water were available adliquid diet for 6 months [37]. Although these data support libitum until rats were 2 months old. At this age, rats werethe view that the HPA axis develops tolerance to the placed into individual cages and assigned to the followingeffects of ethanol, an event that has been claimed to experimental groups.become apparent shortly after the beginning of ethanol (1)Ethanol-treated group: rats were given an aqueoustreatment [24], there are reasons to assume that the ethanol solution as their only available liquid source for 6underlying adaptive mechanisms are incomplete. In fact, it months, i.e., until the age of 8 months. The ethanolhas been shown that after 3 weeks of a daily intra- concentration was progressively increased: starting with aperitoneal injection of alcohol, rats still show indirect signs 5% (v/v) solution, and rising by 1% per day to a final 20%of activation of the HPA axis, namely adrenal hypertrophy (v /v) 2 weeks later. Food was freely available throughout.and thymus involution, and their plasma levels of corticos- The amounts of food and ethanol solution consumed wereterone, although lower than in the first day of treatment, measured every other day. In keeping with previousremain increased relative to control levels [52]. observations [30,31], this experimental model of chronic

Thus, we have hypothesized that the maintenance over ethanol treatment resulted in an average daily ethanollong periods of time of ethanol concentrations that are intake of 9.5 g/kg body weight and in blood alcoholinitially effective in stimulating corticosteroid secretion concentrations of 120 mg/dl in the morning and 90 mg/dlmay reveal alterations in the HPA response that cannot be in the evening.demonstrated in acute and short-term studies. To investi- (2)Withdrawal group: rats were ethanol treated over 6gate this possibility, we have used a chronic ethanol months and then switched to tap water for a further 2ingestion regimen that has proved to produce persistently months. The shift from ethanol treatment to water intakehigh blood alcohol concentrations throughout the day. Our was performed gradually over a 2-week period by progres-first aim was to estimate, using stereological methods, the sive reduction of the ethanol concentration in the drinkingtotal number of neurons in the medial parvocellular solution by 1% per day. Food pellets were freely availabledivision of the PVN (PVNmp). In effect, previous in- throughout the experiment.vestigations have suggested that the development of toler- (3)Pair-fed controls: rats were given an amount ofance to the effects of ethanol might result from changes pellet food that was equivalent to the mean amount of foodtaking place at the hypothalamic level [36], and there is consumed by ethanol-treated rats plus an additional quanti-evidence that chronic ethanol treatment induces neuronal ty that provided a caloric intake equal to that supplied bydegeneration in the hypothalamus [15,30,51]. Our second the quantity of ethanol solution drunk by ethanol-treatedaim was to examine the effects of prolonged ethanol intake rats. The additional number of pellets was calculatedon the synthesizing activity and peptide content of PVNmp taking into account the caloric value of ethanol (1 g57

21

S.M. Silva et al. / Brain Research 954 (2002) 82–9384

kcal) and the mean volume of alcohol consumed by were anesthetized as described above and perfused with aethanol-treated rats (10 ml ethanol57.9 g). fixative solution containing 4% paraformaldehyde in phos-

In all groups, liquids consumed were supplemented with phate buffer (pH 7.6). Brains were removed, immersed in300 mg/100 ml of vitamins (Vitamin Diet Fortification the same fixative solution for 2 h, and maintained over-Mixture, ICN Cleveland, OH) and 500 mg/100 ml of night in a solution of 10% sucrose in phosphate buffer. Theminerals (Salt Mixture, ICN Cleveland, OH). All pro- blocks containing the right and left hypothalami werecedures were carried out in compliance with the European mounted on a Vibratome and serially sectioned at 50mm inCommunities Council Directives of 24 November 1986 the coronal plane. Alternate sections were separately(86/609/EEC) and Portuguese Act no. 129/92. The collected in phosphate-buffered saline (PBS) in order tomaterial used in this study was collected between 09:00 obtain two independent sets of sections from each brain;and 10:00 h. The hypothalami were either embedded in each set was then processed independently for CRH or VPglycolmethacrylate (n56 per group), for the estimation of immunostaining using the avidin–biotin technique withtotal neuron numbers in the PVNmp, or processed for diaminobenzidine (DAB) as the chromogen, as previouslyimmunocytochemical detection of CRH- and VP-contain- described [30,31]. The antiserum against CRH (Peninsulaing neurons (n55 per group) or for the analysis of CRH Laboratories, Belmont, CA) and the antiserum against VPand VP mRNA expression by in situ hybridization (n54 (gift from Dr. R. Buijs, The Netherlands Institute for Brainper group). Research, Amsterdam) were used at the dilution of 1:5000.

2 .2. Blood sampling and corticosterone measurement 2 .3.3. In situ hybridization histochemistryRats were anesthetized and perfused as for the immuno-

Corticosterone concentrations were measured in rats cytochemical studies. The brains were removed, stored inused for immunocytochemical and in situ hybridization the same fixative for 1 h, and immersed in crescentstudies. One week before killing, blood samples (500ml) RNAse-free sucrose solutions (5, 10 and 20%, 1 h each,were taken from the dorsal vein of the tail, between 09:00 and 30% overnight) at 48C. Coronal sections, 20-mmand 10:00 h, and collected in Eppendorf tubes. After thick, were cut on a cryostat at220 8C. The brain sectionscentrifugation, the resulting plasma was preserved at were alternately collected in order to form two series: one220 8C until the time of assay. Corticosterone concen- was used for CRH and the other for VP in situ hybridiza-trations were determined by radioimmunoassay, using a kit tion. Sections were thaw-mounted on poly-L-lysine-coatedsupplied by ICN Biomedicals (Costa Mesa, CA). All slides, warmed at room temperature, and stored at220 8Csamples were assayed in a single run, the intra-assay until use. Prior to hybridization, sections were fixed withcoefficient of variation being|5%. 4% paraformaldehyde in 0.12 M PBS (pH 7.4) for 6 min at

room temperature, rinsed twice with PBS and once with2 .3. Tissue preparation TEA–HCl (0.1 M TEA–HCl–0.9% NaCl, pH 8.0), and

then acetylated in 0.25% acetic anhydride in TEA–HCl for2 .3.1. Conventional histological procedures 10 min at room temperature. After a brief wash with

Rats were anesthetized with 6% chloral hydrate (1 23SSC (SSC50.15 M NaCl and 0.015 M sodium citrate,ml /100 g body weight) and perfused with a solution pH 7.2), the sections were dehydrated in serial ethanolcontaining 1% paraformaldehyde and 1% glutaraldehyde in solutions (70, 80, 90, 96 and 100%, 1 min each), defatted0.12 M phosphate buffer (pH 7.2). The brains were in chloroform (5 min), immersed again in absolute ethanol,removed from the skulls, weighed and postfixed for 15 and allowed to dry. Synthetic 48-based oligodeoxynucleo-days in fresh fixative. After removal of the frontal and tide probes directed against rat CRH, bases encodingoccipital poles, the blocks containing both the right and amino acids 496–543 [19], and rat VP, bases encodingleft hypothalami were dehydrated through a graded series amino acids 477–524 [39], were used. The probes were

35of ethanol solutions, embedded in glycolmethacrylate labeled with [ S]dATP (1200 Ci /mmol; DuPont NEN,(hydroxyethylmethacrylate, Technovit 7100, Kulzer, Wehr- Bad Homburg, Germany) using terminal transferaseheim, Germany), and sectioned in the coronal plane at 40 (Roche Molecular Biochemicals, Mannheim, Germany).mm, as described in detail elsewhere [21,31]. The sections For detection of CRH and VP mRNAs, sections were

6were collected, mounted serially, and stained with a hybridized with 1310 c.p.m. of the labeled probes in aGiemsa solution modified for use in glycolmethacrylate- buffer composed of 50% deionized formamide, 43SSC,embedded material [58]. Finally, the sections were cover- 13Denhardt’s solution, 10% dextran sulfate, 0.1% dieth-slipped with Histomount mounting medium. ylpyrocarbonate-treated water and 100 mM dithiothreitol

(DTT). Sections were protected with coverslips and incu-2 .3.2. Immunohistochemistry bated, for 20 h, in a humidified chamber at 428C. After

Under chloral hydrate anesthesia, colchicine (10ml of a removal of the coverslips, the sections were washed0.25% solution in physiological saline) was stereotaxically sequentially, once in 43SSC (15 min) at room tempera-injected into the lateral ventricle. After 48 h, the animals ture, four times in 13SSC (15 min each) at 408C, once in

22


0.53SSC and once in 0.13SSC (30 min each) at room sampled using an interframe distance of 130mm (x-axis)temperature, and finally in distilled water. They were then by 140mm (y-axis) for CRH neurons and of 120mm (x-dehydrated through graded ethanol solutions in 300 mM andy-axes) for VP neurons; the area of the counting

2ammonium acetate, and finally in absolute ethanol. Slides frames and the height of the disector was 1500mm and 10were dipped in NTB2 nuclear emulsion (Kodak, Roches-ter, NY) diluted in 0.5% glycerol in distilled water (1:1)for 7 weeks (CRH mRNA) or 4 days (VP mRNA). Afterdevelopment with D-19 developer (Kodak) and fixation,the sections were counterstained with thionin (0.1%),dehydrated through an alcohol series (70, 95 and 100%, 1min each), cleared with Histoclear, and coverslipped withPermount. To assess the specificity of labeling, controlsections were co-incubated with unlabeled probe. Nolabeling above background level was detected in thesesections.

2 .4. Stereological analyses

The subdivisions of the PVN were delineated and namedaccording to Swanson and Kuypers [53]. Accordingly, themedial parvocellular division (PVNmp) was identified as adensely packed group of small cells, which is locatedmedial to the posterior magnocellular division and extendsthroughout the caudal half of the PVN (Fig. 1). The totalnumber of Giemsa-stained neurons in the PVNmp wasestimated using the optical fractionator method [21,31,58].The estimates were obtained either from the right or leftPVNmp and thus represent unilateral values. An equalnumber of right and left nuclei were used for each groupanalyzed. All sections in which the PVNmp was visualizedwere used for the estimations. In each section, the fields ofvision were systematically sampled using a step size of140mm along thex-axis and 150mm along they-axis. The

2disector used had a counting frame area of 600mm at thetissue level and a fixed height of 15mm. An average of170 neurons per nucleus was counted; the coefficient oferror of the estimates was 0.08. The estimations wereperformed, at magnification of32000, using the CAST—Grid system software (Olympus DK, Denmark) and aHeidenhain MT-12 microcator (Heidenhain, Germany).

The same stereological method was employed to esti-mate the total number of CRH- and VP-immunoreactiveneurons in the PVNmp. The sampling schemes used wereas described above, with the following modifications:alternate sections were used; microscope fields were

Fig. 1. Photomicrographs of Giemsa-stained coronal sections of theparaventricular nucleus (PVN) to show the location of the medialparvocellular division (mp) relative to other PVN divisions. The sectionsare arranged in a rostrocaudal sequence (A–C). In all sections, theperiphery of the PVN is indicated by a dotted line and the boundaries ofthe PVN divisions by dashed lines. (A) Section through the rostral part ofthe PVNmp. (B) Section through the mid rostrocaudal extent of thePVNmp. (C) Section through the caudal part of the PVNmp. dp, dorsalparvocellular division; lp, lateral parvocellular division; pm, posteriormagnocellular division; V, third ventricle. Scale bars5150 mm.

23


2mm, respectively, for CRH neurons and 6000mm and 15 munoreactive neurons and on the CRH and VP mRNAmm, respectively, for VP neurons. An average of 140 levels. In this case, Rao’sR statistical parameter wasCRH-immunoreactive neurons and 110 VP-immunostained calculated [28]. Whenever appropriate, the Tukey HSDneurons were counted; the CE of the estimates was 0.09 post hoc test was performed to examine the differencesand 0.10, respectively. Immunostaining of the perikaryal between the groups. Differences were considered to becytoplasm with a relatively unstained nucleus was the significant ifP,0.05. Data on body and brain weights arecriteria for identification of CRH- and VP-containing presented as means with their coefficients of variationneurons. Medial parvocellular VP-immunoreactive neurons (CV5S.D. /mean), and corticosterone concentrations arewere discriminated from magnocellular VP-immuno- shown as means and S.E.M.reactive neurons on the grounds of neuronal size andmorphology [54].

3 . Results2 .5. Quantitative analysis of in situ hybridization results

3 .1. Effect of ethanol treatment and withdrawal on bodyBrain paste standards containing serial dilutions of and brain weights35[ S]UTP, used for quantification of mRNA signals, were

prepared concurrently to ensure that optical density was At sacrifice, the mean body weight was 655 g (0.05) forfound within the linear range of the standard curve. controls, 604 g (0.09) for ethanol-treated rats, and 646 gSemiquantitative densitometric analyses of hybridization (0.05) for withdrawn rats. As revealed by ANOVA, thesignals for the mRNAs of interest were done over the variations observed in body weights were dependent on theconfines of cells within the PVNmp by using a computer- effect of treatment (F(2,42)53.79, P50.030). Ethanol-assisted image analyzer (Optimas-Bioscan) fitted with a treated and withdrawn rats did not significantly differ fromLeica axioplan microscope and a Sony Hyper HAD Digital control rats with respect to their body weights. However,color video camera. For each mRNA analyzed, signals withdrawn rats were heavier than ethanol-treated rats (P5were measured in an average of 150 cells per animal,0.02).sampled throughout the entire rostrocaudal extent of the Chronic ethanol treatment and withdrawal had no effectPVNmp. The identification of CRH or VP mRNA-con- on brain weights (F(2,42)50.22, P50.805). The meantaining cells was based on the presence of silver grains inbrain weights were 1.50 g (0.04) for controls, 1.49 g (0.06)the overlying emulsion layer and was dependent on their for ethanol-treated rats, and 1.49 g (0.03) for withdrawndensities in relation to background labeling. Medial par- rats.vocellular VP-synthesizing neurons were differentiatedhistologically from magnocellular neurons on the basis of

3 .2. Effect of ethanol treatment and withdrawal ontheir size, their relatively low level of VP expression, andcorticosterone concentrationstheir small and dense-staining nuclei. Cells expressing

CRH or VP mRNA were first delineated under bright-fieldSerum corticosterone concentrations, expressed in ng/illumination, using a340 objective lens, and the mean

ml, were 81.6 (8.3) for controls, 88.3 (7.1) for ethanol-optical density of the hybridization signals was thentreated rats, and 90.6 (7.6) for withdrawn rats. There weremeasured under dark-field illumination. All gray levelno significant differences in corticosterone concentrationsmeasurements were corrected for background, which wasamong groups (F(2,24)50.37,P50.694).measured in a similar size area adjacent to each cell. Data

were expressed in gray scale values of 1–256.3 .3. Effect of ethanol treatment and withdrawal on

2 .6. Statistical analysis neuron numbers

The precision of the individual estimates was evaluated The total number of parvocellular neurons in theas the coefficient of error (CE), as described by Gundersen PVNmp, estimated from Giemsa-stained sections (Fig.et al. [14]. Normality of distribution of the data was 2A), did not differ among the groups studied (F(2,15)5confirmed using the Kolmogorov–Smirnov test of normali- 0.33;P50.724).ty. To discern main effects, body and brain weights, Conversely, the total number of CRH- and VP-immuno-hormonal concentrations and total number of Giemsa- reactive neurons was significantly influenced by treatmentstained neurons in the PVNmp were assessed using a (RaoR(4,22)54.78,P50.006). Chronic ethanol treatmentone-way analysis of variance (ANOVA). Treatment was produced a significant decrease (21%) in the number ofused as the independent variable. Because neurons produc- CRH-immunostained neurons that was partially reverseding CRH and VP are both located within the PVNmp, a by withdrawal (Figs. 2B and 3A,B). In fact, the totalmultivariate one-way ANOVA was used to test the effect number of CRH-immunoreactive neurons was 11% greaterof treatment on the total number of CRH- and VP-im- in withdrawn than in ethanol-treated rats, but this differ-

24


Fig. 2. Graphic representation of the estimates of neuron numbers obtained from the PVNmp of control (CONT), ethanol-treated (CET) and withdrawn(W) rats. Horizontal bars represent mean values. (A) Total number of Giemsa-stained neurons. The total number of PVNmp neurons does not differ amongthe groups studied. (B) Total number of neurons immunoreactive for CRH. The number of CRH-immunoreactive neurons is smaller in ethanol-treated thanin control rats. (C) Total number of neurons immunoreactive for VP. The number of VP-immunoreactive neurons is smaller in ethanol-treated than incontrol rats. Tukey’s post hoc tests: *P50.03, **P50.01, compared with control rats.

ence was not statistically significant (Fig. 2B). As a result, significant variations in the gene expression of CRH andno significant difference was also detected between with- VP in the PVNmp (RaoR(4,16)53.20,P50.041). Thedrawn and control rats. Similarly, ethanol-treated rats had CRH mRNA levels were 42% lower in ethanol-treatedsignificantly fewer (16%) VP-immunoreactive neurons than in control rats (Figs. 3C,D and 5A). Two months afterthan controls (Figs. 2C and 4A,B). However, 2 months withdrawal, there was a 25% increment over the valuesafter withdrawal, the number of these neurons was in- shown by rats submitted to ethanol treatment for 6 months,creased by 16% relative to ethanol-treated rats and, which, however, did not reach a statistical significant level.consequently, no significant difference was detected be- Consequently, no significant difference was detected be-tween withdrawn and control rats (Fig. 2C). tween withdrawn and control rats, despite the fact that the

CRH mRNA levels were still 28% lower in the former3 .4. Effect of ethanol treatment and withdrawal on than in the latter group (Fig. 5A).mRNA levels The variations induced by chronic ethanol treatment and

withdrawal in the VP mRNA levels, although quantitative-Chronic ethanol treatment and withdrawal produced ly smaller and not statistically significant, were of the same

25


Fig. 3. Photomicrographs from immunostained (A,B) and emulsion-dipped (C,D) sections of the PVN illustrating CRH-producing neurons fromrepresentative control (A,C) or ethanol-treated (B,D) rats. The medial parvocellular division (mp) of the PVN contains fewer CRH-immunoreactiveneurons in the ethanol-treated rat (B) than in the control (A). In (C) and (D), arrowheads point to cells expressing CRH mRNA. The grain density in theoverlying emulsion layer is markedly smaller in the ethanol-treated (D) than in the control (C) rat. pm, posterior magnocellular division of the PVN;V,third ventricle. Scale bars5100 mm in (A,B), 10 mm in (C,D).

type as those described for CRH mRNA levels. In fact, 4 . Discussionethanol treatment produced a 14% decrease in the VPmRNA levels, which was totally reversed by withdrawal Considerable evidence has accumulated in the literature(Figs. 4C,D and 5B). indicating that hypothalamic parvocellular neurons are less

26


Fig. 4. Photomicrographs from immunostained (A,B) and emulsion-dipped (C,D) sections of the PVN illustrating VP-producing neurons fromrepresentative control (A,C) or ethanol-treated (B,D) rats. The dashed lines indicate the border between the medial parvocellular division (mp) and theposterior magnocellular division (pm) of the PVN. The arrows point to parvocellular VP-immunoreactive neurons in the PVNmp. These neurons are lessnumerous in the ethanol-treated (B) than in the control (A) rat. In (C) and (D), arrowheads indicate parvocellular neurons labeled for VP mRNA. Bycomparison with the control rat (C), the grain density is reduced in the ethanol-treated rat (D). V, third ventricle. Scale bars5100mm in (A,B), 10mm in(C,D).

vulnerable to the effects of prolonged ethanol exposure previously reported for other parvocellular cell groups ofthan magnocellular neurons. The results of the present the hypothalamus, namely the suprachiasmatic nucleusstudy strongly reinforce this view because we have found [31]. In contrast, studies performed in humans [15] and inno significant variations in the estimates of total neuron rodents [30,51] have shown that the magnocellular neuronsnumbers in the PVNmp, similar to that which has been of the PVN and supraoptic nucleus undergo marked

27


chronic alcohol intake leads to a significant reduction inthe total number of neurons immunoreactive for CRH(21%) and for VP (14%), and to a decline in the mRNAcontent of both peptides (42 and 16%, respectively). Theseeffects are comparable to those previously observed in thesuprachiasmatic nucleus of rats submitted to the sameregimen of alcohol intake, in which significant reductionsin the number of neurons producing VP, vasoactive intesti-nal polypeptide, somatostatin and gastrin releasing peptide,and in their mRNA levels, were also found [31]. Theobservation that chronic ethanol treatment elicits the sametype of molecular changes in the PVNmp and in thesuprachiasmatic nucleus might lead to the suggestion thatalcohol acts as a nonspecific depressant of parvocellularpeptidergic neurons. However, contrariwise to what waspreviously documented for the suprachiasmatic nucleus[31], we have found that 2 months after withdrawal fromalcohol the total number of CRH- and VP-immunostainedneurons was increased by 11 and 16%, respectively,relative to ethanol-treated rats, and that this increment islikely to be attributable to enhanced peptide synthesis,given that a parallel increase in the CRH (25%) and VP(19%) mRNA levels was also noticed in withdrawnrelative to ethanol-treated rats. Although these variationsdid not reach a statistical significant level, they weresufficiently large to result in the nonappearance of signifi-cant differences between withdrawn and control rats.

Because the alterations we have detected in the geneexpression and peptide levels in the PVNmp are, in part atleast, restored after withdrawal, it might be argued thatthey might result from ancillary effects of alcohol con-sumption, such as dehydration. Although we cannot safelydiscard such a possibility, because we have not included inthis study a water-deprived control group, there are reasonsto assume that changes in osmolality are not a likelyexplanation for the alterations we have found. In fact, wehave previously shown that water deprivation for 12months leads to persistently high circulating levels of VP,whereas the chronic ethanol regimen used in this study isassociated with normal plasma levels of VP, despite the

Fig. 5. Graphic representation of the optical density of CRH (A) and VP increased plasma osmolality [30]. Furthermore, although(B) mRNA levels in control (CONT), ethanol-treated (CET) and with- water deprivation for 48 h is associated with normal VPdrawn (W) rats. The values are expressed as arbitrary optical density

mRNA levels in parvocellular neurons of the PVN [11],units. Horizontal bars represent mean values. The CRH mRNA levelsthe maintenance of osmotic stimulation for 7 days resultswere significantly lower in ethanol-treated than in control rats. There werein significantly increased VP mRNA levels [4], whereasno significant changes in VP mRNA levels after ethanol treatment or

withdrawal. Tukey’s post hoc tests: *P50.01, compared with control rats. the opposite occurs after chronic ethanol treatment.Another possible explanation for the alterations herein

degeneration when exposed for long periods to alcohol. It reported is that corticosterone, whose plasma levels areis also noteworthy that the pattern of reaction to prolonged increased by acute and short-term alcohol exposure, mayethanol exposure, although similar for each cell type, act via negative feedback mechanisms to inhibit thefundamentally differs between magnocellular and par- hypophysiotropic neurons located in the PVN. In fact,vocellular neurons: the magnocellular neurons enhance there is evidence that plasma corticosterone providestheir activity in order to compensate for neuronal loss inhibitory feedback at CRH mRNA expression by acting[30,46,51], whereas parvocellular neurons react by de- directly on CRH-secreting neurons and via inputs to thesecreasing neuropeptide synthesis and expression. neurons [20], and that the ability of alcohol to activate the

Actually, the estimates herein obtained reveal that HPA axis is sensitive to corticosteroid feedback [40].

28


However, the finding that after 6 months of ethanol also been noticed in situations in which the concentrationstreatment the plasma levels of corticosterone are within of glucocorticoids are increased and their ability to inhibitnormal levels essentially rules out such a possibility. ACTH secretion is not impaired, such as during lactation.

It seems therefore likely that the changes we have found It is therefore conceivable that the alterations we havein CRH- and VP-producing neurons in response to chronic described in the synthesis of CRH and VP might representethanol intake might result from direct effects of alcohol adaptive changes in order to maintain normal levels ofupon PVNmp neurons. Supporting this possibility is the corticosterone in rats submitted to prolonged alcoholobservation that the enhancing effects of alcohol on the intake. However, the observation that the response of theactivity of PVNmp neurons are transient and that the HPA axis to a subsequent challenge with alcohol [24,43],activity of the HPA axis changes as a function of the or to immune and non-immune signals [22,25], is bluntedduration of alcohol treatment. In fact, previous studies in rats exposed to high concentrations of alcohol forhave shown that the expression of the immediate-early shorter periods than the one used in this study indicatesgene markers of neuronal activation NGFI-B andc-fos in that ethanol exposure actually leads to a functional break-the PVNmp peaks 1–2 h after acute exposure [35,42], but down at some level of the HPA axis. Data herein obtainedabates thereafter to become undetectable after 6 days of suggest that such alterations certainly involve the hypo-ethanol exposure [25,35]. In the same line, it has been thalamic corticotropic and vasopressinergic systems.reported that the CRH and the VP mRNA levels are both It is known that CRH and VP are released from axons inincreased after acute exposure [34] whereas, after more the external zone of the median eminence and that theyprolonged exposures (7–8 days), the CRH mRNA levels synergistically stimulate ACTH secretion by interactingare increased by 2-fold [45] or unchanged [22], and the VP with CRH type I and VP type V1b receptors in themRNA levels are reduced to half [12,13,18]. Our results pituitary corticotrophs [2]. Although hypophysial portalextend these observations by showing that, by comparison concentrations of CRH and VP have not been measured inwith rats exposed to alcohol for 1 week, the situation has this study, there are reasons to assume that, duringchanged considerably in rats continuously exposed to prolonged ethanol treatment, higher levels of VP than ofalcohol during 6 months. We have found that chronic CRH reach the anterior pituitary to modify the control ofethanol treatment leads to a 42% reduction in the CRH ACTH secretion. First, we have found that the synthesis ofmRNA levels and to a small (16%), and not significant, CRH is significantly reduced in the PVNmp, and there isdecline in the VP mRNA levels measured in the PVNmp. evidence that changes in CRH mRNA levels usuallyTogether, these data indicate that the alcohol effects on the correlate with CRH release [27]. Second, earlier studiesactivity of the CRH- and VP-producing parvocellular have shown that in rats submitted to 8 days of alcoholneurons in the PVN are strongly time-dependent: with the exposure, VP stores in the external zone of the medianelongation of the exposure to alcohol there is a progressive eminence are increased, whereas those of CRH are de-dampening in the synthetic activity of CRH neurons, creased, despite the fact that opposite changes occur inwhereas the reverse occurs with VP neurons, which their mRNA levels measured in the parvocellular PVNpartially resume their activity. [25]. Finally, VP may be released by axons of magnocellu-

Our data also show that prolonged alcohol intake is lar neurons of the PVN and supraoptic nucleus passingassociated with a greater decrease in the total number of through the internal lamina of the median eminence [3,17],neurons immunoreactive for CRH than for VP (21 vs. and there is evidence that these neurons are engaged in the14%), and with a significant decay in the CRH mRNA production of increased amounts of VP in conditions ofcontent (42%). Despite the presence of lower VP mRNA prolonged alcohol intake [30,46,51]. An alternate but notlevels in ethanol-treated rats than in controls (16%), this incompatible hypothesis is that in ethanol-treated rats onlydifference was not statistically significant. Because in small amounts of CRH and VP are required to maintainnormal conditions approximately 50% of the CRH neurons normal corticosterone levels. If that would prove to be thein the PVN also express VP [48,59], the finding of case, then some kind of adaptation might occur at thedissociation between CRH and VP synthesis suggests that anterior pituitary. Supporting this view is the finding thatthese two peptides are differentially regulated during alcohol causes an increased sensitivity of the pituitary tochronic alcohol intake. Data gathered from a wide variety CRH [23] and does not alter the pituitary mRNA levels ofof experimental situations, including chronic stress CRH type I receptors even after 6 days of exposure [43].[7,16,33], osmotic stimulation [4], lactation [57] and In addition, although in rats continuously exposed toadrenalectomy [59], have provided evidence that VP plays alcohol vapors for 2 weeks the production of pro-a pivotal role in the maintenance of the activity of the HPA opiomelanocortin (POMC), the precursor molecule ofaxis under conditions of continuous or repeated stimula- ACTH, by the pituitary gland is decreased [6], there aretion. Although such alterations have been most frequently reports showing that, in rats fed an ethanol liquid diet forobserved when glucocorticoids appear to be less efficient 2–3 weeks, the rate of biosynthesis and release of POMCto elicit negative feedback on the HPA axis, namely under by the anterior and neurointermediate lobes of the pituitarychronic stress conditions or after adrenalectomy, they have is increased [10,50]. Finally, the observation that the

29


[9] F.W. Ellis, Effect of ethanol on plasma corticosterone levels, J.increments in CRH and VP content and mRNA levelsPharmacol. Exp. Ther. 153 (1966) 121–127.induced by withdrawal were not associated with parallel

[10] C. Gianoulakis, W.D. Hutchison, H. Kalant, Effects of ethanolvariations in the circulating levels of corticosterone are in treatment and withdrawal on biosynthesis and processing ofkeeping with data from earlier reports showing that, 15 proopiomelanocortin by the rat neurointermediate lobe, Endocrinol-days after withdrawal from an ethanol liquid diet, the ogy 122 (1988) 817–825.

[11] V. Grinevich, X.-M. Ma, J. Verbalis, G. Aguilera, Hypothalamicbiosynthesis and release of POMC has returned to normalpituitary adrenal axis and hypothalamic–neurohypophysial respon-levels [10].siveness in water-deprived rats, Exp. Neurol. 171 (2001) 329–341.In summary, our results show that, in basal conditions,

[12] K. Gulya, J.R. Dave, P.L. Hoffman, Chronic ethanol ingestionchronic alcohol intake has profound consequences for the decreases vasopressin mRNA in hypothalamic and ex-HPA axis. These involve changes that take place at the trahypothalamic nuclei of mouse brain, Brain Res. 557 (1991)

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reversed 2 months after withdrawal. Therefore, the analysis ˆ[14] H.J.G. Gundersen, E.B.V. Jensen, K. Kiev, J. Nielsen, The efficiencyof our results in view of previously published data also of systematic sampling in stereology—reconsidered, J. Microsc. 193

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[20] M.E. Keller-Wood, M.F. Dallman, Corticosteroid inhibition ofACTH secretion, Endocr. Rev. 5 (1984) 1–24.

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IIII..

Sexually dimorphic response of the hypothalamo-pituitary-adrenal axis to

chronic alcohol consumption and withdrawal

33

Research Report

Sexually dimorphic response of thehypothalamo–pituitary–adrenal axis to chronic alcoholconsumption and withdrawal

Susana M. Silva, M. João Santos-Marques, M. Dulce Madeira⁎

Department of Anatomy, Porto Medical School, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Article history:Accepted 24 September 2009Available online 30 September 2009

In males, long-term alcohol consumption provokes neurochemical changes in the medialparvocellular division of the PVN (PVNmp) that are partially reversed by withdrawal.Because gonadal steroids modulate the activity of the hypothalamo–pituitary–adrenal axis,we analyzed the possibility that the repercussions of chronic alcohol consumption andwithdrawal on the anatomy and neurochemistry of the PVNmp might differ between thesexes. Male and femaleWistar rats were examined after ingesting a 20% alcohol solution for6months or after 2months of withdrawal from 6months of alcohol consumption. The levelsof gonadal steroids and the basal concentrations of corticosterone were also evaluated.Chronic alcohol consumption and withdrawal did not alter the global cytoarchitectonicfeatures of the PVNmp in rats of both sexes. However, alcohol consumption was associatedwith a decrease in the number of vasopressin (VP) neurons only in females and ofcorticotropin releasing hormone (CRH) neurons in males and females. Further, the responseto withdrawal was sexually dimorphic because in males there was a partial recovery of thenumber of CRH neurons whereas in females there was a further loss of VP and CRH neurons.Corticosterone levels were unchanged by alcohol consumption, but they were decreased bywithdrawal in females. Alcohol consumption and withdrawal did not alter estrogen andprogesterone concentrations in females, but decreased testosterone levels in males. Thesefindings show that the response of CRH and VP neurons to excess alcohol is gender-specific,with females being more vulnerable during alcohol consumption and, most notably, afterwithdrawal.

© 2009 Elsevier B.V. All rights reserved.

Keywords:AlcoholWithdrawalHypothalamo–pituitary–adrenal axisGonadal steroids

1. Introduction

Research performed during the past two decades has progres-sively exposed that the course and the consequences ofalcoholism are different in men and women. When comparedto men, women develop alcohol-related complications afterdrinking smaller amounts of ethanol, are at a higher risk of

developing liver disease at any given level of alcohol intakeand aremore vulnerable to the development of cardiovasculardiseases and myopathy (Mann et al., 2003; Rehm et al., 2003).Alcoholic women also appear to have higher sensitivity forbrain damage than alcoholic men, and moderate drinkingseems to confer cognitive benefits to men but not to women(Sohrabji, 2002; Sullivan et al., 2002; Zuccalà et al., 2001). In

B R A I N R E S E A R C H 1 3 0 3 ( 2 0 0 9 ) 6 1 – 7 3

⁎ Corresponding author. Fax: +351 22 5513617.E-mail address: [email protected] (M.D. Madeira).

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ava i l ab l e a t www.sc i enced i r ec t . com

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addition, and as opposed to alcoholic men, the volume of theleft hippocampal formation (Agartz et al., 1999) and the cross-sectional area of the corpus callosum (Hommer et al., 1996) arereduced in alcoholic women, who also develop brain atrophycomparable to alcoholic men after shorter duration of alcoholconsumption (Hommer et al., 2001; Mann et al., 1992, 2005).Despite the recent and rapid increase in human studies usingbrain imaging techniques and the finding of sex differences inthe pharmacotherapy of alcoholism (Baros et al., 2008; Garbuttet al., 2005; Hernandez-Avila et al., 2006), neuropathologicalstudies using females are infrequent and, consequently, it isstill not know how alcoholism differentially affects the centralnervous system of both sexes.

The few available experimental studies that have usedmales and females failed to demonstrate a significantinfluence of sex in the changes induced by chronic alcoholconsumption in the thickness of the frontal cortex and corpuscallosum (Savage et al., 2000), density of choline acetyltrans-ferase neurons in themedial septal region (Savage et al., 2000),total number and volume of supraoptic neurons, and densityand size of vasopressin-producing neurons of the supraopticnucleus (Madeira et al., 1993). Yet, sex differences have beenrecognized in the cognitive processes affected by ethanol(Savage et al., 2000) and in the alcohol-induced variations inthe brain levels of neurosteroids (Janis et al., 1998) and in thedensity and size of the supraoptic neurons that synthesizeoxytocin (Madeira et al., 1993). There is also evidence thatalcohol consumption alters the activity of the hypothalamic–pituitary–adrenal (HPA) axis in a gender-specific way. Theplasma corticotropin (ACTH) and β-endorphin levels are lowerin women than in men and, in both sexes, lower in heavydrinkers than in non-drinkers; however, the decrease pro-voked by alcohol ingestion is more pronounced in womenthan in men (Gianoulakis et al., 2003). In rodents, there arestudies showing that alcohol administration enhances theactivity of the HPA axis, with females secreting more ACTHand corticosterone than males in response to acute (Ogilvieand Rivier, 1997; Rivier, 1993; Silveri and Spear, 2004) andshort-term (Silveri and Spear, 2004) exposures. However, thereis clear evidence that ethanol modifies the activity of theneurons producing neurohormones in a way that is strictlydependent on the length of the exposure (reviewed in Madeiraand Paula-Barbosa, 1999). This is the case of the corticotropinreleasing hormone (CRH)- and vasopressin (VP)-producingneurons of the hypothalamic paraventricular nucleus (PVN),whose synthesizing capacity is depressed in rats submitted tolong-term ethanol consumption (Silva et al., 2002). However,this study focused on males with no mention of how theresponse might be in females.

The remarkable sex differences in the activity of the HPAaxis, either in basal conditions or after stimulation, led us tohypothesize that its response to chronic alcohol consumptionwould be sexually dimorphic as well. We addressed thispossibility in the present study by assessing, in males and infemales consuming ethanol for 6 months, the total number ofneurons that produce the ACTH secretagogues VP and CRH,which are located in the medial parvocellular division of thePVN (PVNmp). Given the relevance of functional recovery fromalcohol dependence and the existing evidence, generated byclinical and preclinical studies, that gender influences the

behavioral and neuronal responses to ethanol withdrawal (fora review, see Wiren et al., 2006), we have additionallyincorporated in this study male and female rats that, after6 months of alcohol consumption, went through a 2-month-long withdrawal period.

2. Results

2.1. Animals and treatments

The amount of solid diet consumed by control and ethanol-treated rats (Fig. 1A)was influencedby treatment [F1,74=786.10,p<0.001], length of the experiment [F6,444=22.80, p<0.001] andsex [F1,74=22.80, p<0.001]. Alcohol consumption was associat-ed with a significant reduction of the amount of food eaten byrats of both sexes. The sharpest decrease occurred during thefirst month of treatment, at the end of which males ate 38%and females 25% less food (p<0.001) than the respectivecontrols. Thereafter, ethanol-treated females consumed arelatively constant amount of food whereas ethanol-treatedmales slowly, but progressively increased the amountthey consumed (p<0.001). Consequently, at the end of theexperiment, the difference between the amount of foodconsumed by ethanol-treated and control male rats (14%),although significant (p<0.001), was reduced relative to earlierexperimental months. Withdrawal was associated with asignificant and progressive increase over the 2 months of theamount of solid food consumed by rats of both sexes (p<0.001;Fig. 1A).

The amount of liquid diet ingested by control andethanol-treated rats (Fig. 1B) varied as a function oftreatment [F1,74=883.15, p<0.001], length of the experiment[F6,444= 127.86, p<0.001] and sex [F1,74=178.16, p<0.001].During the first month of alcohol consumption, the volumeof fluid ingested dropped by 58% (p<0.001) in males and by48% (p<0.001) in females relative to the amount of wateringested by the respective controls. Thereafter, ethanol-treated rats ingested a relatively constant amount of ethanolsolution, whereas control rats progressively reduced(p<0.001 for males and females), from the age of 3 months(second month of treatment) onwards, the amount of waterconsumed. As a result, at the end of the period of alcoholconsumption the differences between ethanol-treated andcontrol rats were smaller, but still significant (p<0.001).Withdrawal resulted in a rapid and significant increase(p<0.001) in the volume of water ingested by rats of bothsexes (Fig. 1B).

The mean daily ethanol consumption during the entireexperiment, expressed in g/day/kg b.w. (SD), was 8.1 (0.7) inmales and 10.1 (2.1) in females, which indicates thatfemales ingested significantly more (24%; p<0.001) ethanolrelative to body weight than males (Table 1). Despite thereduction in food consumption provoked by alcohol treat-ment, the total daily energy intake relative to body weight(Table 1) was significantly higher in ethanol-treated than incontrol rats (males, p<0.001; females, p=0.03). In control aswell as in ethanol-treated groups the total caloric intakerelative to body weight was significantly higher in females(p<0.001 and p=0.01, respectively). Moreover, the caloric

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intake relative to body weight provided by solid food,although significantly higher in control females than incontrol males (p<0.001), did not significantly differ betweenethanol-treated male and female rats (p=0.24; Table 1).Ethanol accounted for 23% of the total caloric intake inmales and for 26% in females.

2.2. Body and brain weights

At the end of the experiment, body weights of males,expressed in g (SD), were 529 (20) for control, 401 (47) forethanol-treated and 459 (41) for withdrawn rats; in females,they were 300 (29), 257 (25) and 286 (21), respectively.Differences in body weights were dependent on the effect oftreatment [F2,72 =46.05; p<0.001] and sex [F1,72 =619.93;p<0.001]; a significant treatment×sex interaction was alsodetected [F2,72=11.51; p<0.001].

Brain weights varied only as a function of sex [F1,72=6.41;p<0.05] as, in all groups, they were heavier in males than infemales. No effect of treatment [F2,72=0.10; p=0.91] and nosignificant treatment×sex interactionwasdetected [F2,72=1.31;p=0.28]. The brain weights of males, expressed in g (SD), were1.54 (0.09) for control, 1.51 (0.10) for ethanol-treated and 1.49(0.08) for withdrawn rats; in females, they were 1.39 (0.19), 1.46(0.20) and 1.46 (0.14), respectively.

2.3. Blood alcohol concentrations

As shown in Fig. 1C, blood alcohol concentrations varied as afunction of the time of day [F2,124=551.62; p<0.001] and, at alltimes analyzed, they were similar in males and in females[F1,124=3.18; p=0.77]; no significant interaction between timeof day and sex was detected [F2,124=1.08; p=0.34]. Bloodalcohol concentrations were higher in the evening than inthe morning and, at the time of death, i.e., 6 to 7 h after lightonset, rats of both sexes had lower, but measurable levels ofalcohol in the blood.

2.4. Corticosterone concentrations

ANOVA revealed a main effect of treatment on serum corti-costerone concentrations [F2,72=13.53; p<0.001]. Male and

Fig. 1 – (A) Effect of alcohol consumption and withdrawal onthe amount of solid diet consumed bymale (M) and female (F)rats. (B) Effect of alcohol consumption and withdrawal on theamount of liquid diet consumed by male and female rats.(C) Diurnal variation of blood alcohol concentrations. In A andB symbols represent means and vertical bars SD. In C,columns representmeans and vertical bars SEM of 26 (assaysin blood samples collected at 10:00 h and 22:00 h) or 13(assays in blood samples collected at 14:00 h) rats per group.Tukey's post hoc tests: *p<0.001 compared to 10:00 h;+p<0.001 compared to 14:00 h.

Table 1 – Energy intake.

Groups Energy(Kcal/d/Kg b.w.)

Total Energy(Kcal/d/Kg b.w.)

Food Fluid

C M 204 (4) – 204 (4)F 241 (18)ϕϕ – 241 (18)ϕϕ

ET M 186 (19)⁎ 57 (5)# 243 (23)⁎⁎

F 192 (17)⁎⁎ 70 (15)# 262 (29)⁎,ϕ

W M 189 (16) – 189 (16)+

F 195 (13)⁎⁎,+

– 195 (13)⁎⁎,+

b.w.=body weight; C=control; ET=ethanol-treated; F= female;M=male; W=withdrawal Values represent mean (SD). Tukey'spost hoc tests: ⁎p<0.05; ⁎⁎p<0.001 compared to C; +p<0.001 comparedto ET; ϕp<0.05; ϕϕp<0.001 compared to M. Student's t-test:#p<0.0001.

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female rats consuming alcohol for 6 months had corticoste-rone concentrations that were not significantly different fromthose of controls (Fig. 2A). Withdrawal did not alter the levelsof corticosterone in males, but it was associated with astatistically significant decrease in females. ANOVA failed toexpose a significant influence of sex on corticosterone levels[F1,72=0.61; p=0.44], but it revealed a significant treatment×-sex interaction [F2,72=18.52; p<0.001]. In control groups,females had the highest levels whereas in withdrawn groupsthe opposite occurred. No sex-related differences were foundin ethanol-treated groups.

2.5. Sex steroid hormone levels

In males (Fig. 2B), alcohol consumption provoked a sig-nificant decrease in testosterone levels, a variation that wasnot reversed by withdrawal [F2,36=11.14; p<0.001]. Converse-ly, in females (Figs. 2C, D), 6 months of alcohol consump-tion and withdrawal did not induce significant variations inestrogen [F2,36=0.30; p=0.74] and progesterone [F2,36=1.69;p=0.20] concentrations. Ethanol-treated and withdrawn ratsmaintained regular, but slightly elongated estrous cycles.

2.6. PVNmp volume and total number of neurons

ANOVA revealed that the volume of the PVNmp (Fig. 3A)was influenced by treatment [F2,36=3.34; p=0.047] and sex[F1,36=92.92; p<0.001]; no significant treatment×sex interac-tion was found [F2,36=0.41; p=0.69]. The volume of the PVNmpwas larger in males than in females, in control as well as inexperimental groups. The total number of neurons in thePVNmp (Fig. 3B) was not influenced by treatment [F2,36=0.36;p=0.70] or sex [F1,36=1.24; p=0.27]; in addition, no significantinteraction between these parameters [F2,36=0.61; p=0.55] wasfound.

2.7. Number of CRH neurons

The total number of CRH-immunoreactive neurons (Fig. 4) wassignificantly influenced by treatment [F2,30=209.47; p<0.001],but not by sex [F1,30=0.23; p=0.64]; however, ANOVA revealeda significant sex×treatment interaction [F2,30=29.05; p<0.001].Control females had significantly more CRH neurons thancontrol males. Alcohol consumption provoked a significantreduction in the number of CRH neurons that was more

Fig. 2 – Serum corticosterone (A), testosterone (B), estrogen (C) and progesterone (D) levels in control (C), ethanol-treated(ET) and withdrawn (W) rats. Columns represent means and vertical bars SEM of 13 rats per group. Tukey's post hoc tests:*p<0.01, **p<0.001 compared to C rats; +p<0.001 compared to ET female rats.

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marked in females (48%) than in males (40%). Consequently,no sex-related difference was found in the number of CRHneurons in ethanol-treated groups. Interestingly, withdrawalhad opposite effects in the number of CRH neurons: in males,it was associated with a significant increase (22%) relative tothe values of ethanol-treated male rats whereas in females itprovoked a further decrease (19%) to 42% of the number foundin control females. As a result, withdrawn females had 30%fewer CRH neurons than withdrawn males. Despite therecovery observed in withdrawn males, the number of CRHneurons did not reach control levels.

2.8. Number of VP neurons

The total number of VP-immunoreactive neurons in thePVNmp (Fig. 5) was significantly influenced by treatment[F2,30=31.42, p<0.001]. Despite the lack of a significant effect ofsex [F1,30=0.13; p=0.72], we found a significant treatment×sexinteraction [F2, 30=14.39; p<0.001]. Control females hadsignificantly more VP neurons (18%) than control males.

Alcohol consumption was associated with a decrease in thenumber of neurons that, however, was significant only infemales (24%). Consequently, the number of VP neurons didnot differ between male and female ethanol-treated rats.Withdrawal did not alter the number of VP neurons in males,but in females it aggravated the alcohol effects leading to afurther decrease (30%) in the number of neurons. As a result,withdrawn males did not differ from control males withrespect to the number of VP neurons, as opposed to femalesthat had approximately half the number of control femalerats. As opposed to controls, withdrawn females had signi-ficantly less (30%) VP neurons than withdrawn males.

3. Discussion

In a previous study performed in males, we have demonstrat-ed that chronic alcohol consumption does not provoke thedeath of parvicellular neurons in the PVN (Silva et al., 2002). Inaddition to confirming these observations, data now obtainedin the PVNmp show that chronic alcohol consumption doesnot lead to cell death in the female PVNmp as well. Moreover,it does not produce significant changes in the volume of thePVNmp in both sexes. Despite the lack of major alterations inits global cytoarchitectonic organization, the PVNmp of maleand female rats consuming alcohol for 6 months hassignificantly fewer CRH neurons, and that of females alsosignificantly less VP neurons than in controls. Depression inneuropeptide synthesis is most probably the leading cause ofthese alterations because, in addition to the absence of cellloss in the PVNmp, there is evidence that excess alcohol leadsto parallel variations in themRNA and protein levels of severalhypothalamic neuropeptides, including CRH and VP (Madeiraet al., 1997; Silva et al., 2002).

The comparative analysis of our results with data availablein the literature suggests that the decrease in the number ofCRH and VP neurons might reflect the adaptation of the HPAaxis to ethanol exposure, a view defended by several authorsbased on the changes developing after relatively short periodsof alcohol consumption (Lee and Rivier, 1997; Ogilvie et al.,1998; Spencer and McEwen, 1990; Zhou et al., 2000). In fact,whereas the acute administration of ethanol to malesincreases CRH and VP mRNA levels in the parvicellular PVNneurons (Ogilvie and Rivier, 1997; Rivier and Lee, 1996), theelongation of ethanol exposure to periods ranging from a fewdays to 3 weeks depresses the activity of these neurons. Afterthis type of treatment, CRH mRNA levels are either increased(Rivier et al., 1990) or within normal levels (Zhou et al. 2000),the hypothalamic CRF content is reduced (Rivier et al., 1984)and the VP mRNA levels are decreased (Gulya et al., 1991;Sanna et al., 1993). The basal plasma corticosterone levelsfollow the hypothalamic alterations as they are markedlyincreased after acute ethanol exposure (Ogilvie and Rivier,1997; Rivier, 1993; Rivier et al., 1984; Zhou et al., 2000) andeither increased relative to ad libitum controls (Rivier et al.,1984; Zhou et al., 2000) or unchanged relative to pair-fedcontrols (Janis et al., 1998; Ogilvie et al., 1997; Patterson-Buckendahl et al., 2005; Rivier et al., 1984) when the treatmentlasts for 1–2 weeks. It should be acknowledged, however, thatthere is one report showing that after 5 weeks of daily alcohol

Fig. 3 – Graphic representation of the estimates of volume (A)and total number of neurons (B) in the PVNmp of control (C),ethanol-treated (ET) and withdrawn (W) rats. Columnsrepresent means and vertical bars SD. Tukey's post hoc tests:*p<0.001 compared to male rats.

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consumption corticosterone levels do not differ from ad libitumcontrols but are moderately increased relative to pair-fedcontrols (Rasmussen et al., 2000). However, the elongation ofthe period of alcohol consumption to 6 (present study) or8 (Little et al., 2008) months leads to a normalization of

corticosterone levels relative to both ad libitum (presentresults) and pair-fed (Little et al., 2008) control rats. As opposedto males, the acute administration of ethanol to females doesnot modify the mRNA levels of CRH and VP in the parvocel-lular PVN, despite the sharp raise it induces in corticosterone

Fig. 4 – Effect of alcohol consumption and withdrawal on the CRH immunoreactivity of the PVNmp. (A–F) Photomicrographs ofcoronal sections taken at mid levels of the PVN of control (A, D), ethanol-treated (B, E) and withdrawn (C, F) male (A, B, C) andfemale (D, E, F) rats. Note that the density of CRH neurons is reduced in ethanol-treated rats of both sexes and still further inthe withdrawn female rat (F). Scale bar=100 μm. (G) Graphic representation of the total number of CRH-immunoreactiveneurons in the PVNmp of control (C), ethanol-treated (ET) and withdrawn (W) rats. Columns represent means and vertical barsSD. Tukey's post hoc tests: *p<0.001 compared to sex-matched C rats; +p<0.05, ++p<0.01 compared to sex-matched ET rats;ϕp<0.001 compared to male rats.

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levels (Ogilvie and Rivier, 1997; Rivier, 1993). Similar to males,however, female rats fed an alcohol diet for 10 days haveunchanged CRH mRNA levels in the parvocellular PVN (Lee

and Rivier, 1994) and normal corticosterone levels (Lee andRivier, 1993, 1994). Therefore, the finding in our study ofnormal corticosterone levels in parallel with reduced numbers

Fig. 5 – Effect of alcohol consumption and withdrawal on the VP immunoreactivity of the PVNmp. (A–F) Photomicrographs ofcoronal sections taken at mid levels of the PVN of control (A, D), ethanol-treated (B, E) and withdrawn (C, F) male (A, B, C) andfemale (D, E, F) rats. The boundaries between the PVNmp (mp) and the posterior magnocellular (pm) and dorsal parvocellular(dp) divisions of the PVN are indicated by dashed lines. In the photomicrographs obtained from male rats (A, B, C), no markeddifferences can be seen in the density of parvicellular VP neurons between control, ethanol-treated and withdrawn rats.Conversely, in females (D, E, F) the cell density is reduced in ethanol-treated and withdrawn rats. The withdrawn rat has thelowest cell density in the PVNmp. Scale bar=100 μm. (G) Graphic representation of the total number of VP-immunoreactiveneurons in the PVNmp of control (C), ethanol-treated (ET) and withdrawn (W) rats. Columns represent means and vertical barsSD. Tukey's post hoc tests: *p<0.01, **p<0.001 compared to sex-matched C rats; +p<0.01 compared to sex-matched ET rats;ϕp<0.01 compared to male rats.

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of CRH and VP neurons might suggest that after long-termalcohol consumption the adaptation of the HPA axis takesplace, in part at least, at the hypothalamic level and involvesreduced synthesis of CRH in both sexes and of VP in females.

If the alterations detected in rats submitted to long-termalcohol consumption would merely reflect the adaptation ofthe HPA axis to excess alcohol it would be expectable that theywould tend to fade away after withdrawal and that, 2 monthslater, they would be virtually non-existent. Yet, that was notthe case in rats of both sexes. Withdrawn males had signi-ficantly more CRH neurons than ethanol-treated male ratsthat, however, did not reach control levels. In females,withdrawal aggravated the alterations induced by alcoholconsumption and further reduced the number of CRH and VPneurons to about half the number in controls. Moreover, thischange in femaleswasassociated to a decline in corticosteroneconcentrations to levels lower than in control and ethanol-treated rats. Therefore, our data indicate that alcohol, whenconsumed for long periods, exerts long-lasting neurotoxiceffects in the PVNmp that, in females, are associated with adeficient activity of the HPA axis even in basal conditions.

Recently published studies have shown that male ratsingesting alcohol for 8 months followed by 2 months of with-drawal have normal circulating levels of corticosterone, whichfits our own observations in males, but have high corticoste-rone concentrations in regions of the brain such as thehypothalamus and the hippocampus (Brooks et al., 2008; Littleet al., 2008). The same studies also demonstrated that theavailable cytosol type II binding is decreased and the nuclearglucocorticoid receptor protein increased in homogenates ofthe prefrontal cortex of mice 2 weeks after withdrawal from28 weeks of alcohol consumption. The presence of highcorticosterone concentrations in the hypothalamus and inthe hippocampus, one of the extrahypothalamic sites thatmediates the inhibitory influence of glucocorticoids on theHPA axis (for a review, see Herman et al., 2005), would explainthe decrease in the number of CRH-immunoreactive neuronsobserved in withdrawn rats. The fact that the tissue levels ofcorticosterone raise only after withdrawal, and not duringalcohol consumption (Little et al., 2008), would contribute tounderstand the additional loss of CRH neurons noticed inwithdrawn females. However, it does not explain the partialrecovery seen in withdrawn males as well as the presence ofreduced numbers of CRH neurons in alcohol-treated rats ofboth sexes. Nor does it provide an explanation for the co-existence of reduced numbers of VP neurons and normal orlow corticosterone levels in ethanol-treated and withdrawnfemales, respectively, considering that even in the presence ofhippocampal lesions VP neurons remain susceptible toglucocorticoid feedback (Herman et al., 1989).

Since the HPA axis is sensitive to sex steroid hormones, thepresence of gender differences in the response of CRH and VPneurons to alcohol consumption and withdrawal raises thepossibility that gonadal steroids might be involved in deter-mining the neural and hormonal consequences of theseconditions. Studies that have analyzed the influence of sexsteroids in the plasma ACTH and corticosterone levels (Binga-man et al., 1994; Figueiredo et al., 2007; Lunga and Herbert,2004; Seale et al., 2004a,b) and in the CRH and VP mRNAexpression in the PVN (Bingaman et al., 1994; Figueiredo et al.,

2007; Patchev et al., 1995; Seale et al., 2004a,b) have providedevidence that testosterone inhibits and estrogen activates theHPA axis. Our results show that alcohol consumption over6 months reduces plasma testosterone to half the levelsmeasured in control males and does not alter estrogen andprogesterone levels in females. We should mention at thispoint that these data are in agreement with results fromhuman studies (reviewed in Gill, 2000; Emanuele and Ema-nuele, 2001) and investigations in rats submitted to relativelyshort periods of alcohol exposure (Adams and Cicero, 1991;Colantoni et al., 2000; Emanuele et al., 2001; Yin et al., 2000).However, ethanol-treated males have corticosterone levelsand numbers of VP neurons similar to those of controls andreduced numbers of CRH neurons, which is in conflict with theeffects of testosterone on these parameters. We noticed thesame incongruity in ethanol-treated and withdrawn females,as they had reduced numbers of CRH and VP neurons andnormal or low corticosterone concentrations despite thepresence of normal circulating levels of estrogen. Although,under basal conditions, CRH mRNA levels correlate positivelywith estrogen during the physiological variations that occurover the estrous cycle (Critchlow et al., 1963) and after singleadministrations of estradiol (Patchev et al., 1995), the oppositeoccurs when under the influence of persistent high levels ofestrogen (Lunga and Herbert, 2004; Paulmyer-Lacroix et al.,1996). However, this is an unlikely explanation for the changesnoticed in ethanol-treated andwithdrawn females because, inboth cases, the estrous cycles, although elongated, wereregular. It is worth pointing out that female rats have beenreported to develop persistent diestrus after being treatedwithan ethanol liquid diet for just a few days (Rettori et al., 1987).The changes in estrous cyclicity found in our study do notconform to these observations, but they are consistent withthose noticed in studies in which rats were fed ethanol liquiddiets during several weeks or months (see Emanuele et al.,2001; see also, Sanchis et al., 1985). Therefore, our data indicatethat the neurochemical and hormonal alterations that occurafter long-term alcohol consumption and withdrawal in thePVNmp of male and female rats are not likely to be a directconsequence of the ethanol- and withdrawal-induced varia-tions in the levels of sex steroid hormones.

In the experimental model used in this study, males andfemales also differ with respect to the amount of alcoholingested. Although males drunk a higher volume of ethanolsolution, females consumed 24%more ethanol relative to bodyweight than males, similarly to what has been noticed in ratsfed an alcohol diet for just a few weeks (Adams, 1995; Almeidaet al., 1998; Lancaster and Spiegel, 1992). Despite this fact, ourdata show that blood alcohol levels were similar in both sexesat all times of day analyzed. The influence of gender in bloodalcohol levels is controversial. Some studies in humans and inexperimental animals have shown that females have higherlevels thanmales (Desroches et al., 1995; Middaugh et al., 1992;Mumenthaler et al., 1999; Rivier, 1993) whereas others haveactually demonstrated that they are identical in both sexes(Lancaster and Spiegel, 1992; Livy et al., 2003; Mumenthaleret al., 1999; Ogilvie and Rivier, 1997; Savage et al., 2000).Because females have increased alcohol bioavailability, theabsence of gender differences in blood alcohol concentrationshas been mainly ascribed to the faster disappearance rate of

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ethanol in females (reviewed in Mumenthaler et al., 1999;Ramchandani et al., 2001). The accelerated rate of ethanolmetabolism enhances the generation of acetaldehyde andother toxic products (Quintanilla et al., 2007), which cancontribute to explain the increased alcohol toxicity in femalesand, thus, the gender-related differences in the consequencesof alcohol consumption and withdrawal.

Alcohol interferes with nutrition and, therefore, anotherpossible reason for the sex-related differences herein de-scribed is that alcohol consumption might have provoked agreater nutritional deficit, and consequently an additionalstress, in females. However, our results show that therepercussions of alcohol consumption on body weights wereless drastic in females than in males since, at the end of theexperiment, ethanol-treated females were just 14% lighterthan control females whereas inmales this difference reached24%. That nutritional factors are not likely to play a major rolein the changes provoked by alcohol consumption over6 months is also supported by studies showing that ratspair-fed for periods ranging from 1 week to 2 months do notdiffer from ad libitum control rats with respect to thecirculating levels of corticosterone (Ogilvie et al., 1997;Patterson-Buckendahl et al., 2005), estrogen and progesterone(Emanuele et al., 2001). Further, even though little is knownabout the effects on the HPA axis of food restriction extendingover weeks ormonths, the few available studies have reportedeither unchanged CRH and VP mRNA levels (Johansson et al.,2008) or discrete decreases in CRH mRNA levels, with malesbeing more affected than females, after approximately2 weeks (Brady et al., 1990), and both an increase in themorning and a decrease in the evening of the CRH mRNAlevels measured after 24 days of food restriction (Girotti et al.,2009). Thus, although the combined effects of alcohol inges-tion and nutrient restriction might have provoked moreserious effects in females than in males, our data do notsupport the view that the male-female differences in theneural consequences of alcohol consumption and withdrawalmight be dependent on the nutritional restriction associatedwith alcohol consumption.

In summary, in the present study we demonstrate that thealterations induced by 6 months of alcohol consumption andsubsequent withdrawal in the basal activity of the HPA axisdiffer between the sexes and, in both situations, are moresevere in females. Our results further show that after 2monthsof withdrawal, the changes provoked by alcohol consumptionin the PVNmp are partially restored in males, but are furtheraggravated in females. The alterations herein describedmightcontribute to understand the attenuated basal and stimulatedadrenocortical concentrations (Adinoff et al., 2005) and thedecreased sensitivity of the HPA axis to stressful stimulinoticed in abstinent alcoholics (Adinoff et al., 1990; Lovallo etal., 2000).

4. Experimental procedures


Male and female Wistar rats were housed three per cage andmaintained throughout the experiments under 12 h light/

dark cycles (lights on at 07:00 h) and temperature of 22 °C.Solid diet (4RF21/C Mucedola, Milan, Italy) and water wereavailable ad libitum until rats were 2-month-old. At this age,rats were placed into individual cages and randomly assignedto one of the following groups: (1) Ethanol-treated: rats weregiven an aqueous ethanol solution as their only availableliquid source for 6 months, i.e., until the age of 8 months. Theethanol concentration was progressively increased; startingwith a 5% (v/v) solution and rising by 1% per day to a final 20%(v/v) 2 weeks later. Food was freely available throughout. (2)Withdrawal: rats were treated with ethanol over 6 months, asdescribed above, and then switched to tap water for a further2 months. The shift from ethanol treatment to water intakewas performed gradually over a 2-week period by progres-sively reducing the ethanol concentration in the drinkingsolution by 1% per day. Food pellets were freely availablethroughout the experiment. (3) Control: rats had free access totap water and pellet food for the duration of the experiment.Groups used for the stereological analysis of the PVNcomprised 7 males and 7 virgin females, whereas groupsused for immunocytochemical studies were composed of 6males and 6 virgin females. Because in a preliminary studywe did not find any difference between 8- and 10-month-oldrats in the stereological parameters herein analyzed, we didnot include in the study an age-matched control group forwithdrawn rats. Female groups were formed by pooling ratsat random stages of the estrous cycle. The estrous cycle wasmonitored by daily collection of vaginal smears and histo-logical examination during the first week of each month oftreatment and at the day of killing.

Food and fluid intake was measured every other day andthe amounts consumed were calculated. The handling andcare of the animals were conducted according to EuropeanCommunities Council guidelines in animal research (86/609/EEC) and Portuguese Act 129/92. All efforts were made tominimize the number of animals used and their suffering.Rats were killed between 13:00 and 14:00 h.

4.2. Blood alcohol concentrations

Blood samples (500 μL) were collected once per month by tailnicks, 2 h after the beginning of both the light and dark periodsand, at the time of killing, directly from the heart intoEppendorf tubes. After complete clot formation, sampleswere centrifuged twice at 2000 rpm for 10 min. Serum wasremoved, collected into aliquots and stored undiluted at−80 °C until analysis. Serum ethanol levels were measuredusing a commercial kit (K620-100; BioVision, Mountain View,CA, USA) with a detection limit of 0.4 ppm of ethanol.

4.3. Hormone assays

The levels of testosterone, 17β-estradiol and progesterone wereassayed in serumobtained as described above from trunk bloodcollectedat the timeof killing. For corticosteronequantification,serum samples were assayed in duplicate using an ELISA kit(AssayPro, St. Charles, MO, USA) with a minimum level ofdetection of 40 pg/mL and intra- and inter-assay coefficients ofvariation of 5.0 and 7.0%, respectively. Testosterone, estradiolandprogesteroneconcentrationsweremeasuredbyusing solid-

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phase competitive chemiluminescent enzyme immunoassaykits (IMMULITE®1000 Analyzer, Siemens Medical SolutionsDiagnostics, Amadora, Portugal) with an analytical sensitivityof 15ng/dL for testosterone, 15pg/mLforestradiol and0.1ng/mLfor progesterone.

4.4. Tissue preparation

Rats used for the estimation of total neuron numbers andvolume of the PVNmp were anesthetized by intraperitonealinjection of a solution (3 mL/kg b.w.) containing 1% sodiumpentobarbital and4%chloral hydrate inphysiological saline, andkilled by transcardiac perfusion of a fixative solution containing1% paraformaldehyde and 1% glutaraldehyde in 0.12 M phos-phate buffer, at pH7.2. Thebrainswere removed fromtheskulls,weighed and post-fixed for 15 days in fresh fixative. Afterremoval of the frontal and occipital poles, the blocks containingboth the right and left hypothalami were dehydrated through agraded series of ethanol solutions, embedded in glycolmetha-crylate (hydroxyethylmethacrylate, Technovit 7100, Kulzer andCo., Wehrheim, Germany) and sectioned in the coronal plane at40 μm. The sections were mounted serially and stained with aGiemsa solution modified for use in glycolmethacrylate-em-bedded material (West et al., 1991). Finally, the sections werecoverslipped with Histomount mounting medium.

Rats used for the immunocytochemical studies wereanesthetized by sequentially injecting, at intervals of 10 min,solutions in physiological saline of 0.25% acepromazine(176 μL/kg b.w., subcutaneous; Laboratórios Vitória, Amadora,Portugal), followed by 2% xylazine (132 μL/kg b.w., intramus-cular; Sigma) and, finally, 10% ketamine (500 μL/kg, intramus-cular; Laboratórios Pfizer, Seixal, Portugal). Then, rats wereplaced on a stereotaxic apparatus with bregma and lambda inthe same horizontal plane and the calvaria was exposed byperforming a midline incision in the skin of the skull. Holeswere drilled unilaterally, 1.1 mm posterior to the bregma and1.7 mm lateral to the midline, and a 10-μL Hamilton syringe(901N; Hamilton Bonaduz AG, Bonaduz, Switzerland) waslowered into the right lateral ventricle until 5.2 mm from thesurface of the skull. Colchicine (C9654, Sigma-Aldrich) wasdissolved in 0.25% physiological saline (50 nmol/μL, pH 7.4)and injected gradually (2 μL every 2 min) until the totalamount of 10 μL was delivered. The needle was left in place foran additional 10 min before being slowly withdrawn. Forty-eight hours after the infusion, rats were anesthetized byintraperitoneal injection of sodium pentobarbital and chloralhydrate, as described above, and injected intracardially with0.1 mL of heparin solution (containing 10 units USP per mL),followed by 1 mL of 1% sodium nitrate in saline. Then, theywere perfused transcardially with 150 mL of 0.1 M phosphatebuffer, pH 7.6, for vascular rinse, followed by 250 mL of afixative solution containing 4% paraformaldehyde in phos-phate buffer, at pH 7.6. The brains were removed from theskulls, immersed for 2 h in the same fixative and maintainedovernight in a solution of 10% sucrose in phosphate buffer, at4 °C. After trimming away the frontal and occipital poles, theblocks of tissue containing both hemispheres were mountedon a vibratome and serially sectioned in the coronal planethrough the PVN at 40 μm. After random selection of the firstsection between the two most rostral sections thus obtained,

alternate sections were separately collected in phosphatebuffered saline in order to obtain two independent sets fromeach brain: one was used for CRH immunostaining and theother for VP immunostaining. Sections were processed forimmunocytochemistry using the avidin–biotin techniquewithdiaminobenzidine as the chromogen, as previously described(Madeira et al., 1993, 1997; Silva et al., 2002). The antiseraagainst CRH and VP (purchased from Bachem, Weil am Rhein,Germany) were used at the dilution of 1:5,000. Tominimize thevariation in staining, immunohistochemical procedures werecarried out in parallel in sets of rats of the different experi-mental groups.

4.5. Stereological analyses

The cytoarchitectonic criteria used for the parcellation of thePVN and the terminology herein employed are those proposedby Swanson and Kuypers (1980). Accordingly, the medialparvocellular subdivision of the PVN (PVNmp) was identifiedas a densely packed cell group that is located medial to theposterior magnocellular division and extends throughout thecaudal half of the PVN. The volume of the PVNmp wasestimated by using the principle of Cavalieri and point-counting techniques (Gundersen and Jensen, 1987). The totalnumber of Giemsa-stained neurons in the PVNmp wasestimated in all sections in which the nucleus was visualizedby using the optical fractionator method (Madeira et al., 1997;Silva et al., 2002;West et al., 1991). In each section, the fields ofvisionwere systematically sampled using a step size of 150 μmalong the x-axis and 160 μm along the y-axis. The disectorused had a counting frame area of 600 μm2 at the tissue leveland a fixed height of 15 μm. On average, 170 neurons werecounted per nucleus; the coefficient of error of the estimateswas 0.08.

The total numbersofCRH-andVP-immunoreactiveneuronsin the PVNmp were estimated by using the same stereologicalmethod. However, the microscope fields were sampled inalternate sections using interframe distances of 120 μm (x-andy-axis) for CRH neurons and of 140 μm (x-and y-axes) for VPneurons; the area of the counting frameswas 4082 μm2 for CRHneuronsand6970μm2 forVPneurons; theheight of thedisectorwas 10 μm for both neuronal populations. The criteriaemployed for the identification of CRH-and VP-immunoreac-tive neurons was the immunostaining of the perikaryalcytoplasm with a relatively unstained nucleus. ParvicellularVP-immunoreactive neuronswere discriminated frommagno-cellular VP-immunoreactive neurons based on their morphol-ogy and size (Fig. 5; Swanson and Sawchenko, 1983). Onaverage, 140 CRH-immunoreactive neurons and 110 VP-immunostained neurons were counted per animal; the CE ofthe estimates was 0.09 and 0.10, respectively. All the estima-tionswere performed, atmagnification of ×2000, using the C.A.S.T.-Grid system software (Olympus DK A/S, Denmark) and aHeidenhain MT-12 microcator (Heidenhain GmbH, Germany).

4.6. Statistical analyses

Statistical comparisons between the amount of solid food andvolume of ethanol solution or water consumed by ethanol-treated and control rats during the experiment were per-

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formed by analysis of variance (ANOVA) with repeatedmeasures using treatment and sex as the independentvariables and length of the experiment as repeated measures.Comparisons between withdrawn rats and ethanol-treatedrats on the last month of treatment were done using theStudent's t-test for independent variables. A one-way ANOVAwith treatment as the independent variable was used toanalyze sex steroid hormones, and a two-way ANOVA withtreatment and sex as the independent variableswas applied tothe remaining data. Whenever significant results were foundfrom the overall ANOVA, pair-wise comparisons were madewith the post hoc Tukey's honest significant difference test.Differences were considered to be significant if p<0.05.

Acknowledgments

The authors thank Professor M.M. Paula-Barbosa for helpfulcomments on the manuscript. This work was supported by agrant from Centro de Morfologia Experimental (Unit 121/94)from Fundação para Ciência e a Tecnologia.

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IIIIII..

Effects of chronic alcohol consumption and withdrawal on the response of

the male and female hypothalamic-pituitary-adrenal axis to acute immune

stress

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Research Report

Effects of chronic alcohol consumption and withdrawalon the response of the male and femalehypothalamic–pituitary–adrenal axis to acute immune stress

Susana M. Silva⁎, M. Dulce MadeiraDepartment of Anatomy, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Article history:Accepted 7 January 2012Available online 25 January 2012

The hypothalamic–pituitary–adrenal (HPA) axis plays a central role in the response tostress, and its activity is sexually dimorphic and modulated by sex steroids. Recent workindicates that HPA axis functioning is disturbed by chronic alcohol consumption andsubsequent withdrawal in rats of both sexes, but particularly in females. To examine theinfluence of sex steroid hormones in HPA axis response to acute stress after ingestion ofa 20% ethanol solution over 6 months and subsequent withdrawal (2 months), intactmales, and estradiol- and oil-injected ovariectomized females received a single intraperito-neal injection of lipopolysaccharide (LPS). Six hours after LPS administration, corticosteroneconcentrations were increased in all male groups; however, in ethanol-treated rats theyremained below those of control and withdrawn rats. mRNA levels of corticotrophin-releasing hormone (CRH) increased, and were identical in all groups after LPS stimulation,whereas those of vasopressin, although increased, remained below control levels. LPS stim-ulation elevated corticosterone concentrations in all oil-injected female groups, but did notalter those of estradiol-injected females. In oil- and estradiol-injected ethanol-treatedfemales, CRH mRNA levels did not change in response to LPS stimulation, whereas thoseof vasopressin increased, but stayed below control levels. In withdrawn oil- and estradiol-injected females, CRH and vasopressin gene expression increased, but did not reach controllevels. These data show that prolonged alcohol consumption produces long-lasting,possibly irreversible, changes in the neuroendocrine system that regulates the productionof corticosteroids, and that these consequences are more profound in females, particularlywhen estrogen levels are low.

© 2012 Elsevier B.V. All rights reserved.

Keywords:HPA axisChronic alcohol consumptionWithdrawalLPSSex steroid

1. Introduction

It is widely known, from both human research and studieswith experimental animals, that the acute exposure to etha-nol leads to a transient, but powerful activation of the maincomponents of the hypothalamic–pituitary–adrenal (HPA)

axis (for a review, see Madeira and Paula-Barbosa, 1999).Specifically, a single alcohol administration increases theblood levels of adrenocorticotrophic hormone (ACTH) and cor-ticosterone, and enhances the synthesis of corticotrophin-releasing hormone (CRH) and vasopressin (VP) by neurons ofthe medial parvocellular division of the hypothalamic para-

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⁎ Corresponding author. Fax: +351 22 5513617.E-mail address: [email protected] (S.M. Silva).

0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2012.01.013

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

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ventricular nucleus (PVNmp), the central site that regulatesthe activity of the HPA axis (Whitnall, 1993). There are alsodata indicating that more prolonged exposures to ethanollead to the development of tolerance in laboratory animals.In particular, it has been shown that when the exposure ismaintained for a few days or weeks, the plasma concentra-tions of corticosterone are lower than those detected afteracute exposures, and the mRNA levels of CRH are eitherincreased or unchanged, and those of VP are decreased(reviewed in Madeira and Paula-Barbosa, 1999). In addition,studies using experimental models of alcohol consumptionover months have shown that corticosterone concentrationsreturn to normal levels, but the synthesis, and consequentexpression, of CRH and VP in the PVNmp is reduced (Silva etal., 2002, 2009). In theory, these changes might merely reflectthe adaptation of the HPA axis to alcohol exposure (Ogilvieet al., 1998; Spencer and McEwen, 1990). However, the obser-vation that withdrawal does not abrogate the effects ofchronic alcohol consumption on the number of CRH- andVP-producing neurons in the PVNmp (Silva et al., 2002, 2009)suggests that the down-regulation of these neuropeptidesmight actually result from ethanol effects on cellular function.

To explore this possibility, we have tested the hypothesisthat rats submitted to prolonged alcohol consumption andto subsequent withdrawal are unable to mount an adequateresponse of the HPA axis to stressors. It is well known thatacute stress activates the HPA axis by increasing the synthesisof CRH and VP in the parvocellular neurons of the PVN and therelease of ACTH by the pituitary, leading to the production ofglucocorticoids (Harbuz and Lightman, 1992; Itoi et al., 2004;Kovács and Sawchenko, 1996; Ma et al., 1997; Tsigos andChrousos, 2002). The endotoxin lipopolysaccharide (LPS) is asystemic stressor that induces the production of endogenouscytokines, which, in turn, stimulate the synthesis and therelease of CRH and VP into the hypophysial portal blood,with subsequent pituitary-adrenal activation and increasedglucocorticoid secretion (Beishuizen and Thijs, 2003; Rivestet al., 1995). In the present experiment, we have administereda single intraperitoneal injection of LPS to rats consumingalcohol for 6 months and to rats withdrawn from chronic alco-hol consumption for 2 months, and measured the impact ofthe immune stimulation on the mRNA levels of CRH and VPin the PVNmp and on the circulating levels of corticosterone.

There is ample evidence that the activity of the HPA axis issexually dimorphic in basal conditions and after stimulation,with females producing more corticosterone and having moreCRH and VP neurons in the PVNmp than males (Figueiredoet al., 2007; Kudielka and Kirschbaum, 2005; Silva et al., 2009).The vulnerability of the HPA axis to excess alcohol and to etha-nol withdrawal also differs between the sexes. Females secretemore corticosterone than males in response to acute andshort-term alcohol exposures (Ogilvie and Rivier, 1997) and,unlikemales, females have reduced circulating levels of cortico-sterone after withdrawal from chronic alcohol consumptiontogether with a further depression in CRH and VP expressionby PVNmp neurons (Silva et al., 2009). There are also studiesshowing that, in rodents, the HPA response to endotoxinand to cytokines is enhanced by gonadectomy and attenuatedby estradiol and testosterone replacement (Kudielka andKirschbaum, 2005; Seale et al., 2004a, 2004b; Spinedi et al.,

1992). Therefore, in this study we have analyzed in parallel theinfluence of LPS on the functioning of the HPA axis duringchronic alcohol consumption and after withdrawal in malesand in females. To examine the degree to which the sexualdimorphism of the HPA axis response to LPS is influenced bythe estrous cycle, and specificallywhether circulating estrogensare responsible for the sexually dimorphic response, femaleswere ovariectomized and injected 2 weeks later, before LPSadministration, with either estradiol benzoate or vehicle.

2. Results

2.1. Ethanol consumption and blood alcohol levels

The variations in the amount of liquid diet ingested over theperiod of alcohol consumption were very similar to thosedescribed in an earlier study (Silva et al., 2009): the volumeof fluid ingested by ethanol-treated rats decreased to abouthalf the amount of water drunk by controls during the firstmonth of alcohol consumption and, then, remained relativelysteady. The mean daily ethanol consumption over the entireexperimental period, expressed in g/day/ kg bw, was signifi-cantly higher (p<0.001) in females (10.9±0.04) than in males(8.2±0.02).

Two hours after light onset, blood alcohol concentrationswere 25±0.3 mg/dl in males and 23±0.4 mg/dl in females; 2 hafter light offset, they were 76±0.4 mg/dl in males and 70±0.4 mg/dl in females.

2.2. Estrous cycle pattern

Chronic alcohol consumption only slightly changed the estrouscycle pattern until the fourth month of alcohol consumption,at which time most females exhibited regular, but elongatedcycles, relative to control females. After 5 months of alcoholconsumption, only 33% of the females showed estrous cyclessimilar to those of controls. The differences included elongationof metestrous and diestrous phases, leading to an increasedtime interval betweenproestrous surges. Inwithdrawn females,the occurrence of regular cycles was reduced, with some fe-males being in persistent estrous (13%) and others completelyacyclic (25%). However,whenpresent, the cycleswere also elon-gated (63%).

2.3. Uterine and relative adrenal weights

Uterine weights, expressed in g, were 0.42±0.09 in oil-injectedrats and 1.29±0.29 in EB-injected rats. These differences werestatistically significant (p<0.001).

The relative adrenal gland weights and the results of the re-spective statistical analyses are shown in Table 1. The relativeweight of adrenal glands was higher in females than in malesin all groups studied. There were no significant differences inadrenal weights between oil- and EB-injected rats in all groupsstudied, except between unstressed withdrawn groups, inwhich adrenal glands were heavier in oil- than in EB-injectedrats. In unstressed rats, adrenal glandswere significantly heavi-er in ethanol-treated groups than in the respective controls and,after withdrawal, they returned to control levels in all groups,

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except oil-injected females. After LPS administration, the adre-nal glands were heavier in ethanol-treated and withdrawnrats than in the respective controls in all groups studied.

2.4. Serum corticosterone concentrations

ANOVA revealed significant effects of group, treatment and LPSon serum corticosterone concentrations; significant group×treatment, group×LPS and treatment×LPS interactions werealso detected (Table 2).

In males under basal conditions, corticosterone concentra-tions did not differ between control, ethanol-treated andwithdrawn rats (Fig. 1). LPS administration increased cortico-sterone concentrations in all groups. However, 6 h after theadministration, corticosterone concentrations were reducedin ethanol-treated males relative to those of control and with-drawn males.

In femalesunder basal conditions, corticosterone concentra-tions did not differ between control and ethanol-treated rats,but were significantly lower in withdrawn than in control rats,both in the oil- and EB-injected groups; in the EB-injectedgroup, they were also lower in withdrawn than in ethanol-treated females (Fig. 1). LPS administrationdid not change corti-costerone concentrations in all EB-injected rats, but significant-ly increased them in oil-injected control andwithdrawn rats. Asa result, after LPS stimulation, corticosterone concentrationsdid not differ between control, ethanol-treated and withdrawn

rats of the oil- as well as of the EB-injected group; however, inoil-injected rats, there was a small difference, of borderlinesignificance (p=0.05), between ethanol-treated and controlfemales.

In basal conditions, corticosterone concentrations werehigher in control and ethanol-treated females of the oil- andEB-injected groups than in the respective male groups; in with-drawn groups, the concentrations were similar in oil-injectedfemales and in males, and were, in both groups, lower than inEB-injected females.

2.5. Total number of nuclei expressing c-Fos

The estimations were done separately in the right and leftPVNmp, and the mean number per animal was calculated.Because the numbers were similar in oil- and EB-injectedfemales, data from these animals were pooled together. Inmales, the number of nuclei in the PVNmp positive for c-Fosestimated at 2, 4, 6 and 8 h after LPS administration was4822 (221), 5878 (168), 7367 (238) and 3892 (131), respectively(Fig. 2). In females, it was 4512 (179), 6409 (124), 8393 (144)and 4199 (177), respectively.

2.6. CRH mRNA expression

The values of CRHmRNA expression in the PVNmp are shownin Fig. 3 and the results of ANOVA in Table 2.

Table 1 – Relative adrenal weights (means±SD) in basal and LPS-injected rats and respective analysis of variance.

Relative adrenal gland weights (mg/kg bw)

Basal LPS

Male Oil EB Male Oil EB

C 105±31 199±24ϕ 235±24ϕϕ 137±38 308±53+, ϕϕ 273±53ϕϕEt 201±31⁎⁎⁎ 336±66⁎⁎, ϕϕ 384±56⁎⁎⁎, ϕϕ 169±26 455±58⁎⁎⁎, ++, ϕϕ 449±44⁎⁎⁎, ϕϕ

W 122±14## 422±64⁎⁎⁎, ϕϕ 264±48##, ϕϕ, δ 203±36⁎⁎, +++ 406±42⁎, ϕϕ 360±58⁎, #, +, ϕϕ

Source of varianceL T G L×T L×G T×G L×T×GF1,90=39.7 F2,90=70.6 F2,90=205.8 F2,90=0.1 F2,90=2.6 F4,90=10.7 F4,90=7.2p<0.001 p<0.001 p<0.001 p=0.91 p=0.08 p<0.001 p<0.001

Significance is denoted as: ⁎p<0.05, ⁎⁎p<0.01 and ⁎⁎⁎p<0.001 vs respective C rats; #p<0.05 and ##p<0.01 vs Et of the same group; +p<0.05, ++p<0.01and +++p<0.001 vs respective unstressed groups (Basal); ϕp<0.01 and ϕϕp<0.001 vs males; δp<0.001 vs oil-injected females. bw, body weight; EB,EB-injected females; G, group; Oil, oil-injected females; L, LPS; T, treatment.

Table 2 – Three-way analysis of variance showing the effects of LPS administration on corticosterone concentrations, andon CRH and VP mRNA levels.

Source of variance

L T G L×T L×G T×G L×T×G

Corticosterone F1,90=107.3 F2,90=7.9 F2,90=168.9 F2,90=11.7 F2,90=23.5 F4,90=7.3 F4,90=1.1p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p=0.37

CRH mRNA F1,90=117.3 F2,90=136.6 F2,90=13.7 F2,90=9.1 F2,90=6.8 F4,90=10.2 F4,90=11.3p<0.001 p<0.001 p<0.001 p<0.001 p<0.01 p<0.001 p<0.001

VP mRNA F1,90=1083.2 F2,90=259.4 F2,90=25.7 F2,90=21.4 F2,90=0.8 F4,90=18.2 F4,90=1.2p<0.001 p<0.001 p<0.001 p<0.001 p=0.47 p<0.001 p=0.31

G, group; L, LPS; T, treatment.

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In unstressed males, prolonged alcohol consumption pro-voked a significant reduction in CRH expression (27%), andwithdrawal reversed this effect. Although not statistically sig-nificant, this increase (22%) resulted in the absence of signifi-cant differences between withdrawn and control rats. LPSinjection significantly raised CRH mRNA levels in controland ethanol-treated rats, but not in withdrawn rats. Sincethe increase was relatively larger in ethanol-treated rats

than in the remaining groups, after LPS administration therewere no significant differences in CRH mRNA levels amongmale groups.

In unstressed oil- and EB-injected females, prolongedalcohol consumption provoked a significant reduction in CRHmRNA levels (31%and40%, respectively) thatwas further aggra-vated by withdrawal. Consequently, the levels were lower inwithdrawn than in control rats of the oil- and EB-injected (44%

Fig. 1 – Serum corticosterone concentrations in control (C), ethanol-treated (Et) and withdrawn (W) males, and oil- andEB-injected ovariectomized females, in unstressed conditions (Basal) and at 6 h after LPS administration (LPS). Columnsrepresent means and vertical bars 1 SEM. Significance is denoted as: ⁎p<0.05 vs respective C rats; #p<0.05 and ##p<0.001 vs Etrats of the same group; +p<0.05, ++p<0.01 and +++p<0.001 vs respective unstressed group; ϕp<0.01 and ϕϕp<0.001 vsmale rats;δp<0.001 vs oil-injected female rats.

Fig. 2 – Representative photomicrographs of c-Fos positive cells in the medial parvocellular subdivision of the hypothalamicparaventricular nucleus (PVNmp) of adult male (A–D) and female (E–H) rats killed at 2 h (A, E), 4 h (B, F), 6 h (C, G) and 8 h(D, H) following intraperitoneal administration of LPS (25 μg/100 g body weight). V, third ventricle. Scale bar=200 μm.

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and 48%, respectively) groups. In contrast tomales, after LPS ad-ministration, CRHmRNA levels remained significantly reducedin ethanol-treated and withdrawn rats of the oil- (37% and 18%,respectively) and EB-injected (33%and19%, respectively) groupsrelative to controls. Interestingly, in oil- and EB-injected groups,the administration of LPS increased CRH mRNA levels in with-drawn, but not in ethanol-treated rats.

In unstressed control rats, CRH mRNA levels did not differbetween males and oil-injected females and were, in bothgroups, lower than in EB-injected females. Conversely, inunstressed ethanol-treated rats no sex- or EB-related differ-ences were found, whereas in unstressed withdrawn rats CRHmRNA levels were lower in oil- and EB-injected females than

in males. After LPS administration, there were no male–femaledifferences in CRH mRNA levels, except among ethanol-treated rats, in which the expression was higher in males thanin both groups of females.

2.7. VP mRNA expression

The values of VP mRNA expression in the PVNmp are shownin Fig. 4 and the results of ANOVA in Table 2.

In unstressedmales, chronic alcohol consumption induceda significant reduction in VP mRNA levels (20%) that wasreversed by withdrawal. However, after LPS administration,the levels were significantly lower in ethanol-treated (29%),

Fig. 3 – Graphic representation of the optical density of CRH mRNA levels in control (C), ethanol-treated (Et) and withdrawn(W) male, and oil- and EB-injected ovariectomized rats, in unstressed conditions (Basal) and at 6 h after LPS administration(LPS). The values are expressed as arbitrary optical density units. Columns represent means and vertical bars 1 SD.Significance is denoted as: ⁎p<0.05, ⁎⁎p<0.01 and ⁎⁎⁎p<0.001 vs respective C rats; #p<0.01 vs Et rats of the same group; +p<0.01and ++p<0.001 vs respective unstressed groups; ϕp<0.05 and ϕϕp<0.001 vs male rats; δp<0.05 vs oil-injected female rats.

Fig. 4 – Graphic representation of the optical density of VP mRNA levels in control (C), ethanol-treated (Et) and withdrawn(W)male, and oil- and EB-injected ovariectomized rats, in unstressed conditions (Basal) and at 6 h after LPS administration (LPS).The values are expressed as arbitrary optical density units. Columns represent means and vertical bars 1 SD. Significance isdenoted as: ⁎p<0.05 and ⁎⁎p<0.001 vs respective C rats; #p<0.05 and ##p<0.01 vs Et rats of the same group; +p<0.001 vsrespective unstressed groups; ϕp<0.05, ϕϕp<0.01 and ϕϕϕp<0.001 vsmale rats; δp<0.05 and δδp<0.01 vs oil-injected female rats.

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as well as in withdrawn (21%) rats, than in controls becausethe relative increase induced by LPS was greater in controlthan in ethanol-treated and withdrawn rats.

In unstressed females, chronic alcohol consumption re-duced VP mRNA levels, but significantly only in EB-injectedrats, an effect that was further aggravated by withdrawal.Consequently, oil- and EB-injected withdrawn females hadsignificantly lower VP mRNA levels than the respective con-trols (31% and 42%, respectively). Similar to males, LPS injec-tion evoked a significant increase in VP mRNA levels in allfemale groups. Despite this fact, after LPS administration,the levels remained lower in ethanol-treated and withdrawnfemales than in controls, both in oil- (22% and 29%, respec-tively) and EB-injected (30% and 32%, respectively) groups.

In unstressed control rats, VP mRNA levels did not differbetween males and oil-injected females and were, in bothgroups, lower than in EB-injected females. These differenceswere still present after LPS administration. In unstressedethanol-treated rats, the levels did not differ between oil-and EB-injected females, and were, in both groups, higherthan in males. No effects of sex and EB treatment were appar-ent among unstressed withdrawn rats or between LPS-injected ethanol-treated and withdrawn rats.

3. Discussion

Data of the present study show that the response of the HPAaxis to an acute immune challenge during prolonged alcoholconsumption and ethanol withdrawal is dependent on genderand, in females, relies on the circulating levels of estrogens. Inmales, corticosterone release in response to a single intraper-itoneal injection of LPS is attenuated during prolonged alcoholconsumption and within control levels after withdrawal.Females do not increase corticosterone concentrations inresponse to LPS administration during alcohol consumptionirrespective of the circulating levels of estrogens and, afterwithdrawal, they do it when estrogen levels are low, but notwhen they are elevated. The results also show that the LPS-stimulated release of corticosterone depends only in part onthe activation of the PVNmp neurons producing CRH and VP.

In line with data from previous studies in which the sameexperimental model of alcohol consumption and withdrawalwas used (Silva et al., 2002, 2009), ethanol-treated and with-drawn male rats had basal corticosterone concentrationsthat did not differ from those of controls. Also in keepingwith those studies, CRH and VP mRNA levels in the PVNmphad declined after 6 months of alcohol consumption, butwere within control levels 2 months following withdrawal. Inall male groups studied, LPS injection evoked a significantincrease in corticosterone concentrations measured at 6 h.In controls, this effect was associated with activation ofPVNmp neurons producing CRH and VP, which is in accor-dance with data from earlier investigations (Grinevich et al.,2001; Harbuz and Lightman, 1992). In ethanol-treated males,the response to LPS was identical, but unlike CRH, whoseexpression increased to control levels, VP mRNA levelsremained lower than in LPS-injected controls. This is not like-ly to result from ethanol-induced changes in the levels of thevarious cytokines whose secretion is induced by LPS because

there is evidence that the increase in hypothalamic tumornecrosis factor-α and blood cytokines is unaffected by alcoholconsumption (Taylor et al., 2002). A significant reduction inthe hypothalamic production of interleukin-1β has howeverbeen reported (Taylor et al., 2002), but this cytokine does notseem to undergo a robust increase after LPS administration(Hadid et al., 1999; Kakizaki et al., 1999). Therefore, thedown-regulation of VP neurons might just be a consequenceof the enhanced negative feedback sensitivity of PVNmp neu-rons that underlies the adaptation of the HPA axis to pro-longed alcohol consumption. This does not preclude thepossibility that changes in other components of the HPA axismight also contribute to the presence of reduced corticoste-rone levels in ethanol-treated rats after LPS stimulation. Infact, there are studies showing that, after a few days of alcoholconsumption, the pituitary sensitivity to VP is reduced (Leeand Rivier, 1995) and the adrenal cortex response to LPS is at-tenuated (Mohn et al., 2011). Present data also show that theresponse of the HPA axis to the immune challenge inducedby LPS administration is related to the duration of alcohol ex-posure. Whereas males exposed to ethanol for 8–10 days re-lease more corticosterone than controls in response to LPS,despite the blunted activation of parvicellular PVN neurons(Lee et al., 2000), males consuming ethanol for 6 monthshave lower corticosterone concentrations and VP mRNAlevels than controls after LPS administration.

Inmales, withdrawal from prolonged alcohol consumptionenhanced corticosterone production in response to LPSadministration. The blood levels were higher than in the re-spective non-stressed group and LPS-injected ethanol-treated rats. Withdrawn male rats have approximately halfthe serum testosterone concentrations of control males(Silva et al., 2009), and there is experimental evidence thatthe removal of endogenous androgens by castration increasesLPS-stimulated, but not basal, corticosterone concentrations(Evuarherhe et al., 2009; Papadopoulus and Wardlaw, 2000;Seale et al., 2004a; Spinedi et al., 1992; Williamson et al.,2005). However, the lack of inhibitory influence of testoster-one on the activity of the HPA axis cannot be the sole explana-tion for the presence of higher corticosterone concentrationsin withdrawn than in ethanol-treated rats after LPS adminis-tration because testosterone concentrations are also reducedin rats consuming alcohol for long periods (Silva et al., 2009).In addition, there are studies reporting that the influence oftestosterone in corticosterone production is mediated by acti-vation of CRH and VP neurons in the PVN (Evuarherhe et al.,2009; Seale et al., 2004a, 2004b; Spinedi et al., 1992), an effectthat we were unable to detect in this study. In fact, our datashow that CRH and VPmRNA levels did not significantly differbetween withdrawn and ethanol-treated rats after LPS stimu-lation. These findings, together with the observation that inLPS-injected withdrawn rats the adrenal glands were 66%heavier than in the respective non-stressed group and 48%heavier than in LPS-injected control males, changes thatwere not apparent in LPS-injected ethanol-treated rats, sug-gests that the increased production of corticosterone in with-drawn males results from LPS effects at extra-hypothalamicsites. This possibility is supported by existing data showingthat orchidectomy enhances the stimulatory effects ofinterleukin-1β (Lee and Rivier, 1995; Rivier, 1994) and LPS

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(Hadid et al., 1995) on ACTH release and, conversely, testoster-one inhibits ACTH-stimulated corticosterone output from dis-persed inner adrenocortical cells (Nowak et al., 1995).

In females under basal conditions, corticosterone concen-trations were within control levels after 6 months of alcoholconsumption and reduced following withdrawal, which is inline with earlier observations made in intact females (Silvaet al., 2009). Also in conformity with the same study (Silva etal., 2009), alcohol consumption provoked a significant de-crease in CRH mRNA levels and a smaller, yet not significant,decrease in VPmRNA levels, and withdrawal aggravated theseeffects leading to significant reductions in CRH and VP mRNAlevels relative to those of control females. Although similar inoil- and EB-injected females, the reductions in CRH and VPgene expression induced by alcohol consumption and with-drawal were more evident in EB-injected females due to theenhancing effects of estrogens on CRH and VP mRNA levelsin controls (Li et al., 2003; Patchev et al., 1995). It deserves tobe mentioned at this point that females were gonadally intactuntil two weeks before the end of the experiments, and weretreated, for two days before being killed, with either estradiolbenzoate or vehicle. Our data show that estradiol treatmentincreased CRH and VP gene expression in control, but not inethanol-treated and withdrawn females, which indicatesthat the changes induced by prolonged alcohol consumptionand withdrawal at the hypothalamic level were not influ-enced by the short-term administration of estradiol.

LPS stimulation increased corticosterone concentrations inoil-injected control and withdrawn females, but not in ethanol-treated females. These results show that, like in males, femaleswith low estrogen levels are unable to release an adequateamount of corticosterone in response to a systemic immunechallenge after prolonged alcohol consumption, as opposed towhat happens after short exposures in which, despite theblunted ACTH production, corticosterone concentrationsincrease as much as in controls (Lee and Rivier, 1993). In clearcontrast with oil-injected females, the HPA axis of EB-injectedfemales did not respond to the immune challenge triggered byLPS administration in all groups studied. This observation is atodds with some (Seale et al., 2004a, 2004b; Watanobe andYoneda, 2003), but not all prior reports, which havemostly dem-onstrated that estrogens attenuate, or inhibit, the release ofcorticosterone in response to endotoxin and/or cytokines stimu-lation in humans (Beishuizen and Thijs, 2003; Puder et al., 2001)and experimental animals (Brown et al., 2010; Rivier, 1994;Spinedi et al., 1992) due to the inhibitory influence they exert,when in high concentrations, on the peripheral and centralsecretion of pro-inflammatory cytokines (reviewed in Straub,2007).

In females consuming alcohol for 6 months, LPS stimula-tion evoked no response in CRH neurons and significantly in-creased VP gene expression to levels that were, however,lower than in the respective controls. These effects were sim-ilar in oil- and EB-injected females and, thus, independent ofthe circulating levels of estrogens. If the down-regulation ofPVNmp neurons in females consuming alcohol for 6 monthswould merely reflect neuroadaptation to the chronic stressprovoked by excess alcohol, it might be plausible, then, to ex-pect that females would exhibit an amplified response of VPneurons to a novel stress, as repeatedly observed in other

models of chronic stress (Aguilera et al., 2008; Grinevich etal., 2001; Ma et al., 1999). Therefore, the finding in ethanol-treated rats of a blunted activation of VP neurons suggeststhat prolonged ethanol exposure, rather than leading just toadaptation of the HPA axis, disrupts the function of PVNmpneurons and/or of the neural afferents that regulate theiractivity. The changes induced by prolonged alcohol consump-tion in CRH and VP gene expression seem to be long-lastingbecause they are still apparent 2 months after withdrawal, inbasal conditions as well as after LPS stimulation. In addition,the increase in corticosterone concentrations provoked byLPS stimulation in withdrawn females was associated withactivation of VP neurons, like in controls, but also of CRH neu-rons. The increase in CRH mRNA levels after LPS administra-tion in withdrawn females, an effect that was not apparentin control females, points for a dysfunction of PVNmp neu-rons and/or dysregulation of their neural afferents more pro-found than expected based on data from ethanol-treatedrats. In fact, there is evidence that glucocorticoid receptor con-centrations in brain regions that modulate the activity ofPVNmp neurons are increased after withdrawal from pro-longed alcohol consumption, but not during alcohol con-sumption (Little et al., 2008), which might result in greaternegative feedback than expected from plasma corticosteroneconcentrations (Herman et al., 2005).

In this study, we have used c-Fos expression as a tool to ex-amine neuronal activity in the PVNmp in response to LPSstimulation. The estimates of total number of immunoreac-tive nuclei for c-Fos showed that activation was highest at6 h after LPS injection in rats of both sexes, which is in linewith earlier observations (Grinevich et al., 2001; Rivest et al.,1995). At this time point, CRH and VP neurons were both acti-vated in males, whereas in females only VP neurons showedincreased activity. It is possible that the preferential activa-tion of VP neurons in females depends on the length of thepost-LPS period used in this study. In fact, in the few availablereports on the effects of LPS on the activity of the HPA axis infemales it was shown that CRH and VP mRNA levels are bothincreased at 3 h after a single intraperitoneal injection(Harbuz and Lightman, 1992; Nappi et al., 1997; Seale et al.,2004a, 2004b). In addition, prior studies in males revealedthat CRH levels reach their peak 3 h after intraperitoneal ad-ministration of LPS (Kakizaki et al., 1999) and that the expres-sion of Fos by PVN neurons is contemporaneous with thetranscriptional activation of the VP gene and occurs 2 h afterthe activation of CRH transcription (Kovács and Sawchenko,1996). Our data in males is consistent with this observationas we found out that at the time point of maximal c-Fos ex-pression there was a 77% increase in VP mRNA levels, where-as the increase in CRH expression did not exceed 23%. We arenot aware of any similar investigation in females and thedesign of our study does not allow determining whether, ornot, the sequential activation of CRH and VP neurons is tem-porally different in males and in females. Nevertheless, thepresent data show that at the time point of maximal activa-tion of the PVNmp by LPS, the neurochemical phenotype ofits neurons differs between the sexes. Although speculatively,it is possible that the preferential response of VP neurons at6 h after LPS stimulation in females, but not in males, just re-flects the basal state of hyperactivation of the HPA axis in

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females, characterized by higher plasma corticosterone con-centrations and relative adrenal weights than in males.

In summary, present data demonstrates that prolongedalcohol consumption attenuates the release of corticosteronein response to an acute immune stress. The improvement ofthe corticosterone response after withdrawal together withthe weak activation of PVNmp neurons suggests that extra-hypothalamic mechanisms may be compensating for hypo-thalamic dysfunction. In general, the corticosterone responseto LPS is similar in males and oil-injected females. In females,the short-term increase in estrogen levels annuls the cortico-sterone response to LPS and blunts the activation of hypotha-lamic neurons.

4. Experimental procedures


Male and female Wistar rats were housed three per cage andmaintained throughout the experiments under 12 h light/dark cycles (lights on at 07:00) and temperature of 22 °C.Solid diet (4RF21/C Mucedola, Milan, Italy) and water werefreely available until rats were 2-month-old. At this age, ratswere randomly assigned to the following experimentalgroups: (1) Ethanol-treated rats were given an aqueous etha-nol solution as their only available liquid source for 6 months,i.e., until the age of 8 months. The ethanol concentration wasprogressively increased, starting with a 5% (v/v) solution andrising by 1% per day to a final 20% (v/v) 2 weeks later. Foodwas freely available throughout. (2) Withdrawn rats wereethanol-treated over 6 months, as described above, and thenswitched to tap water for a further 2 months. The shift fromethanol treatment to water intake was performed graduallyover a 2-week period by progressively reducing the ethanolconcentration in the drinking solution by 1% per day. Foodpellets were freely available throughout the experiment.(3) Control rats had free access to tap water and pellet foodfor the duration of the experiment. Food and fluid intakewere measured every other day and the amounts consumedper cage calculated; based on these values, the average dailyfood and fluid consumption per animal was estimated. Onlyregularly cycling rats were included in the study. During thefirst week of each month of treatment, the estrous cycle wasmonitored by daily collection of vaginal smears and histolog-ical examination.

Two weeks before the end of the experiments, females wereovariectomized bilaterally under deep anesthesia induced bysequentially injecting promethazine (10 mg/kg body weight,subcutaneous), xylazine (2.6 mg/kg bodyweight, intramuscular)and ketamine (50 mg/kg bw, intramuscular). Starting twelvedays later, rats were injected subcutaneously with either estra-diol benzoate (EB; 10 μg dissolved in 0.1 ml sesame oil; all fromSigma-Aldrich Company Ltd., Madrid, Spain) or sesame oil(0.1 ml) on two consecutive days (Figueiredo et al., 2007; Kisleyet al., 2000). Twenty-four hours after the last injection, half ofthe rats in each group received bacterial endotoxin lipopolysac-charide (from Escherichia coli, Serotype 055:B5, Sigma, L-2880, Lot067H4056, Sigma, St Louis, MO, USA) diluted in 0.3 ml of sterilepyrogen-free saline (0.9%), at a dose of 25 μg/100 g bw by

intraperitoneal injection (Castanon et al., 2001; Konsman et al.,1999; Lacroix and Rivest, 1997). The remaining rats in eachgroup were injected at the same time with 0.3 ml saline.

Rats (n=6 per group) were killed 6 h after LPS or saline ad-ministration. This time interval was chosen because, asrevealed on a pilot study in which the total number of neu-rons expressing c-Fos were counted in the PVNmp of ratskilled at 2, 4, 6 and 8 h after LPS administration, it allows forhigher activation of PVNmp neurons (see Results section andFig. 2). Before decapitation, animals were gently anesthetizedby intraperitoneal injection of a solution (3 ml/kg bodyweight)containing sodium pentobarbital (10 mg/ml) and chloral hy-drate (40 mg/ml) in physiological saline to ensure that the pro-cedure would be done with as little stress as possible. Afterdecapitation, brains were quickly removed, frozen on liquidnitrogen and stored at −80 °C. Then, the uteri and the adrenalglands were rapidly dissected and weighed.

The handling and care of the animals were conductedaccording to European Communities Council guidelines in an-imal research (2010/63/EU) and Portuguese Act 129/92. All ef-forts were made to minimize the number of animals usedand their suffering.

4.2. Biochemical assays

To determine blood alcohol concentrations, blood samples(500 μl) were collected once per month by tail nicks, 2 h afterthe beginning of the light and of the dark periods, directly intoEppendorf tubes. For measurement of corticosterone concen-trations, trunk blood samples (500 μl) were collected immedi-ately after decapitation into Eppendorf tubes. After completeclot formation, samples were centrifuged twice at 2000 rpm for10 min. Serumwas removed, collected into aliquots and storedundiluted at −80 °C until analysis. Serum ethanol levels weremeasured using a commercial kit (K620-100; BioVision,Mountain View, CA, USA) with a detection limit of 0.4 ppm ofethanol. Corticosterone concentrations were measured byradioimmunoassay using 125I RIA Kit (MP Biomedicals, Orange-burg, NY, USA). Sensitivity range: 7.7–1000 ng/ml, inter-assayvariation: 6.5%, intra-assay variation: 4.4%.

4.3. c-Fos immunohistochemistry and quantification

Male rats, and oil- and EB-injected females (n=12 per group)were injected intraperitoneally with LPS (25 μg/100 g bodyweight, diluted in 0.3 ml saline). At 2, 4, 6 and 8 h after the injec-tion, three rats of each group were intraperitoneally anesthe-tized as described above. They were perfused transcardiallywith 250ml of 0.1 M phosphate buffer, pH 7.6, for vascularrinse, followed by 400 ml of a fixative solution containing 4%paraformaldehyde in phosphate buffer, at pH 7.6. Brains wereremoved from the skulls, immersed for 1 h in the same fixative,andmaintained overnight in a solution of 10% sucrose in phos-phate buffer, at 4 °C. The blocks containing the right and lefthypothalamiwere isolated,mounted ona vibratome and serial-ly sectioned in the coronal plane through the PVN, at 40 μm.Alternate sections were collected in phosphate buffer saline(PBS) and immunostained for visualization of c-Fos. The adja-cent sections were Nissl-stained and used for identifying theboundaries of the PVNmp. For c-Fos immunostaining, sections

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were treated with 3% H2O2 for 10min to inactivate endogenousperoxidase, washed twice in PBS, and immersed in a 10% solu-tion of goat normal serum in PBS for 30 min at room tempera-ture. They were incubated for 48 h, at 4 °C, in a rabbit anti-c-Fos polyclonal antibody (Santa Cruz Biotechnology, Inc. CA,USA), at a dilution of 1:5000 in PBS, and then washed twiceand incubated for 1 h, at room temperature, with biotinylatedgoat anti-rabbit antibody (Vector Laboratories, Burlingame, CA,USA), at a dilution of 1:400 in PBS. Sectionswere then incubated,for 1 h at room temperature, with avidin–biotin peroxidasecomplex (Vectastain Elite ABC kit, Vector Laboratories) at a dilu-tion of 1:800 in PBS. Then, sectionswere incubated for 10min in0.05% diaminobenzidine (Sigma), to which 0.01% H2O2 wasadded. Sections were rinsed with PBS for at least 15min be-tween each step. To increase the tissue penetration 0.5% TritonX-100 was added to the PBS used in all immunoreactions andwashes. Sections were mounted on gelatin-coated slides, air-dried and dehydrated in a series of ethanol solutions (50%,70%, 90% and 100%) and coverslipped using Histomount(National Diagnostics, Atlanta, GA, USA).

The total number of nuclei expressing c-Fos was estimatedusing the optical fractionator method, as described in detailelsewhere (Silva et al., 2002, 2009).

4.4. In situ hybridization histochemistry

Serial 14-μm-thick coronal sections of the hypothalamicregion containing the PVN were cut on a cryostat at −20 °Cand collected in order to form three independent series, onefor detection of CRH mRNA, the other for detection of VPmRNA and the remaining for Nissl staining. The sectionswere thaw-mounted on poly-L-lysine coated glass slides andstored at −80 °C. Prior to hybridization, sections were fixed,for 5 min, in 4% paraformaldehyde at 4 °C, washed in 1×PBS,dehydrated in serial ethanol solutions (70, 80, 90, 96 and100%, 1 min each), and allowed to dry. Synthetic 48-based oli-godeoxynucleotide probes directed against rat CRH, basesencoding amino acids 496–543, and rat VP, bases encodingamino acids 477–524, were used. The probes were labeledwith [35S]dATP (1200 Ci/mmol; DuPont NEN, Bad Homburg,Germany) using terminal transferase (Roche Molecular Bio-chemicals, Mannheim, Germany). For detection of CRH and VPmRNAs, sections were hybridized with 1×106 c.p.m. of thelabeled probes in a buffer composed of 50% deionized formam-ide, 4×SSC, 1×Denhardt's solution, 10% dextran sulfate, 0.1%diethylpyrocarbonate-treated water and 100 mM dithiothreitol(DTT) (100 μl probe/slide). Sections were protected with cover-slips and incubated, for 20 h, in a humidified chamber with50% formamide, 4×SSC, at 42 °C. To test thehybridization signalspecificity, a few sectionswere incubatedwith a 100-fold excessof unlabelled probe with the corresponding 35S-labeled probe.After removal of the coverslips, the sections were washedsequentially, twice in 2×SSC (15 min each) at room temperatureand 1×SSC (30 min each) at 50 °C, and once in 1×SSC, 0.5×SSCand 0.1×SSC (3 min each) at room temperature. After beingdehydrated and air-dried, sections were apposed to BiomaxMR film (Kodak, Rochester, NY) for 28 days (for CRH mRNA) or7 days (for VP mRNA). The autoradiographic films were devel-oped in Kodak D19 developer at 19 °C for 5 min, and fixed inKodak Fixer at 24 °C for 10min.

4.5. Analysis of CRH and VP mRNA signals onautoradiograms

In situ hybridization autoradiograms were analyzed underdark-field illumination using a computer-assisted image ana-lyzer (Leica QWin) fitted with a Leica DMR microscope and aLeica DC 300F color video camera. CRH and VP mRNA signalswere measured bilaterally in eight sections of the PVNmpper animal. In each section, the boundaries of the PVNmpwere defined based on cytoarchitectonic features visualizedin the adjacent Nissl-stained sections. Measurements weredone, at final magnification of ×100, by using a frame of18057 μm2 that was placed in five randomly selected positionswithin the PVNmp. Data were expressed in gray scale valuesof 1 to 256. All gray level measurements were corrected forbackground.

4.6. Statistical analyses

Ethanol intake, blood alcohol levels and serum corticosteroneconcentrations are expressed as mean±SEM, and the remain-ing data as the mean±SD of the values. The influence of EBinjection on uterine weight and of sex on ethanol intake wasanalyzed using the Student's t-test for independent variables.Remaining data were analyzed using a three-way analysis ofvariance (ANOVA) to determine the effects of group (males,and EB- and oil-injected ovariectomized rats), treatment (con-trol, chronic alcohol consumption and withdrawal) and LPS(LPS injection). Whenever significant results were foundfrom the overall ANOVA, pair-wise comparisons were madewith the post hoc Tukey's HSD test. The level of significancewas set at 0.05.

Acknowledgments

This work is supported by National Funds through FCT— Fun-dação para a Ciência e a Tecnologia within the scope of theStrategic Project Centro de Morfologia Experimental (CME/FM/UP) — 2011–2012 and Project PEst-OE/SAU/UI0121/2011.We are grateful to Dr. Conceição Gonçalves for help with bio-chemical measurements. We also thank Professors M. M.Paula-Barbosa for helpful comments on the manuscript andArmando Pinto for statistical advise. The authors have noconflict of interests to declare.

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IIVV..

Prolonged alcohol intake leads to irreversible loss of vasopressin and

oxytocin neurons in the paraventricular nucleus of the hypothalamus

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Brain Research 925 (2002) 76–88www.elsevier.com/ locate /bres

Research report

Prolonged alcohol intake leads to irreversible loss of vasopressin andoxytocin neurons in the paraventricular nucleus of the hypothalamus

*Susana M. Silva, M. Dulce Madeira, Carlos Ruela, Manuel M. Paula-BarbosaˆDepartment of Anatomy, Porto Medical School, Alameda Hernani Monteiro, 4200-319 Porto, Portugal

Accepted 24 October 2001

Abstract

Previous data revealed that numerous neurons in the supraoptic nucleus degenerate after prolonged ethanol exposure, and that thesurviving neurons increase their activity in order to prevent dramatic changes in water metabolism. Conversely, excess alcohol does notinduce cell death in the suprachiasmatic nucleus, but leads to depression of neuropeptide synthesis that is further aggravated bywithdrawal. The aim of the present study is to characterize the effects of prolonged ethanol exposure on the magnocellular neurons of theparaventricular nucleus (PVN) in order to establish whether or not magnocellular neurons display a common pattern of reaction to excessalcohol, irrespective of the hypothalamic cell group they belong. Using conventional histological techniques, immunohistochemistry andin situ hybridization, the structural organization and the synthesis and expression of vasopressin (VP) and oxytocin (OXT) in themagnocellular component of the PVN were studied under normal conditions and following chronic ethanol treatment (6 or 10 months) andwithdrawal (4 months after 6 months of alcohol intake). After ethanol treatment, there was a marked decrease in the number of VP- andOXT-immunoreactive magnocellular neurons that was attributable to cell death. The surviving neurons were hypertrophied and the VPand OXT mRNA levels in the PVN unchanged. Withdrawal did not alter the number of VP- and OXT-producing neurons or the geneexpression of these peptides. These results substantiate the view that after prolonged ethanol exposure numerous neurons of thehypothalamic magnocellular system degenerate, but the mRNA levels of VP and OXT are not decreased due to compensatory changesundergone by the surviving neurons. 2002 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Neuronal death

Keywords: Paraventricular nucleus; Alcohol; Withdrawal; Vasopressin; Oxytocin; Degeneration

1. Introduction (3 days to 3 weeks) globally down-regulates their activity.However, continuous alcohol treatment for periods longer

The administration of alcohol to rodents modifies neuro- than 1 month leads to changes in neuronal activity thatnal activity in several regions of the brain. In the hypo- display a region-specific pattern. In effect, there is evi-thalamus, such effects have been mostly examined through dence that parvocellular neurons in the suprachiasmaticthe evaluation of alterations in the synthesis and expression nucleus become down-regulated [28], whereas magnocel-of neuropeptides involved either in the control of neuroen- lular neurons in the supraoptic nucleus turn out to bedocrine mechanisms or in neurotransmission /neuromodu- activated [27,39].lation, and by the measurement of hormone concentrations The supraoptic nucleus shares common features with thein the plasma (reviewed in Ref. [25]). From these studies it magnocellular component of the paraventricular nucleus ofbecame apparent that changes in neuronal activity correlate the hypothalamus (PVN). Clear similarities are manifest inwith the duration of alcohol exposure: while acute expo- that both are composed of magnocellular neurons thatsure activates hypothalamic neurons, short-term exposure synthesize vasopressin (VP) and oxytocin (OXT) and

project directly to the neurohypophysis to release neuro-hormones into the general circulation [4]. However, they*Corresponding author. Tel.: 1351-22-509-6808; fax: 1351-22-550-differ in several dimensions, most of which are intimately5640.

E-mail address: [email protected] (M.M. Paula-Barbosa). related to the afferent inputs to their constituent neurons

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )03261-9

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S.M. Silva et al. / Brain Research 925 (2002) 76 –88 77

and the efferent connections they establish. For example, it until rats were 2 months old. At this age, rats were placedhas been shown that the number of GABAergic boutons into individual cages and assigned to the following ex-terminating on magnocellular neurons [19] and the gluta- perimental groups.mate decarboxylase activity [49] are both greater in the (1) Ethanol-treated groups: rats were given an aqueousPVN than in the supraoptic nucleus, and that the vas- ethanol solution as their only available liquid source for 6opressinergic and the oxytocinergic neurons projecting to or 10 months, i.e., until the age of 8 or 12 months. Thethe brainstem and spinal cord are confined to the PVN ethanol concentration was progressively increased: starting[12,13,40,46]. In addition, the GABA receptor subunits with a 5% (v/v) solution, and rising by 1% per day to aA

synthesized and expressed by magnocellular neurons differ final 20% (v/v) 2 weeks later. Food was freely availablebetween both nuclei [9], and glucocorticoid receptors are throughout. The amounts of food and ethanol solutiononly found in neurons of the supraoptic nucleus [20]. consumed were measured every other day. In keeping withActually, studies using chronic electroconvulsive shocks previous observations [27,28,43], this experimental model[14], kainic acid-induced seizures [44], immobilization of chronic ethanol treatment resulted in an average dailystress [18], adrenalectomy [7], hypovolemic stimuli [38] ethanol intake of 9.5 g/kg body weight and in bloodand food deprivation [30] have shown that magnocellular alcohol concentrations of 120 mg/dl in the morning and 90neurons of the supraoptic nucleus and of the PVN are not mg/dl in the evening.homogeneous populations. Similarly, data from other (2) Withdrawal group: rats were ethanol treated over 6investigations suggest that neurons from both nuclei might months and then switched to tap water for a further 4also differ with respect to their sensitivity to alcohol. For months. The shift from ethanol treatment to water intakeexample, the VP mRNA levels are increased in the PVN was performed gradually over a 2-week period by progres-and unaltered in the supraoptic nucleus 3 h after acute sive reduction of the ethanol concentration in the drinkingadministration of ethanol [31], and are decreased in the solution by 1% per day. Food pellets were freely availablePVN but returned to normal levels in the supraoptic throughout the experiment.nucleus after 24 h of withdrawal from short-term alcohol (3) Control groups: rats had free access to standardexposure [16]. In addition, osmotic stimulus, which is an laboratory diet and tap water for the duration of the study,ineffaceable side effect of alcohol consumption, elevates i.e., until the age of 8 or 12 months. Because we haveVP mRNA levels more in the supraoptic nucleus than in previously shown that the reduced intake of pellet food bythe PVN [10], and triggers the expression of BDNF mRNA ethanol-treated rats does not interfere with the structure ofin the latter but not in the former nucleus [5]. hypothalamic nuclei [27,28], pair-fed controls, i.e., rats

The chronic ethanol ingestion regimen used in our whose caloric intake was controlled by reference to thestudies has been shown to induce neuronal degeneration in ethanol-treated group, were not included in this study. Inthe supraoptic nucleus, and hypertrophy of the surviving all groups, liquids consumed were supplemented with 300neurons [27] and of the organelles involved in protein mg/100 ml of vitamins (Vitamin Diet Fortification Mix-synthesis [39]. To examine whether the regional vul- ture, ICN Cleveland, OH) and 500 mg/100 ml of mineralsnerability of hypothalamic neurons to prolonged ethanol (Salt Mixture, ICN Cleveland, OH).exposure relates to the morphological neuronal type (mag- All procedures were carried out in compliance with thenocellular versus parvocellular), the neuronal circuits in European Communities Council Directives of 24which they are incorporated or the physiological actions of November 1986 (86/609/EEC) and Portuguese Act no.the neuropeptides they produce (neurohormone versus 129/92. Body weights were recorded every 15th day, andneurotransmitter /neuromodulator), we have extended our on the day of sacrifice. The brains used in this inves-observations to the magnocellular component of the PVN. tigation were collected between 09:00 and 10:00 h. TheThe analyses were performed in rats submitted to chronic hypothalami were either embedded in glycolmethacrylateethanol treatment and withdrawal using conventional his- (n56 per group), for the characterization of the mor-tological methods, immunocytochemistry and in situ hy- phometric parameters of the magnocellular component ofbridization histochemistry. the PVN, or processed for immunocytochemical detection

of VP- and OXT-containing neurons (n55 per group) orfor the analysis of VP and OXT mRNA expression by in

2. Materials and methods situ hybridization (n55 per group).

2.1. Animals and treatments 2.2. Conventional histological procedures

Male Wistar rats (Gulbenkian Institute of Science, Rats were deeply anesthetized with chloral hydrate (i.p.,Oeiras, Portugal) were maintained throughout the experi- 1 ml /100 g body weight, 6% solution) and perfusedment under standard laboratory conditions: 12-h reverse transcardially with a fixative solution containing 1%light /dark cycle (lights on at 07:00 h) and temperature of paraformaldehyde and 1% glutaraldehyde in 0.12 M228C. Food pellets and water were available ad libitum phosphate buffer, pH 7.2. The brains were removed from

65

78 S.M. Silva et al. / Brain Research 925 (2002) 76 –88

the skulls, weighed and postfixed for 15 days in fresh zation, the non-specifically bound probe was removed byfixative. The blocks containing both the right and left one wash in 43 SSC for 15 min at room temperature,hypothalami were dehydrated, embedded in gly- followed by two 30-min rinses in 13 SSC and two 30-mincolmethacrylate (hydroxyethylmethacrylate, Technovit rinses in 0.13SSC at 408C and, finally, by one rinse in the7100, Kulzer, Wehrheim, Germany), and sectioned in the washing solution for 5 min. To block non-specific staining,coronal plane at 40 mm, as described in detail elsewhere sections were afterwards immersed in the blocking solution[28]. The sections were collected, mounted serially, and for 1 h at room temperature. The probes were detected bystained with a Giemsa solution modified for use in incubation with sheep anti-digoxigenin–alkaline phospha-glycolmethacrylate-embedded material [50]. tase antibody (Digoxigenin Detection Kit; Roche Molecu-

lar Biochemicals, Mannheim, Germany) diluted 1:500 in2.3. Immunohistochemistry the blocking solution for 1 h at room temperature. Sections

were then rinsed for 1 h in the washing solution and,Animals were anesthetized as described above and killed subsequently, immersed for 5 min in the detection solution.

by transcardiac perfusion of a fixative solution containing After overnight incubation in the dark at room tempera-4% paraformaldehyde in phosphate buffer, at pH 7.6. After ture, alkaline phosphatase activity was demonstrated usingremoval from the skulls, the brains were immersed in the 5-bromo-4-chloro-3-indolyl phosphatase and nitrobluesame fixative for 2 h, and maintained overnight in a tetrazolium (NBT/BCIP, Roche Molecular Biochemicals).solution of 10% sucrose in phosphate buffer. The blocks The sections were then briefly washed in PBS, dehydratedcontaining the right and left hypothalami were mounted on in graded acetone solutions (50, 70, 90 and 100%, 15 sa Vibratome and serially sectioned in the coronal plane at each), cleared twice in xylene and mounted with Permount.50 mm. Alternate sections were separately collected in The control reactions consisted of hybridization of thephosphate-buffered saline (PBS) in order to obtain two sections with the hybridization buffer alone and pretreat-independent sets of sections from each brain: one set was ment of the sections with RNAse (50 mg/ml, Amersham–used for VP immunostaining, the other for OXT immuno- Pharmacia Biotech, Uppsala, Sweden). None of thesestaining. Sections were processed for immunocytoch- procedures resulted in a specific signal.emistry, using the avidin–biotin technique withdiaminobenzidine (DAB) as the chromogen, as previously 2.5. Data analysisdescribed [26]. The antisera against VP and OXT (giftfrom Dr. R. Buijs, The Netherlands Institute for Brain The cytoarchitectonic criteria used for the parcellation ofResearch, Amsterdam) were used at the dilutions of 1:5000 the PVN and the terminology employed are those proposedand 1:15 000, respectively. originally by Swanson and Kuypers [45], and later further

characterized in several studies of the same group2.4. In situ hybridization histochemistry [40,42,46]. Accordingly, the magnocellular component of

the PVN consists of three subdivisions, i.e., anterior,Rats were anesthetized and perfused as for the immuno- medial and posterior, that can be discriminated on the basis

cytochemical studies. The brains were removed, stored in of cell size, location in the nucleus, peptide localizationthe same fixative for 1 h, maintained overnight in RNAse- and projecting sites. The analyses performed in this studyfree 10% sucrose solution at 48C, and sectioned in the were confined to the posterior division, named the poste-coronal plane at 40 mm using a Vibratome. Sections rior magnocellular division (PVNpm; Fig. 1), because itcontaining the PVN were alternately sampled in order to contains VP- and OXT-producing neurons, contrary to theobtain two independent sets of sections from each brain: anterior and medial divisions that are composed almostone was used for detection of VP mRNA and the other for exclusively by OXT-producing neurons [40].detection of OXT mRNA. Prior to hybridization, sections The volume of the PVNpm was estimated by using thewere treated with proteinase K (1 mg/ml; Sigma, St. principle of Cavalieri and point-counting techniques [11].Louis, MO) for 10 min to increase the intensity of the The total numbers of Giemsa-stained neurons, and of VP-labeling and, subsequently, rinsed twice with PBS for a and OXT-immunoreactive magnocellular neurons weretotal of 10 min at room temperature. The hybridization estimated using the optical fractionator [22,28,50]. Thebuffer consisted of 50% deionized formamide (Sigma), 43 mean somatic and nuclear volumes of the magnocellularsodium chloride / sodium citrate (SSC), 13 Denhardt’s neurons located in the PVNpm were evaluated by means ofsolution (Sigma), 1% N-lauroylsarcosine (Sigma) and 10% the optical rotator [22,48]. All the estimations weredextran sulfate (Sigma) in 0.1% diethylpyrocarbonate- performed, at magnification of 32000, using the C.A.S.T.treated H O (Sigma). Hybridizations were carried out — Grid system software (Olympus DK, Denmark) and a2

under steady-state conditions overnight at 408C with 39- Heidenhain MT-12 microcator (Heidenhain, Germany).tailed with digoxigenin (MWG-Biotech, Ebesberg, Ger- Hybridization signals from the magnocellular neurons ofmany) AVP [37] and OXT [17] probes in concentrations of the PVNpm were digitized at magnification of 32000100 ng/ml of the hybridization buffer. Following hybridi- using the same software, and processed using the Scion

66


Fig. 1. Photomicrographs of Giemsa-stained glycolmethacrylate-embedded sections through the paraventricular nucleus (PVN) of rats from the control (A),ethanol-treated (B) and withdrawal (C) groups. The PVN is delineated by a dotted line and the boundaries of the posterior magnocellular (pm), medialparvocellular (mp) and dorsal parvocellular (dp) divisions by dashed lines. Photomicrographs shown in (D–F) are from the same sections as those depictedin (A–C), respectively, with focus on the PVNpm to illustrate the enlargement of magnocellular neurons following ethanol treatment (E) and withdrawal(F). V, third ventricle. Scale bars5100 mm in (A–C), 30 mm in (D–F).

67


Image for Windows (version beta 4.02; Scion Corporation, (F(1,50)51.05, P50.31) or withdrawal (F(2,40)52.75,Frederick, MD). Densitometry of the digitized images was P50.08) on brain weights were detected.used to integrate and compare the optical densities ofhybridization signals, after correction for the background. 3.2. Morphological features of the PVNpmAll the estimations were performed on both sides of thebrain and the data were pooled for each animal. Prolonged ethanol treatment produced significant varia-

A two-way analysis of variance (ANOVA) was per- tions in the volume of the PVNpm (F(1,20)547.29, P,24formed on data from 8- and 12-month-old control and 5310 ) that were not influenced by the duration of

ethanol-treated rats to discern the effects of ethanol alcohol consumption (F(1,20)50.86, P50.36). Identicaltreatment and duration of alcohol consumption. The effects effects were also detected for the total number of PVNpm

24of withdrawal were assessed by one-way ANOVA tests neurons (treatment, F(1,20)578.77, P,5310 ; durationapplied to data obtained by in situ hybridization and to of treatment, F(1,20)50.31, P50.58), as well as for their

24stereological data estimated from 12-month-old control, mean somatic (treatment, F(1,20)5314.90, P,5310 ;8-month-old ethanol-treated and withdrawn rats. Whenever duration of treatment, F(1,20)50.51, P50.49) and nuclear

24appropriate, post hoc analyses were performed using the (treatment, F(1,20)5173.08, P,5310 ; duration ofTukey HSD test. Throughout the text, data are presented as treatment, F(1,20)51.35, P50.26) volumes. By compari-means with their coefficients of variation (CV5S.D. / son with controls, the volume of the PVNpm was increasedmean). Differences were considered to be significant if by 68 and 71% in rats submitted to ethanol treatment for 6P,0.05. and 10 months, respectively (Fig. 2A). Rats submitted to

ethanol treatment for 6 and for 10 months did not differwith respect to the total number of PVNpm neurons;however, both groups had 35% fewer neurons than the

3. Results respective controls (Fig. 2B). The opposite effects ofethanol treatment on neuron numbers and on the volume of

3.1. Body and brain weights the PVNpm were, partially at least, explained by variationsin neuronal size. Actually, the mean somatic volume of

The mean body and brain weights are presented in Table PVNpm neurons was, on average, 3.8 times larger in1. Body weights were influenced by ethanol treatment ethanol-treated rats than in controls (Figs. 1 and 2C).

24(F(1,50)546.03, P,5310 ) and duration of alcohol Identical variations were detected in the mean nuclear24consumption (F(1,50)519.70, P55310 ). Control rats volumes, which were 2.5 times larger in ethanol-treated

were significantly heavier than age-matched ethanol- rats than in controls (Fig. 2C).treated rats. Although 12-month-old rats were heavier than Withdrawal after 6 months of ethanol treatment did not8-month-old rats, the differences between both age groups produce significant variations in the volume of the PVNpmwere statistically significant for control rats, but not for and in the total number of its neurons relative to ratsethanol-treated rats (P50.07). Withdrawn rats were heavier submitted to ethanol treatment for an identical period.than rats submitted to ethanol treatment during the same Fitting with these data, we found that the volume of theperiod, but they weighed less than age-matched controls PVNpm was larger (52%) and the total number of neurons

24(F(2,40)554.99, P,5310 ). smaller (34%) in withdrawn than in age-matched controlNo significant effects of ethanol treatment (F(1,50)5 rats (F(2,15)58.08, P,0.01 and F(2,15)544.33, P,53

243.35, P50.07), duration of alcohol consumption 10 , respectively). In addition, the somatic and nuclearvolumes of PVNpm neurons were slightly, but not sig-nificantly, smaller (15 and 3%, respectively) in withdrawn

Table 1 rats than in rats treated with ethanol for an identical period;Mean body and brain weights of control, ethanol-treated an withdrawn

however, they were significantly greater (3 and 2.3 times,rats, at 8 and 12 months of agerespectively) in withdrawn rats than in age-matched con-

Control rats Ethanol-treated Withdrawn rats 24trols (somatic volume, F(2,15)540.58, P,5310 ; nu-rats 24clear volume, F(2,15)527.09, P,5310 ).

Body weight (g)8 months 722 (0.12)‡ 594 (0.10)* –

3.3. Immunohistochemical studies12 months 836 (0.09) 672 (0.12)** 747 (0.05)**†

Brain weight (g) Ethanol treatment produced significant reductions in the8 months 1.51 (0.04) 1.48 (0.03) – 24total number of VP- (F(1,16)539.48, P,5310 ) and12 months 1.53 (0.05) 1.50 (0.04) 1.48 (0.05)

OXT-immunoreactive neurons (F(1,16)535.09, P,53Data are expressed as mean (CV). Tukey’s post hoc tests: *P,0.01, 2410 ), which were not influenced by the length of ethanol**P,0.001, compared with age-matched controls; †P,0.001, compared

exposure (F(1,16)50.31, P50.59 and F(1,16)53.16, P5with 8-month-old ethanol-treated rats; ‡P,0.01, compared with 12-month-old rats. 0.09, respectively). After 6 and 10 months of ethanol

68


Fig. 2. Graphic representation of the morphometric data obtained from the PVNpm of control (CONT), ethanol-treated (CET) and withdrawn (W) rats,aged 8 (8M) and 12 months (12M). Columns represent means and vertical bars 1 S.D. (A) Volume estimates. The volume of the PVNpm is larger inethanol-treated rats than in age-matched controls. In withdrawn rats, the volume is larger than in controls, but it does not differ from that of 8-month-oldethanol-treated rats. (B) Neuron number estimates. The total number of neurons in the PVNpm is smaller in ethanol-treated and withdrawn rats than incontrols. (C) Mean somatic and nuclear volumes of PVNpm neurons. The volumes are larger in ethanol-treated and withdrawn rats than in controls. Thevolumes do not differ between withdrawn and ethanol-treated rats. Tukey’s post hoc tests: *P,0.02, **P,0.001, compared with control rats.

treatment, the number of VP-immunostained neurons was significantly influenced by ethanol treatment and with-24reduced by 30 and 32%, respectively (Fig. 3A), whereas drawal (F(2,12)541.10, P,5310 for VP neurons and

24the number of OXT-immunoreactive neurons was de- F(2,12)5139.41, P,5310 for OXT neurons). Thecreased by 26 and 22%, respectively (Fig. 3B). Withdrawal mean volumes of VP-immunoreactive neurons were 1987

3 3did not induce significant variations in the total number of mm (0.10) in controls, 4362 mm (0.10) in ethanol-treated3VP- and OXT-immunoreactive neurons, as shown by the rats and 3576 mm (0.15) in withdrawn animals, whereas

absence of differences between withdrawn rats and rats those of OXT-immunoreactive neurons were 1966 (0.04),3submitted to alcohol consumption for an identical period 3349 (0.06) and 3029 mm (0.04), respectively. Therefore,

(Fig. 3A,B). As a result, the number of VP- and OXT- the average change in the size of VP-immunoreactiveimmunoreactive neurons was significantly smaller in with- neurons was a 2.2-times increase (P,0.001) in ethanol-drawn rats than in age-matched controls (VP neurons, treated rats and a 1.8-times enlargement (P,0.001) inF(2,12)518.71, P,0.001; OXT neurons, F(2,12)513.09, withdrawn rats as compared to control values; in addition,P,0.001). in withdrawn rats cell size was 18% smaller (P50.03) than

The somatic size of the neurons identified by immuno- in ethanol-treated rats. Likewise, the somatic size of OXTcytochemistry in rats aged 12 months (Fig. 4) was neurons was 1.7 (P,0.001) and 1.5 (P,0.001) times

69


significantly altered (F(2,12)50.07, P50.936) by chronicethanol treatment and withdrawal (Fig. 6A).

No significant effects of ethanol treatment and with-drawal on the density of the hybridization signals indicat-ing the levels of VP mRNA (F(2,12)51.77, P50.212) andOXT mRNA (F(2,12)50.19, P50.833) in the entirePVNpm were detected (Fig. 6B).

4. Discussion

In the present study we have demonstrated that pro-longed alcohol consumption, regardless of the duration ofthe intake, leads to a loss of 35% of the neurons located inthe posterior magnocellular division of the PVN, and tometabolic activation of the surviving neurons. Thesealterations are similar to those previously observed in thesupraoptic nucleus [27], which indicates that magnocellu-lar neurons, irrespective of the hypothalamic cell groupthey incorporate, represent an homogeneous neuronalpopulation as regards the vulnerability they display whencontinuously exposed, for long periods, to ethanol. Thesimilarity of the alcohol effects on the supraoptic nucleusand on the magnocellular neurons of the PVN suggests thatthe neural afferents and efferents do not play a critical rolein determining ethanol neurotoxicity. Supporting this viewis the finding that VP- and OXT-producing neurons displayan identical sensitivity to alcohol despite differences intheir connectivity pattern. In effect, afferents to the PVNtend to respect the topography of the VP and OXT neuronsand seem to innervate these neurons differentially. This is,for example, the case of the noradrenergic afferents [41,47]

Fig. 3. Graphic representation of the total number of vasopressin (VP)- and of the projection from the subfornical organ [42],and oxytocin (OXT)-immunoreactive neurons estimated from the PVNpm

which are mainly directed at VP-producing cells, asof control (CONT), ethanol-treated (CET) and withdrawn (W) rats, agedopposed to serotoninergic [24], galaninergic [23] and8 (8M) and 12 months (12M). Columns represent means and vertical barssomatostatinergic [15] pathways and projections from the1 S.D. The total number of VP- and OXT-immunoreactive neurons is

smaller in ethanol-treated and withdrawn rats than in controls. Tukey’s dorsomedial and arcuate nuclei of the hypothalamus, andpost hoc tests: *P50.01, **P,0.01, **P,0.001, compared with control from the bed nucleus of the stria terminals [42], all ofrats.

which are preferentially distributed to regions containingOXT-producing neurons. However, our results show that

larger in ethanol-treated and in withdrawn rats, respective- degeneration induced by ethanol encompassed both VP andly, than in controls, and it was 10% (P,0.01) smaller in OXT neurons. Although ethanol-treated rats lost twicewithdrawn than in ethanol-treated rats. more VP- than OXT-immunostained neurons, the percentu-

al decrease in neuron numbers was similar for bothsubpopulations, which indicates that the toxic effects of

3.4. In situ hybridization studies ethanol are not specific of a particular neuronal phenotype.There is ample evidence that although VP and OXT have

Ethanol treatment for 10 months and withdrawal pro- different biochemical and physiological properties andduced significant variations (F(2,12)58.54, P50.005) in exert distinct homeostatic functions [4,6], they share somethe density of the hybridization signals indicating the functional aspects, namely the regulation of plasma os-levels of VP mRNA in individual magnocellular neurons molality [3]. Thus, the resemblance of the alcohol effectsof the PVNpm (Fig. 5). The density was increased by 23% upon magnocellular neurons producing VP and OXTin ethanol-treated rats and by 20% in withdrawn animals suggests that neuropeptide function is an important factorrelative to controls (Fig. 6A). The density of the hybridiza- in determining neuronal vulnerability to alcohol. Support-tion signals indicating the levels of OXT mRNA in ing this view are data from earlier investigations showingindividual magnocellular neurons of the PVNpm was not that chronic ethanol treatment does not lead to loss of the

70


Fig. 4. Vasopressin (VP) and oxytocin (OXT) immunoreactivity in the PVN of control (A,D), ethanol-treated (B,E) and withdrawn (C,F) rats. The borderbetween the posterior magnocellular division (pm) and the medial parvocellular division (mp) of the PVN is indicated by a dashed line. Neuronal size islarger in the ethanol-treated rat than in the control and withdrawn rats, and the differences are particularly notorious in VP-immunostained sections (A–C).The density of immunostained neurons is lower in the ethanol-treated and withdrawn rats than in the control rat. This reduction is particularly evident in thesections immunostained for OXT (D–F) due to the relatively low density of OXT-immunoreactive neurons. V, third ventricle. Scale bars5100 mm.

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Fig. 5. Photomicrographs of the PVN of control (A,D), ethanol-treated (B,E) and withdrawn (C,F) rats following in situ hybridization for vasopressin (VP;A–C) and oxytocin (OXT; D–F). The boxes illustrated in (A–C) delineate the areas shown at higher magnification in (G–I), respectively. Neurons fromthe control rat (G) are smaller in size than those of the ethanol-treated rat (H) and withdrawn rat (I). mp, medial parvocellular division of the PVN; pm,posterior magnocellular division of the PVN; V, third ventricle. Scale bars550 mm in (A–F), 30 mm in (G–I).

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quantifications were performed in independent sets ofhistological sections, given the existing evidence that VPand OXT co-localize in some magnocellular neurons [51].By comparison with previous quantitative studies of thePVNpm [40], we have found higher numbers of magnocel-lular neurons immunoreactive for VP and OXT. Althoughthis discrepancy is probably due to differences in cellcounting methods, it is conceivable that they might alsoresult from differences in antisera sensitivity because,according to our estimates, the ratio between the number ofVP and OXT neurons is also higher than that previouslyreported [40].

Despite the severe loss of magnocellular neurons, thevolume of the PVNpm was enlarged in ethanol-treated ratsdue, in part at least, to the hypertrophy of the survivingneurons. The ethanol-induced increase in the volume of theneuronal cell bodies was accompanied by an enlargementof their nuclei, and encompassed both VP- and OXT-producing neurons. A positive correlation between thesomatic size of magnocellular neurons and the volume oftheir nucleoli, nuclei, and cytoplasmic organelles involvedin protein synthesis has been observed in conditions ofincreased hormonal demand, such as during normal de-velopmental growth [26,35], dehydration, osmotic imbal-ance and lactation (for a review see Ref. [13]; see also Ref.[52]). Therefore, the earlier finding that in rats submittedto prolonged ethanol treatment the hypertrophy of supraop-tic neurons reflected the enlargement of the organellesinvolved in protein synthesis, allowed to suggest thatsupraoptic neurons were engaged in the synthesis ofincreased amounts of peptides [27,39]. Data from thepresent study lend support to such a possibility. In fact, ourresults show that despite the ethanol-induced decrease inthe number of VP- and OXT-producing neurons, the

Fig. 6. Graphic representation of the levels of mRNAs coding fordensity of VP and OXT hybridization signals in the entirevasopressin (VP) and oxytocin (OXT) in control (CONT), ethanol-treatedPVNpm does not differ between ethanol-treated and(CET) and withdrawn (W) rats. The values are expressed as arbitrarycontrol rats. This finding suggests that the VP and OXToptical density units. Columns represent means and vertical bars 1 S.D.

(A) Estimates obtained from individual neurons. (B) Estimates obtained mRNA levels per individual neuron are increased infrom the entire PVNpm. Tukey’s post hoc tests: *P,0.02, **P,0.01, ethanol-treated rats, an inference that, in the case of VP-compared with control rats.

synthesizing neurons, is corroborated by the finding thatthe density of the VP hybridization signals in individual

small suprachiasmatic neurons that manufacture VP for neurons was higher in ethanol-treated than in control rats.neurotransmission [28]. No similar difference was observed in the case of OXT-

In previous investigations, the number of neurons in the synthesizing neurons which, however, does not precludePVNpm of the adult male rat has been variously estimated the possibility that the amount of OXT mRNA peras 1300 [32], 1700 [1], 2000 [2] and 4100 [21]. The latter individual neuron might be increased by ethanol treatment,figure is consistent with the 3700 PVNpm neurons as- given that their somatic size was significantly larger insessed in the present study, despite differences in the rat ethanol-treated than in control rats. Together, these ob-strains analyzed and in the quantitative methods employed. servations indicate that chronic ethanol exposure exertsAssuming that magnocellular neurons produce either VP or effects that, although qualitatively similar for VP- andOXT, our estimate of neuron numbers in the PVNpm may OXT-producing neurons, are more exuberant for VP-seem relatively small in comparison with the sum of the producing neurons.number of VP- (|2900) and the number of OXT-immuno- In rats submitted to prolonged ethanol exposure thereactive magnocellular neurons (|1500). The apparent plasma levels of VP are within normal levels, notwith-overestimation of the total number of immunostained standing the increased plasma osmolality [8,27,43], whichneurons in the PVNpm might result from the fact that is by itself a stimulus for the production of VP [13].

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Therefore, the observation that in rats chronically exposed In conclusion, we have shown in this study that pro-to alcohol the osmotic stimulus does not lead to an longed ethanol treatment leads to a decrease in the numberaugmentation of the plasma levels of VP suggests that, in of VP- and OXT-producing neurons in the magnocellularthis condition, magnocellular neurons are unable to com- component of the PVN and that this alteration results frompensate for increased hormonal demands. Hypothetically, cell death. The finding of neuronal hypertrophy andthis might derive from the inability of magnocellular increased VP and OXT mRNA levels in the PVNpm afterneurons to synthesize sufficient amounts of VP or, alter- prolonged ethanol treatment and withdrawal suggests thatnatively, from their incapability to transport and release the surviving magnocellular neurons have the capability toneurohormones in an efficient way. Data obtained in this enhance their activity in order to maintain adequatestudy enable to deny the first possibility given that they circulating levels of neurohormones. The neuronal altera-indicate that the overall mRNA levels of VP and OXT in tions we have noticed in the PVN, including neuronalthe PVNpm of ethanol-treated rats are likely to be in- degeneration, are reminiscent of those previously observedcreased relative to those of controls. In effect, the absence in the supraoptic nucleus in identical experimental con-of differences in the density of VP and OXT hybridization ditions. It is thus tempting to suggest that the hypothalamicsignals between ethanol-treated and control rats associated magnocellular neurons display a common pattern of re-to the larger volume of the PVNpm in ethanol-treated rats action to prolonged ethanol exposure, which is specific ofsuggests that prolonged ethanol treatment leads to an this cell type given that no similar effects have beenincrease in the amount of VP and OXT mRNA in the detected in hypothalamic parvocellular neurons.PVNpm. Therefore, in addition to the cell loss alluded toabove, it seems probable that changes in axonal transportand/or release of peptides, which are known to occur Acknowledgementsfollowing chronic exposure to ethanol [29,33], mightcontribute to explain the absence of increased plasma We thank Mr. Alberto Alfaia for excellent technicallevels of VP in rats chronically exposed to ethanol. assistance. This work was funded by project no. 35767/99

˜ ˆAfter withdrawal from alcohol, no further changes and Unit 121/94 from Fundacao para a Ciencia e aoccurred either in the total number of PVNpm neurons or Tecnologia (FCT).in the total number of neurons immunostained for VP andOXT. Neither was any significant variation detected in thedensity of VP and OXT hybridization signals. Together, Referencesthese findings indicate that withdrawal from alcohol doesnot represent an additional aggression to magnocellular [1] R.C. Bandaranayake, Morphology of the accessory neurosecretory

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77

DDIISSCCUUSSSSÃÃOO GGEERRAALL

79

As experiências relatadas nos trabalhos que integram a presente dissertação tiveram como

objectivo analisar as repercussões sobre a organização estrutural e neuroquímica do PVNmp e

sobre a actividade do eixo HPA - estimada através das concentrações séricas de corticosterona

- da exposição a um factor indutor de stresse crónico, a ingestão prolongada de álcool, e a

resposta que, nestas condições, o eixo HPA consegue elaborar face à exposição aguda a um

outro factor indutor de stresse. Porque os restantes componentes do PVN parecem ser capazes

de actuar tanto a nível local como sobre as estruturas alvo do sistema hipofisiotrófico (revisto

em Herman et al., 2002; Engelmann et al., 2004), o componente magnocelular do PVN foi

também analisado no modelo experimental de ingestão prolongada de álcool. Com o

propósito de avaliar a potencial capacidade de recuperação do eixo HPA após remoção do

factor indutor de stresse crónico, analisaram-se os mesmos parâmetros em animais que, após

ingestão prolongada de álcool, entraram em abstinência. Realizaram-se os estudos

simultaneamente em machos e em fêmeas com a finalidade de avaliar se as hormonas sexuais

são, ou não, factores determinantes da resposta do eixo HPA quer à ingestão prolongada de

álcool e subsequente abstinência quer à exposição aguda a um segundo factor indutor de

stresse.

Verificou-se que a ingestão de uma solução alcoólica a 20% causava abrupta redução

da ingestão alimentar, de tal forma que, findo o 1º mês de tratamento, a quantidade de

alimento sólido ingerido pelos ratos submetidos a alcoolização era significativamente menor

(38% nos machos e 25% nas fêmeas) que nos respectivos controlos. Após este período, a

quantidade de alimentos ingeridos não se alterava nas fêmeas enquanto nos machos

aumentava lenta e progressivamente até ao fim do período experimental. O mesmo se

observou em relação ao volume de solução alcoólica ingerida já que, durante o 1º mês de

tratamento, os ratos do grupo de alcoolização reduziam a ingestão líquida para cerca de

metade da dos controlos. Esta diferença, embora menos marcada, era também evidente no fim

do período experimental. Durante a abstinência alcoólica verificou-se que a quantidade de

alimentos e o volume de água ingerida aumentava progressivamente para níveis idênticos aos

dos controlos, tanto nos machos como nas fêmeas (Trabalho II). Apesar dos machos

80

ingerirem maiores volumes de solução alcoólica, a ingestão média de álcool (calculada em

g/Kg de peso corporal) era maior (24%) nas fêmeas que nos machos, dado ser menor o seu

peso corporal (Trabalhos II e III). Curiosamente, as alcoolémias não diferiram entre machos e

fêmeas ao longo de todo o período experimental, o que está em sintonia com dados de alguns

(Lancaster e Spiegel, 1992; Ogilvie e Rivier, 1997; Savage et al., 2000; Livy et al., 2003),

mas não da totalidade (Middaugh et al., 1992; Rivier, 1993; Desroches et al., 1995) dos

estudos disponíveis na literatura.

Durante o 1º mês de tratamento, a ingestão de álcool não teve qualquer repercussão

sobre o peso corporal das fêmeas, mas reduziu significativamente o dos machos. Nestes, a

diferença de peso corporal entre o grupo de controlo e o de alcoolização aumentou

progressivamente ao longo de todo o período experimental devido, não à redução do peso

corporal dos ratos alcoolizados, mas ao aumento gradual do peso corporal dos controlos. Pelo

contrário, nas fêmeas, as diferenças ponderais apenas se tornaram óbvias durante o segundo

mês de alcoolização. Estas diferenças mantiveram-se depois estáveis não só porque nas

fêmeas do grupo de alcoolização não ocorreram variações adicionais do peso corporal mas

também porque as fêmeas do grupo de controlo da estirpe utilizada nos trabalhos que constam

desta dissertação estabilizam o seu peso corporal a partir dos 4 meses de idade. Assim,

decorridos 6 meses de experiência, os ratos alcoolizados eram mais leves que os controlos e,

em ambos os grupos, os machos mais pesados que as fêmeas (Trabalhos II e III). A

abstinência associou-se a rápido aumento do peso corporal para valores idênticos aos dos

respectivos controlos, tanto nos machos como nas fêmeas (Trabalhos I, II e III).

Trabalhos realizados nas últimas décadas no Departamento de Anatomia permitiram

demonstrar que a ingestão prolongada de uma solução alcoólica a 20% provoca alterações

estruturais e funcionais de natureza e gravidade distintas em diferentes regiões do SNC. No

cérebro do Rato, a par de áreas como o cerebelo (Tavares e Paula-Barbosa, 1983; Tavares et

al., 1983, 1985), o córtex cerebral (Cadete-Leite et al., 1990), a formação do hipocampo

(Borges et al., 1986; Cadete-Leite et al., 1988b; Andrade et al., 1992; Paula-Barbosa et al.,

1993) e o núcleo supra-óptico (Madeira et al., 1993; Ruela et al., 1994), onde as alterações

degenerativas são profundas e variadas, outras há, tais como o córtex pré-frontal (Cadete-

Leite et al., 1988a) e o núcleo supraquiasmático (Madeira et al., 1997), onde a ingestão

crónica de álcool tem repercussões sobretudo a nível funcional. Curiosamente, no PVN

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observam-se os dois tipos de alterações. Com efeito, dados constantes nos Trabalhos I e II da

presente dissertação mostram que a ingestão prolongada de álcool não induz morte neuronal

nem alterações do volume do PVNmp ou dos seus corpos celulares, mas reduz

significativamente o número total dos seus neurónios produtores de CRH e da vasopressina.

Pelo contrário, provoca morte neuronal nas suas divisões magnocelulares, com consequente

redução significativa do número dos seus neurónios que expressam vasopressina e oxitocina

(Trabalho IV).

Embora se saiba que a ingestão de álcool pode interferir com o transporte axonal e a

libertação de produtos de secreção neuronal (Paula-Barbosa e Tavares, 1985; McLane, 1987;

Pérez-Delgado et al., 1995), a observação de que, após 6 meses de ingestão de álcool, os

níveis de mRNA da CRH e da vasopressina se encontram reduzidos (Trabalhos I e III) mostra

que a depressão da actividade metabólica dos neurónios do PVNmp pela ingestão prolongada

de álcool é factor determinante da modificação do padrão neuroquímico do PVNmp. Pelo

contrário, nas divisões magnocelulares do PVN, essa redução parece ser consequente à morte

neuronal já que a expressão do mRNA da vasopressina e da oxitocina não se encontrava

alterada quando avaliada na totalidade do componente magnocelular e estava

significativamente aumentada quando a estimativa era feita por neurónio (Trabalho IV).

Curiosamente, apesar da redução da produção dos neuropeptídeos secretagogos da ACTH no

PVNmp, as concentrações séricas de corticosterona estavam inalteradas após 6 meses de

ingestão de álcool (Trabalhos I, II e III). Dado que na sequência de exposições agudas ou de

curta duração ao álcool (para revisão, ver Madeira e Paula-Barbosa, 1999; ver também

Rasmussen et al., 2000; Zhou et al., 2000) tanto os níveis plasmáticos de corticosterona como

a síntese de CRH e vasopressina se encontram aumentados, a disparidade observada nos

trabalhos incluídos nesta dissertação entre os níveis circulantes de corticosterona e a

expressão quer do peptídeo quer do mRNA da CRH e da vasopressina mostra que o eixo HPA

tem a capacidade de se adaptar à ingestão prolongada de álcool. Que os componentes mais

periféricos do eixo HPA têm essa capacidade é facto já anteriormente conhecido a partir de

estudos realizados no homem, onde se demonstrou que a ingestão prolongada de álcool se

acompanha de diminuição da sensibilidade da adeno-hipófise e do córtex suprarrenal à

estimulação pelos respectivos secretagogos (Wand e Dobs, 1991). Os dados constantes nos

Trabalhos I, II e III não só são consentâneos com esta observação como demonstram que a

progressiva normalização da actividade do eixo HPA, que tem lugar após exposições

prolongadas ao álcool, ocorre também ao nível do componente central que regula a actividade

do eixo HPA, o PVNmp.

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É amplamente reconhecida a existência de diferenças sexuais na actividade do eixo HPA, e os

dados apresentados nos Trabalhos II e III, referentes aos grupos de controlo, estão de acordo

com os disponíveis na literatura. Com efeito, demonstram que as concentrações séricas de

corticosterona são mais elevadas nas fêmeas que nos machos (Trabalho II) e que, em fêmeas

ovariectomizadas, variam em função dos níveis circulantes de estradiol, sendo mais elevadas

nas tratadas com benzoato de estradiol do que nas injectadas com veículo (Trabalho III).

Mostram, ainda, que a ovariectomia reduz, mas não anula, as diferenças sexuais detectadas

nos níveis de corticosterona, o que está também em sintonia com a perspectiva de alguns

autores (Young, 1996; Sibilia et al., 2000) sobre a importância dos efeitos organizacionais dos

esteróides sexuais na expressão das diferenças entre sexos na actividade do eixo HPA.

Os resultados constantes dos Trabalhos II e III revelam também que a ingestão crónica

de álcool não interfere com a expressão daquelas diferenças sexuais já que, tal como nos

grupos de controlo, nos submetidos a ingestão de álcool os níveis séricos de corticosterona

eram mais elevados nas fêmeas que nos machos. Esta observação, em tudo condizente com

dados reportados em estudos realizados em condições de exposição aguda e de curta duração

ao álcool (Rivier, 1993; Silveri e Spear, 2004), é particularmente interessante dado saber-se

que as diferenças sexuais nos níveis de corticosterona são, em grande medida, dependentes

não só da acção estimuladora dos estrogénios, mas também da influência inibidora da

testosterona sobre a actividade do eixo HPA (revisto em Handa et al., 2009; ver também Lund

et al., 2004; Lunga e Herbert, 2004; Seale et al., 2004a,b; Viau et al., 2005; Figueiredo et al.,

2007). Ora, após 6 meses de ingestão alcoólica, os níveis de testosterona encontram-se

reduzidos para cerca de metade dos observados nos controlos e as concentrações séricas de

estrogénios estão inalteradas (Trabalho II). Dado saber-se que a orquidectomia se acompanha

de aumento da síntese de CRH ao nível do PVN (Viau et al., 2003; Seale et al., 2004a),

aquela alteração hormonal poderia justificar a redução proporcionalmente menor do número

de neurónios produtores de CRH e de vasopressina nos machos do que nas fêmeas e,

consequentemente, a ausência de diferenças sexuais no número total destes neurónios em

ratos submetidos a ingestão prolongada de álcool (Trabalho II). A redução dos níveis de

testosterona poderia também fundamentar a anulação, nos grupos submetidos a ingestão

prolongada de álcool, das diferenças observadas entre os níveis de mRNA da CRH estimados

nos machos e nas fêmeas injectadas com estradiol do grupo de controlo. Porém, tal explicação

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não é compatível com o facto de, nos ratos alcoolizados e ao contrário do observado nos

controlos, os níveis de mRNA da vasopressina serem mais elevados nas fêmeas do que nos

machos, até porque a orquidectomia também induz aumento da síntese deste neuropeptídeo

pelos neurónios do PVN (Viau et al., 2003; Seale et al., 2004a). Acresce que, nos grupos de

ratos alcoolizados, não se encontraram diferenças nos níveis de mRNA tanto da CRH como

da vasopressina entre fêmeas injectadas com estradiol ou com veículo, ao contrário do

detectado nos grupos de controlo e do descrito na literatura sobre os efeitos estimuladores dos

estrogénios na expressão pelo PVN do mRNA da CRH e da vasopressina (Lund et al., 2004;

Seale et al., 2004a).

No seu conjunto, estas observações demonstram que a ingestão prolongada de álcool

interfere com os mecanismos de regulação da actividade dos neurónios do PVNmp pelos

esteróides sexuais, mas que, apesar desse facto e talvez por adaptação dos restantes

componentes do eixo HPA, não altera o padrão dimórfico sexual patente nos níveis

circulantes de corticosterona.

Ao contrário do observado nos ratos submetidos a ingestão prolongada de álcool, os estudos

realizados nos ratos abstinentes mostraram que os esteróides sexuais desempenham papel de

relevo na resposta do eixo HPA a esta condição experimental. De facto, nos machos, e ao

contrário do observado nas divisões magnocelulares (Trabalho IV), a abstinência alcoólica

permitiu a recuperação parcial da actividade dos neurónios do PVNmp (Trabalhos I e II), com

aumento do número total de neurónios vasopressinérgicos para valores que não diferiam dos

do grupo de controlo e do número total de neurónios produtores de CRH para valores que,

embora se mantivessem inferiores aos dos controlos, eram superiores, em cerca de 50%, aos

do grupo submetido a alcoolização crónica. Os dados obtidos nos Trabalhos I e III permitiram

ainda demonstrar que esta recuperação resultou do aumento da síntese destes neuropeptídeos

pelos neurónios do PVNmp. Pelo contrário, nas fêmeas, a abstinência alcoólica provocou

agravamento adicional das alterações induzidas pela ingestão prolongada de álcool, de tal

modo que o número total de neurónios produtores de CRH e de vasopressina era cerca de

50% menor nas fêmeas abstinentes que nos controlos (Trabalho II). Tal como se demonstra

no Trabalho III, esta variação é explicável pela depressão acrescida, induzida pela abstinência

alcoólica, da síntese de CRH e de vasopressina. Este efeito da abstinência era, em termos

quantitativos, idêntico nas fêmeas injectadas com estradiol e com veículo, facto que, por si só,

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é particularmente curioso tendo em consideração a reconhecida acção neuroprotectora dos

estrógenios (Brann et al., 2007) e a capacidade que estes esteróides possuem de promover

plasticidade neuronal (revisto em Dumitriu et al., 2010).

A diferença observada entre machos e fêmeas na actividade metabólica dos neurónios

do PVN produtores de CRH e de vasopressina após abstinência alcoólica teve repercussões

nos níveis circulantes de corticosterona. Efectivamente, nos machos, as concentrações séricas

desta hormona não diferiam entre ratos abstinentes e controlos (Trabalhos II e III) enquanto,

nas fêmeas, a abstinência alcoólica estava associada a redução significativa dos níveis

circulantes de corticosterona (Trabalhos II e III). Este efeito era proporcionalmente maior nas

fêmeas injectadas com veículo do que nas tratadas com estradiol (Trabalho III), o que sugere

que, na ausência dos efeitos depressores da alcoolização crónica, os estrogénios são capazes

de promover o aumento da actividade metabólica dos neurónios do PVNmp, ainda que para

níveis inferiores aos dos controlos. Idêntica recuperação se verificou no PVNmp dos machos

abstinentes, onde os níveis de mRNA da vasopressina eram, ainda que não significativamente,

superiores (30%) aos dos ratos do grupo de controlo. Esta observação torna legítimo supor

que, à semelhança do verificado nas fêmeas, na ausência da influência depressora do álcool,

os baixos níveis circulantes de testosterona (Trabalho II) permitem a desinibição da actividade

metabólica dos neurónios do PVNmp, particularmente dos produtores de vasopressina.

A verificação de que a abstinência alcoólica não revertia plenamente as alterações

neuroquímicas induzidas pela ingestão prolongada de álcool no PVNmp dos machos, e até

agravava essas alterações nas fêmeas, contribuiu não só para fragilizar a hipótese, defendida

por vários autores (Spencer e McEwen, 1990; Lee e Rivier, 1997; Rivier e Lee, 2001), de que

tais alterações reflectem apenas a adaptação do eixo HPA ao excesso de álcool, mas também

para alicerçar a convicção, já anteriormente anunciada por outros (Paula-Barbosa et al., 1993;

Cadete-Leite et al., 2003; para revisão, ver Fadda e Rossetti, 1998; Jaatinen e Rintala, 2008),

de que a ingestão prolongada de álcool provoca degenerescência neuronal e a abstinência

alcoólica representa, pelo menos nalgumas regiões do SNC como é o caso do PVNmp das

fêmeas, uma noxa adicional.

Com o objectivo de melhor fundamentar a hipótese de que a ingestão prolongada de álcool

provoca lesões neuronais que não são reversíveis, pelo menos na sua totalidade, pela

85

abstinência alcoólica, julgou-se pertinente analisar a resposta do eixo HPA de ratos

alcoolizados e abstinentes a um estímulo indutor de stresse agudo.

É conhecido que no processo de activação do eixo HPA por factores indutores de

stresse são recrutados circuitos neuronais específicos para cada tipo de estímulo (revisto em

Sawchenko et al., 2000; Pacák e Palkovits, 2001; Herman et al., 2005). Os estímulos

neurogénicos pressupõem integração da informação veiculada através de vias

somatossensoriais ou nociceptivas em estruturas tais como o córtex límbico, a amígdala, o

septo lateral e o núcleo supraquiasmático (para revisão, ver Herman et al., 2005), cuja

estrutura e função se sabe estarem comprometidas após exposição prolongada ao álcool e

subsequente abstinência (Paula-Barbosa et al., 1993; Lukoyanov et al., 1999; Roy e Pandey,

2002). Pelo contrário, a transdução dos estímulos de índole sistémica (fisiológica) é efectuada

por um número relativamente restrito de receptores periféricos e centrais, e não requer

integração cortical superior imediata (revisto em Sawchenko et al., 2000; ver também Dayas

et al., 2001). Pelos motivos expostos, nos estudos que integram a presente dissertação optou

-se por estimular a actividade do eixo HPA com um agente indutor de stresse fisiológico.

Seleccionou-se, para esse efeito, o lipopolissacarídeo (LPS), um fragmento derivado da

parede de bactérias do tipo Gram-negativo (Lee et al., 2000; Turrin et al., 2001; Taylor et al.,

2002) que, para além de activar o sistema imunológico, induz a síntese e a libertação de

citocinas pró-inflamatórias que sinalizam o SNC, tais como as interleucinas 1β (IL-1 β) e 6

(IL-6) (Bluthé et al., 2000; Turrin et al., 2001) e o factor de necrose tumoral α (TNF-α;

Töllner et al., 2000). A sua administração intraperitoneal leva à activação de terminais do

nervo vago (Goehler et al., 1999), com subsequente transmissão da informação sobretudo

para o núcleo do feixe solitário. Este núcleo, verdadeira região nodal de transdução e

integração de sinais inflamatórios (Ericsson et al., 1997; Marvel et al., 2004), estabelece

conexões com outros núcleos do tronco cerebral, designadamente os grupos celulares A1/C1 e

A2 da formação reticular do bolbo ventrolateral, e áreas telencefálicas tais como o bed

nucleus of the stria terminalis, o núcleo central da amígdala (Dayas et al., 2001; Crane et al.,

2003) e, muito particularmente, o PVN (Hollis et al., 2004). Assim, a activação do PVN após

administração intraperitoneal de substâncias pró-inflamatórias faz-se de modo directo, sem

que haja envolvimento importante de regiões corticais superiores, através de vias

catecolaminérgicas provenientes dos grupos celulares A1/C1 e A2 do bolbo ventrolateral e do

núcleo do feixe solitário (Kapcala et al., 1996; Dayas et al., 2001), e também indirecto via

receptores localizados nos órgãos circunventriculares (revisto em Benarroch, 2005).

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A quantificação dos neurónios positivos para c-Fos mostrou que o período de máxima

activação do PVNmp pelo LPS ocorria aproximadamente 6 horas após a sua administração

intraperitoneal, pelo que todos os resultados reportados no Trabalho III foram determinados

em ratos que foram sacrificados após esse intervalo de tempo. Verificou-se que, nos machos,

a injecção intraperitoneal de LPS aumentava os níveis de corticosterona em todos os grupos

estudados, mas de modo menos acentuado nos ratos alcoolizados, como o demonstra o facto

de, nestes animais, as concentrações séricas de corticosterona serem significativamente

menores que nos controlos e nos ratos abstinentes. Curiosamente, observou-se o mesmo tipo

de variação nas fêmeas ovariectomizadas não suplementadas com estradiol, mas não nas que

possuíam níveis circulantes de estradiol elevados. Nestas, a injecção intraperitoneal de LPS

não provocou qualquer variação nas concentrações séricas de corticosterona. Verificou-se

também que os efeitos da injecção de LPS sobre a actividade dos neurónios produtores de

CRH e vasopressina eram específicos de cada sexo. Enquanto nos machos os níveis de

mRNA da CRH eram idênticos em todos os grupos analisados, os da vasopressina, embora

aumentassem em todos os grupos em resposta à injecção de LPS, permaneciam

significativamente reduzidos nos ratos alcoolizados e nos abstinentes. Pelo contrário, nas

fêmeas alcoolizadas e abstinentes, os níveis de mRNA da CRH e da vasopressina mantinham

-se reduzidos relativamente aos das fêmeas do grupo de controlo, e estes efeitos não eram

influenciados pelos níveis circulantes de estradiol.

Quando os efeitos da administração de LPS sobre a transcrição génica ao nível do

PVNmp são analisados na globalidade, pode concluir-se que os machos diferem das fêmeas

na medida em que, naqueles, há activação preferencial dos neurónios produtores de CRH,

enquanto nestas são os neurónios vasopressinérgicos os que mais activamente respondem a

este estímulo. Embora tanto a CRH como a vasopressina sejam secretagogos da ACTH, sabe

-se desde há muito que, em condições basais ou em resposta a estímulos agudos, a CRH é a

principal moduladora da actividade do eixo HPA, actuando a vasopressina como um

potenciador da sua acção (Antoni, 1993). Porém, estudos realizados em diversos laboratórios

têm vindo a produzir dados que atribuem à vasopressina papel determinante na activação do

eixo HPA em animais de experiência que, estando submetidos a stresse crónico, são expostos

a um estímulo agudo indutor de stresse (Ma et al., 1999; Grinevich et al., 2001; Jiang et al.,

2004). Face a estes dados, é lícito conjecturar que a resposta preferencial dos neurónios

vasopressinérgicos nas fêmeas, mas não nos machos, após exposição aguda ao LPS possa

espelhar o estado de maior activação, mesmo em condições basais, do eixo HPA das fêmeas

relativamente ao dos machos, o que está de acordo com a existência, nas fêmeas, quer de

87

níveis de corticosterona mais elevados que nos machos, tanto em condições basais como após

estimulação, quer de glândulas suprarrenais cujo peso é superior ao dos machos (Trabalho

III).

Ao contrário do observado ao nível do PVNmp, e salvaguardando as diferenças

sexuais patentes nas concentrações séricas de corticosterona, a análise global dos dados

relatados no Trabalho III também mostra que a activação do eixo HPA em resposta à

exposição aguda ao LPS, avaliada em termos das variações observadas na produção de

corticosterona, é idêntica nos machos e nas fêmeas que possuem baixos níveis circulantes de

estradiol. Tanto nos machos como nas fêmeas não injectadas com estradiol, os níveis de

corticosterona aumentavam, em todos os grupos experimentais, em resposta ao LPS, embora

nos ratos alcoolizados esse aumento não fosse suficiente para atingir concentrações idênticas

às dos controlos. Tendo em consideração as diferenças, já discutidas, observadas na expressão

génica ao nível do PVNmp entre machos e fêmeas não suplementadas com estrogénios, a

similitude das variações das concentrações séricas de corticosterona sugere que o LPS tem a

capacidade de activar outros componentes do eixo HPA, designadamente a adeno-hipófise e o

córtex da glândula suprarrenal, de modo distinto em machos e em fêmeas. Embora dados de

outros autores tenham já sugerido que o LPS é capaz de activar os componentes mais

periféricos do eixo HPA (revisto em Beishuizen e Thijs, 2003; ver também Mazzocchi et al.,

1995), os dados reportados nesta dissertação são os primeiros a sugerir que esses efeitos são

de magnitude distinta nos dois sexos. Em óbvio contraste com os machos e as fêmeas com

baixos níveis de estrogénios, as fêmeas que possuíam níveis séricos elevados de estradiol não

responderam à estimulação provocada pela injecção de LPS com aumento da produção de

corticosterona, em todos os grupos experimentais estudados. Esta ausência de resposta não

pode ser atribuída a insuficiente sinalização do PVNmp uma vez que as variações observadas

na expressão de CRH e vasopressina eram idênticas às detectadas nas fêmeas injectadas com

veículo. É, pois, possível que, tal como tem sido sugerido em estudos da responsabilidade de

outros autores (revisto em Beishuizen e Thijs, 2003; ver também Puder et al., 2001), os

estrogénios atenuem a libertação de citocinas pró-inflamatórias, tais como a IL-6 e o TNF-α, e

que esta alteração resulte na menor activação dos componentes periféricos do eixo HPA em

resposta à administração de LPS.

No seu conjunto, os dados obtidos nas experiências relatadas no Trabalho III

demonstram que o eixo HPA, tanto dos machos como das fêmeas, não é capaz de aumentar a

sua actividade em resposta à administração de LPS de modo a produzir níveis circulantes de

corticosterona idênticos aos dos controlos. No entanto, após abstinência alcoólica, tanto os

88

machos como as fêmeas são capazes de produzir níveis de corticosterona semelhantes aos dos

controlos após exposição a um factor indutor de stresse agudo. No caso deste factor ser um

estímulo de natureza imune, como é o caso da injecção de LPS, os níveis circulantes de

estrogénios desempenham papel crucial no tipo de resposta, sendo a sua presença um factor

que pesa negativamente na activação do eixo HPA.

89

CCOONNCCLLUUSSÕÕEESS

91

A globalidade dos resultados obtidos nos trabalhos incluídos na presente dissertação e a sua

exegese permitiram concluir que

a) O consumo prolongado de álcool não altera as características citoarquitectónicas do

PVNmp, tanto em machos como em fêmeas. Contudo, deprime a síntese de CRH e

de vasopressina, com consequente redução do número de neurónios

imunorreactivos para estes neuropeptídeos, tanto nos machos como nas fêmeas.

b) Os efeitos da ingestão prolongada de álcool sobre o PVNmp são, nos machos,

parcialmente reversíveis pela abstinência alcoólica. Pelo contrário, nas fêmeas, a

abstinência agrava as alterações neuroquímicas provocadas pelo consumo

prolongado de álcool.

c) Nos ratos alcoolizados de ambos os sexos as concentrações séricas de

corticosterona não diferem das dos controlos. Nos ratos abstinentes, esses níveis

estão inalterados nos machos e deprimidos nas fêmeas.

d) A ingestão prolongada de álcool impede a elaboração de uma resposta adequada do

eixo HPA à estimulação com LPS tanto nos machos como nas fêmeas.

e) Após abstinência alcoólica, os machos e as fêmeas com baixos níveis circulantes de

estrogénios são capazes de aumentar a produção de corticosterona para níveis

idênticos aos dos respectivos controlos. Pelo contrário, nas fêmeas com níveis

circulantes de estrogénios elevados, o eixo HPA não é activado pela administração

de LPS.

92

f) As alterações provocadas no PVNmp pela ingestão prolongada de álcool, pela

abstinência alcoólica e pela estimulação com LPS não se repercutem no parâmetro

que mede a actividade global do eixo HPA, i.e., nos níveis circulantes de

corticosterona.

g) Os componentes periféricos do eixo HPA, i.e., a adeno-hipófise e/ou o córtex

suprarrenal, cuja actividade é também modulada pelos restantes componentes do

PVN, parecem ser sensíveis quer à exposição prolongada ao álcool quer à

estimulação pelo LPS.

93

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RREESSUUMMOO

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O trabalho de investigação que constitui o cerne da presente dissertação teve como objectivo

avaliar as repercussões sobre o eixo hipotálamo-hipófise-suprarrenal (HPA) da exposição a

um factor indutor de stresse crónico, a ingestão prolongada de álcool, e a resposta que, nestas

condições, o eixo HPA consegue elaborar face à exposição aguda a um outro factor indutor de

stresse. Para esse efeito, analisou-se a organização estrutural e neuroquímica da divisão

parvocelular medial do núcleo paraventricular do hipotálamo (PVNmp), através da aplicação

de técnicas de quantificação, algumas das quais de base estereológica, a material incluído em

glicolmetacrilato ou processado por imuno-histoquímica e hibridização in situ, e

determinaram-se os níveis plasmáticos de corticosterona em ratos Wistar que ingeriram uma

solução alcoólica a 20% durante 6 meses. Com o propósito de avaliar a potencial capacidade

de recuperação do eixo HPA após remoção do factor indutor de stresse crónico, analisaram-se

os mesmos parâmetros em animais que, após ingestão prolongada de álcool, se encontravam

em abstinência há 2 meses. Realizaram-se os estudos simultaneamente em machos e em

fêmeas com a finalidade de avaliar se as hormonas sexuais são, ou não, factores determinantes

da resposta do eixo HPA quer à ingestão prolongada de álcool e subsequente abstinência quer

à exposição aguda a um segundo factor indutor de stresse. Porque os níveis circulantes de

estrogénios flutuam ao longo do ciclo ovárico, as fêmeas dos diferentes grupos experimentais

foram, nalguns estudos, analisadas em fases aleatórias do ciclo éstrico e, noutros, sujeitas a

ovariectomia e, depois, tratadas com benzoato de estradiol ou veículo.

Verificou-se que o consumo crónico de álcool não altera as características

citoarquitectónicas do PVNmp, tanto em machos como em fêmeas. Contudo, provoca

depressão da síntese de corticoliberina (CRH) e vasopressina e, consequentemente, redução

do número total de neurónios imunorreactivos para estes neuropeptídeos em ratos de ambos

os sexos. Os resultados obtidos mostraram ainda que os efeitos da ingestão prolongada de

álcool sobre o PVNmp são, nos machos, parcialmente reversíveis pela abstinência alcoólica.

Pelo contrário, nas fêmeas, a abstinência agrava as alterações neuroquímicas induzidas por

esta noxa. Apesar dos efeitos que exerce sobre o PVNmp, a ingestão prolongada de álcool não

alterou as concentrações plasmáticas de corticosterona, tanto nos machos como nas fêmeas.

Após abstinência alcoólica, esses níveis também não sofreram alterações significativas nos

machos, ao contrário do observado nas fêmeas em que estavam significativamente reduzidos.

Mostrou-se, também, que a ingestão prolongada de álcool anula, tanto nos machos

como nas fêmeas, a capacidade que o eixo HPA possui de aumentar a sua actividade em

resposta à exposição aguda ao lipopolissacarídeo (LPS). Após abstinência alcoólica, o eixo

HPA dos machos e das fêmeas que possuem baixos níveis circulantes de estrogénios tem a

capacidade de aumentar a produção de corticosterona para níveis idênticos aos dos

respectivos controlos em resposta à administração de LPS, o que não se verifica nas fêmeas

que possuem níveis circulantes de estrogénios elevados.

Foi também possível concluir que as repercussões da ingestão prolongada de álcool,

abstinência alcoólica e estimulação com LPS sobre a actividade do eixo HPA são apenas

parcialmente explicáveis pelas alterações detectadas ao nível do componente que regula

centralmente a actividade deste eixo, o PVNmp. É, pois, possível que as alterações que

ocorrem ao nível de outras divisões do PVN, designadamente as magnocelulares, e dos

componentes periféricos do eixo HPA, ou seja, a adeno-hipófise e/ou o córtex suprarrenal,

possam também contribuir para determinar a influência que aquelas condições experimentais

exercem sobre as concentrações plasmáticas de corticosterona.

117

AABBSSTTRRAACCTT

119

The main focus of the investigations described in the present thesis was the analysis of the

consequences upon the hypothalamic-pituitary-adrenal (HPA) axis of the exposure to a

chronic stressor, long-term alcohol consumption, and of the response that, in this

experimental condition, the HPA axis builds when secondarily challenged by an acute

stressor. For this purpose, the structural and neurochemical organization of the medial

parvocellular division of the hypothalamic paraventricular nucleus (PVNmp) was examined in

Wistar rats that were fed over 6 months with a 20% ethanol solution, by applying quantitative

techniques, some of which stereological, to glycolmethacrylate-embedded sections of the

PVN and to sections of the PVN that were processed for immunocytochemistry or in situ

hybridization histochemistry. The plasma concentrations of corticosterone were also

determined. To evaluate whether, or not, the normal activity of the HPA axis can be reinstated

after removal of the chronic stressor, the same morphological and biochemical parameters

were estimated in rats that were withdrawn, for 2 months, from prolonged alcohol

consumption. In order to examine if sex steroid hormones modulate the response of the HPA

axis to chronic alcohol consumption and subsequent withdrawal, and also the activation of the

HPA axis of rats under these experimental conditions by an acute stressful stimulus, the

studies were carried out simultaneously in male and in female rats. Because the circulating

levels of estrogens fluctuate over the ovarian cycle, some studies were carried out in groups of

rats that were at random stages of the estrous cycle, whereas others were performed using

females that, after ovariectomy, were treated with either estradiol benzoate or vehicle.

It was found that long-term alcohol consumption does not alter the cytoarchitectonic

characteristics of the PVNmp in males as well as in females. However, it leads to depression

of the synthesis of corticotrophin-releasing hormone (CRH) and vasopressin and,

consequently, it reduces the total number of PVNmp neurons immunoreactive for these

neuropeptides, both in males and in females. Data obtained also show that, in males, the

effects of prolonged alcohol consumption upon the neurochemical features of the PVNmp are

partially reversed by alcohol withdrawal. Conversely, in females, withdrawal aggravates the

effects of prolonged alcohol consumption, leading to a further depression of neuropeptide

synthesis and expression. Despite the changes noticed in the PVNmp, prolonged alcohol

consumption was not associated with changes in the plasma concentrations of corticosterone,

in males as well as in females. After withdrawal, the concentrations remained unchanged in

males, but in females they were significantly reduced relative to those estimated in control

and ethanol-treated rats.

It was also shown that prolonged alcohol consumption prevents the LPS-induced

activation of the HPA axis in males and in females. However, after withdrawal, the HPA of

males and of vehicle-injected females was able to build an adequate response to LPS

exposure, as opposed to estradiol-injected females in which there was no increase in the

plasma concentrations of corticosterone after LPS administration.

It was also possible to conclude that the effects of prolonged alcohol consumption,

withdrawal and LPS administration on the activity of the HPA axis can be only partially

attributable to the changes induced by these conditions at the hypothalamic centre that

regulates the activity of this neuroendocrine axis, the PVNmp. It is thus possible that the

changes provoked by these conditions in other components of the PVN, namely its

magnocellular divisions, as well as on the peripheral components of the HPA axis, that is, the

adenohypophysis and/or the adrenal gland, might also play a role in determining the influence

of those experimental conditions in the plasma concentrations of corticosterone.

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