Influência do estresse de separação materna sobre os comportamentos relacionados ao consumo de etanol em camundongos: análise da neurotransmissão CRFérgica
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Data
2024-03-04
Autores
Bertagna, Natalia Bonetti [UNIFESP]
Orientadores
Cruz, Fábio Cardoso [UNIFESP]
Tipo
Tese de doutorado
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ISSN da Revista
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O transtorno por uso de etanol (TUE) é um transtorno multifatorial, sendo o estresse um importante fator de risco envolvido na iniciação, manutenção e recaída ao consumo de etanol. Experiências adversas na infância podem favorecer alterações moleculares duradouras que, aumentam a vulnerabilidade para o desenvolvimento do TUE. Em roedores, o protocolo de estresse de separação materna (SM) é reconhecido como um modelo preditivo para investigar os efeitos da exposição ao estresse na infância sobre o consumo de etanol na vida adulta. A amídala estendida, formada pela borda do núcleo accumbens (NAc shell), amídala central (CeA) e no núcleo intersticial da estria terminal (BNST), é conhecida pelo seu papel na mediação dos comportamentos relacionados ao estresse e consumo de etanol. A ativação do sistema do fator liberador de corticotrofina (CRF) na amídala estendida, particularmente na amídala (AMY) e no BNST, parecem estar envolvidas nessas respostas. O presente estudo teve como objetivo avaliar os efeitos a longo prazo da SM nos comportamentos de consumo de etanol em camundongos machos e fêmeas, bem como o envolvimento do sistema CRFérgico da AMY e BNST na interação entre estresse de SM e o consumo de etanol nesses roedores. Para isso, camundongos C57BL/6J machos e fêmeas foram separados diariamente de suas mães por três horas do dia pós-natal (DPN) 1 até o DPN 14. Os animais controle foram mantidos nas gaiolas moradia, sob os cuidados maternos até o DPN 21, quando ocorreu o desmame de todas as ninhadas. No primeiro experimento, a partir do DPN 45, os camundongos (Controle: n= 14, 7 machos e 7 fêmeas; SM: n= 14, 7 machos e 7 fêmeas) foram submetidos ao protocolo de consumo involuntário de etanol 20%, durante 3 semanas. Após, foi realizada a aquisição do comportamento de autoadministração operante de etanol em esquema de razão fixa (RF 1-3-5), em sessões de 2 horas, por 3 semanas. Em seguida, foi avaliada a influência da SM sobre a motivação pela busca do etanol 20% utilizando o protocolo de razão progressiva em 3 sessões de 2 horas. O consumo operante ilimitado de etanol 20% foi avaliado em uma sessão de binge com 4 horas de duração. Vinte e quatro horas após a sessão de binge, a busca pelo etanol 20% frente a um estímulo aversivo foi avaliada quando concentrações crescentes de quinino foram adicionadas a solução de etanol. O envolvimento dos subnúcleos da amídala estendida foi avaliado através da expressão da contagem do número de células positivas para a proteína Fos no Nac, AMY e BNST. No segundo experimento, foram utilizados os mesmos protocolos de SM e autoadministração de etanol (SM: n=31; 15 fêmeas e 16 machos; Controle: n=29; 15 fêmeas e 14 machos), porém, após o protocolo de razão progressiva, os animais foram alocados em dois grupos distintos: expostos (Controle: 8 fêmeas, 8 machos; SM: 7 fêmeas, 9 machos) e não expostos (Controle: 7 fêmeas e 6 machos; SM: 8 fêmeas, 7 machos) ao teste de Exposição ao Rato (TER). Após a exposição ao TER, os animais foram submetidos a uma sessão de binge e amostras da AMY e do BNST foram coletadas para análise da expressão dos receptores CRFR1 e CRFR2 e da proteína de ligação ao CRF (CRF-BP), por Western Blotting. Amostras de sangue foram coletadas antes do início do protocolo de autoadministração, depois do TER e do binge, para a análise das concentrações de corticosterona plasmática. No terceiro experimento, a partir do DPN 60, camundongos machos e fêmeas dos grupos controle e SM foram submetidos ao protocolo de livre escolha de etanol ou água, no qual a concentração da solução de etanol foi aumentada a cada 3 dias (5%, 10%, 15% e 20%). Em seguida os animais foram submetidos a uma cirurgia estereotáxica para a implantação de cânulas no BNST. Após esse procedimento foram realizados três dias de consumo voluntário de etanol 20% intercalados por três dias de abstinência, em que um grupo de animais controles e SM recebeu injeções intra-BNST de veículo e CP 376395 (antagonista de receptores CRFR1, doses 3,0 nmol/0,1 μl e 6,0 nmol/0,2 μl) e outro grupo de recebeu veículo e CRF6-33 (inibidor da proteína CRF-BP, doses 0,25 μg/0,1 μl e 0,5 μg/0,2 μl) que foram administrados seguindo o esquema Latin Square. No experimento 1, os resultados demonstram que a SM aumentou a ingestão de etanol em camundongos fêmeas, enquanto diminuiu o consumo em machos no consumo involuntário. Os animais submetidos SM apresentaram maior motivação para buscar etanol durante o protocolo de razão progressiva. Camundongos fêmeas do grupo controle, assim como machos submetidos a SM, foram mais resistentes a reduzir seu consumo em relação aos camundongos machos controle durante a adulteração com quinino. Para a imunohistoquímica, observamos aumento de neurônios imunorreativos para Fos na BLA e CeA em camundongos submetidos ao MS, mas não no NAc, nem no BNST. Já para o experimento 2, a SM induziu efeitos duradouros nas respostas comportamentais a um estressor heterotípico durante a vida adulta e na busca pelo etanol após a exposição ao TER, de maneira sexo-específica. Camundongos machos que permaneceram mais tempo na toca apresentaram maior número de reforços e respostas ao nose poke ativo em relação quando comparados às fêmeas. Camundongos dos grupos SM que não foram submetidos ao TER apresentaram redução da expressão de receptores CRFR1 no BNST e CRF-BP na AMY, enquanto aqueles que foram expostos ao TER apresentaram aumento na expressão de CRFR2 na AMY. No experimento 3, foi observado que a Sm reduziu o consumo de etanol em um protocolo de livre escolha de etanol com concentrações crescentes dessa substância. Esses efeitos não foram influenciados pelo antagonismo de receptores CRFR1 com CP376395. A inibição da proteína CRF-BP não produziu alterações de consumo e preferência de consumo entre os grupos. Esse estudo demonstra que a SM produziu alterações duradouras nos comportamentos relacionados ao consumo de etanol, de modo dependente de sexo e em diferentes protocolos de autoadministração de etanol. Observa-se também que esse estresse influencia comportamentos defensivos e o consumo de etanol em resposta a um estressor heterotípico, na vida adulta. Tais respostas foram associadas a ativação neuronal específica de subnúcleos da amídala estendida, de forma sexo-específica. Destaca-se, ainda, que a separação materna alterou a expressão de proteínas relacionadas ao sistema CRFérgico na amídala estendida. Entretanto, o antagonismo de receptores CRFR1 e a inibição da proteína CRF-BP parecem não influenciar os efeitos do estresse de SM sobre o consumo de etanol.
Alcohol use disorder (AUD) is a multifactorial condition, with stress playing a crucial role in the initiation, maintenance, and relapse of ethanol consumption. Adverse childhood experiences may lead to lasting molecular alterations, increasing vulnerability to the development of AUD. In rodents, the maternal separation (MS) stress protocol is considered a predictive model to investigate the effects of early-life stress on ethanol consumption in adulthood. The extended amygdala, composed of the nucleus accumbens shell (NAc shell), central amygdala (CeA), and the bed nucleus of the stria terminalis (BNST), is known for its role in mediating stress-related behaviors and ethanol consumption. Activation of the corticotropin-releasing factor (CRF) system in the extended amygdala, particularly in the amygdala (AMY) and BNST, appears to be involved in these responses. However, the role of this system in modulating the effects of MS on AUD-related behaviors is not fully understood. This study aimed to assess the long-term effects of MS on ethanol consumption behaviors in male and female mice, as well as the involvement of the CRF system in the AMY and BNST in the interaction between MS and ethanol consumption in adults. C57BL/6J mice were separated from their mothers for three hours daily from postnatal day (DPN) 1 to DPN 14. Control animals were maintained in home cages under maternal care until DPN 21 when weaning occurred. In experiment 1, starting from DPN 45, mice (Control: n= 14, 7 male e 7 female; MS: n= 14, 7 male e 7 female) underwent a 3-week protocol of involuntary 20% ethanol consumption. Subsequently, the acquisition of operant behavior under an increasing fixed ratio (FR 1-3-5) schedule was evaluated in 2-hour sessions for 3 weeks. The influence of MS on motivation to seek 20% ethanol was assessed using the progressive ratio in three 2-hour sessions. Unlimited ethanol consumption was evaluated in a 4-hour binge session. Following, ethanol seeking in response to an aversive stimulus was assessed by different quinine concentrations. The involvement of the extended amygdala subnuclei was evaluated through Fos protein expression. In the second experiment, using the same MS and ethanol self-administration protocols (MS: n=31; 15 female e 16 male; Control: n=29; 15 female e 14 male), animals were divided into two groups after the progressive ratio protocol: those exposed (Control: 8 females, 8 males; MS: 7 females, 9 males) and not exposed (Control: 7 females e 6 males; MS: 8 females, 7 males) to the Rat Exposure Test (RET). After RET exposure, mice underwent a binge session, and samples from the AMY and BNST were collected for Western Blotting analysis of CRFR1 and CRFR2 receptors and CRF-binding protein (CRF-BP). In experiment 3, starting from DPN 60, male and female mice from control and MS groups underwent a free-choice protocol for ethanol or water. The ethanol solution concentration increased every 3 days (5%, 10%, 15%, and 20%). After stereotaxic surgery for cannula implantation in the BNST, three days of voluntary 20% ethanol consumption were followed by three days of abstinence. Following a Latin Square design, intra-BNST injections of vehicle and CP 376395 (CRFR1 receptor antagonist, doses 3.0 nmol/0.1 μl and 6.0 nmol/0.2 μl) or vehicle and CRF6-33 (CRF-BP inhibitor, doses 0.25 μg/0.1 μl and 0.5 μg/0.2 μl) were administered. In experiment 1, results demonstrated that MS increased ethanol intake in female mice, while decreased it in males during involuntary consumption. MS-exposed animals showed greater motivation to seek ethanol during the progressive ratio protocol. Female control mice, as well as male MS mice, were more resistant to reducing their consumption compared to male control mice during quinine adulteration. Immunohistochemistry revealed an increase in Fos immunoreactive neurons in the BLA and CeA in MS mice but not in the NAc or BNST. In experiment 2, MS induced long-lasting effects on behavioral responses to a heterotypic stressor in adulthood and ethanol seeking after RET exposure, in a sex-specific manner. Male mice that spent more time in the home chamber presented more reinforcements and responses to active nose poke than females. MS-induced changes observed in the CRF system were not sex-dependent: Mice from MS groups not subjected to RET showed reduced expression of CRFR1 in the BNST and CRF-BP in the AMY, while those exposed to RET exhibited increased expression of CRFR2 in the AMY. In experiment 3, females and control mice increased their consumption in a free-choice protocol with distinct concentrations. These effects were not influenced by the antagonism of CRFR1 receptors with CP376395. CRF-BP protein inhibition did not produce changes in ethanol consumption and preference between the groups. This study demonstrates that MS induced lasting alterations in ethanol-related behaviors, dependent on sex and under different self-administration protocols. It is also noted that this stress influences defensive behaviors and ethanol consumption in response to a heterotypic stressor in adulthood. Such responses were associated with specific neuronal activation in subnuclei of the extended amygdala in a sex-specific manner. Additionally, maternal separation altered the expression of proteins related to the CRF system in the extended amygdala. However, CRFR1 receptors antagonism and the inhibition of the CRF-BP protein do not seem to influence the observed responses in a free-choice ethanol protocol.
Alcohol use disorder (AUD) is a multifactorial condition, with stress playing a crucial role in the initiation, maintenance, and relapse of ethanol consumption. Adverse childhood experiences may lead to lasting molecular alterations, increasing vulnerability to the development of AUD. In rodents, the maternal separation (MS) stress protocol is considered a predictive model to investigate the effects of early-life stress on ethanol consumption in adulthood. The extended amygdala, composed of the nucleus accumbens shell (NAc shell), central amygdala (CeA), and the bed nucleus of the stria terminalis (BNST), is known for its role in mediating stress-related behaviors and ethanol consumption. Activation of the corticotropin-releasing factor (CRF) system in the extended amygdala, particularly in the amygdala (AMY) and BNST, appears to be involved in these responses. However, the role of this system in modulating the effects of MS on AUD-related behaviors is not fully understood. This study aimed to assess the long-term effects of MS on ethanol consumption behaviors in male and female mice, as well as the involvement of the CRF system in the AMY and BNST in the interaction between MS and ethanol consumption in adults. C57BL/6J mice were separated from their mothers for three hours daily from postnatal day (DPN) 1 to DPN 14. Control animals were maintained in home cages under maternal care until DPN 21 when weaning occurred. In experiment 1, starting from DPN 45, mice (Control: n= 14, 7 male e 7 female; MS: n= 14, 7 male e 7 female) underwent a 3-week protocol of involuntary 20% ethanol consumption. Subsequently, the acquisition of operant behavior under an increasing fixed ratio (FR 1-3-5) schedule was evaluated in 2-hour sessions for 3 weeks. The influence of MS on motivation to seek 20% ethanol was assessed using the progressive ratio in three 2-hour sessions. Unlimited ethanol consumption was evaluated in a 4-hour binge session. Following, ethanol seeking in response to an aversive stimulus was assessed by different quinine concentrations. The involvement of the extended amygdala subnuclei was evaluated through Fos protein expression. In the second experiment, using the same MS and ethanol self-administration protocols (MS: n=31; 15 female e 16 male; Control: n=29; 15 female e 14 male), animals were divided into two groups after the progressive ratio protocol: those exposed (Control: 8 females, 8 males; MS: 7 females, 9 males) and not exposed (Control: 7 females e 6 males; MS: 8 females, 7 males) to the Rat Exposure Test (RET). After RET exposure, mice underwent a binge session, and samples from the AMY and BNST were collected for Western Blotting analysis of CRFR1 and CRFR2 receptors and CRF-binding protein (CRF-BP). In experiment 3, starting from DPN 60, male and female mice from control and MS groups underwent a free-choice protocol for ethanol or water. The ethanol solution concentration increased every 3 days (5%, 10%, 15%, and 20%). After stereotaxic surgery for cannula implantation in the BNST, three days of voluntary 20% ethanol consumption were followed by three days of abstinence. Following a Latin Square design, intra-BNST injections of vehicle and CP 376395 (CRFR1 receptor antagonist, doses 3.0 nmol/0.1 μl and 6.0 nmol/0.2 μl) or vehicle and CRF6-33 (CRF-BP inhibitor, doses 0.25 μg/0.1 μl and 0.5 μg/0.2 μl) were administered. In experiment 1, results demonstrated that MS increased ethanol intake in female mice, while decreased it in males during involuntary consumption. MS-exposed animals showed greater motivation to seek ethanol during the progressive ratio protocol. Female control mice, as well as male MS mice, were more resistant to reducing their consumption compared to male control mice during quinine adulteration. Immunohistochemistry revealed an increase in Fos immunoreactive neurons in the BLA and CeA in MS mice but not in the NAc or BNST. In experiment 2, MS induced long-lasting effects on behavioral responses to a heterotypic stressor in adulthood and ethanol seeking after RET exposure, in a sex-specific manner. Male mice that spent more time in the home chamber presented more reinforcements and responses to active nose poke than females. MS-induced changes observed in the CRF system were not sex-dependent: Mice from MS groups not subjected to RET showed reduced expression of CRFR1 in the BNST and CRF-BP in the AMY, while those exposed to RET exhibited increased expression of CRFR2 in the AMY. In experiment 3, females and control mice increased their consumption in a free-choice protocol with distinct concentrations. These effects were not influenced by the antagonism of CRFR1 receptors with CP376395. CRF-BP protein inhibition did not produce changes in ethanol consumption and preference between the groups. This study demonstrates that MS induced lasting alterations in ethanol-related behaviors, dependent on sex and under different self-administration protocols. It is also noted that this stress influences defensive behaviors and ethanol consumption in response to a heterotypic stressor in adulthood. Such responses were associated with specific neuronal activation in subnuclei of the extended amygdala in a sex-specific manner. Additionally, maternal separation altered the expression of proteins related to the CRF system in the extended amygdala. However, CRFR1 receptors antagonism and the inhibition of the CRF-BP protein do not seem to influence the observed responses in a free-choice ethanol protocol.
Descrição
Citação
BERTAGNA, Natalia Bonetti. Influência do estresse de separação materna sobre os comportamentos relacionados ao consumo de etanol em camundongos: análise da neurotransmissão CRFérgica. 2024. 185 f. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.