Os autores gostariam de agradecer a Anni Lee e Maureen Byrne por sua ajuda no estabelecimento do protocolo de preferência local condicionado estendida.

Todos os procedimentos são realizados em conformidade com o Weill Cornell Medical College Animal Care Institucional e Comitê de Regras de Uso. 1. Preparação e configuração para entrega estereotáxica de vetores virais <ol><li> Se um instrumento, cotonete estéril, ou a mão usando luva estéril é contaminado ao entrar em contato com uma superfície não-estéril, descarte ou re-esterilizar o instrumento utilizando um esterilizador talão quente, descartar o cotonete ou mudar para novas luvas estéreis. </li><li> Coloque a gaiola do rato com roupa de cama em um bloco de calor para aquecer para a recuperação pós-operatória. </li><li> Configure barbeador elétrico para barbear cabeça e etanol e iodo para esterilizar o couro cabeludo. </li><li> Limpe as barras de ouvido e mantenha a boca do stereotax com etanol 70%. </li><li> Usando a 5 mL seringa Hamilton, elaborar 5 mL de vetor viral (1 x 10 6 partículas virais / l) e criado em suporte de seringa em stereotax. </li><li> Almofada de aquecimento por microondas ou virar bloco elétrico em umº lugar em stereotax. Cubra com uma almofada absorvente limpa para reduzir o risco de queimaduras. Monitor de temperatura corporal do rato (entre 97-99,5 ° F) com freqüência durante todo o procedimento. </li><li> Injectar rato (pesando entre 22-35 g) com uma mistura de cetamina (100 mg / kg) e xilazina (20 mg / kg). Uma vez que um rato é anestesiado, ponta da cauda pitada de verificar os reflexos do rato e observe qualquer movimento ou resposta para garantir que o mouse está devidamente anestesiados. </li><li> Raspar a cabeça entre as orelhas e os olhos. </li><li> Limpe a cabeça raspada com a alternância de etanol e iodo aplicadores de algodão estéreis. </li><li> Transferir mouse para pad aquecida em stereotax. </li></ol> 2. Microinjeção estereotáxica de vetores virais <ol><li> Usar luvas estéreis durante toda a cirurgia. Se as luvas estão contaminadas em qualquer ponto ao tocar superfícies não-estéreis, mude as luvas. </li><li> Zero bar dente e escala de barras de ouvido. </li><li> Definir os dentes na barra do dente. </li><li> Aperte focinho. Se mexer ou bigode movimentomento é de notar, a qualquer momento, administrar uma dose de reforço IP (~ 0,05 cc) de uma mistura de cetamina (100 mg / kg) e xilazina (20 mg / kg). </li><li> Parafuso em bares de ouvido. </li><li> Lubrifique os olhos com PuraLube. </li><li> Corte couro cabeludo aberto apenas atrás das orelhas com um bisturi. </li><li> Coloque grampos bulldog nos cantos do couro cabeludo para descascar a pele longe do crânio. Bregma e lambda deve ser facilmente visível. </li><li> Certifique-se de sutura de bregma ao lambda está alinhada reta. Se a sutura é o posicionamento reajuste torto da cabeça. </li><li> Verifique se todas as configurações no stereotax são zerados. </li><li> Coloque a ponta da agulha no lugar em bregma. </li><li> Meça todas as coordenadas para tanto bregma e lambda. Isso inclui coordenadas dorsal / ventral, lateral / medial e anterior / posterior. </li><li> Ajustar a cabeça até que as coordenadas ventrais para bregma e lambda estão dentro de 0,02 milímetros um do outro. </li><li> Uma vez alinhada, a partir bregma ir medialmente para as coordenadas medial / lateral (M / L). Certificar-seos lados esquerdo e direito estão alinhados dentro de 0,02 milímetros um do outro. </li><li> Re-center ponta da agulha no bregma e de lá se mover na direção anterior / posterior (A / P) para atingir a cota anterior / posterior desejado. </li><li> Se necessário, agulha ângulo em uma direção e verifique se o stereotax está bloqueado. Vá para a esquerda M / L de coordenadas (+ / - 1,52) </li><li> Medir a posição ventral do topo do crânio para usar como uma referência, após o furo é perfurado. </li><li> Faça um furo com uma broca estéril na coordenada desejada. Verificar que a agulha pode entrar ininterrupta através do crânio. </li><li> Abaixe a agulha para a coordenada ventral desejado. </li><li> Uma vez que a cota é alcançada, dip 0,015 milímetros abaixo de ~ 10 segundos para criar uma pequena bolsa para o vector viral. </li><li> Injectar o volume desejado de vector virai, a uma taxa de ~ 0,1 mL / min. Espere 3 min para o vetor viral para difundir completamente. Levante a agulha ponta de 0,015 milímetros e esperar um adicional de 2 min. </li><li>Mergulhar o agulha (se fazendo injecções angulares) no sentido oposto. De novo, o registo da posição ventral como uma referência para o topo do crânio. </li><li> Repita os mesmos passos como no primeiro lado para injetar vetor viral no lado oposto (se fazendo injeções bilaterais). </li><li> Aplique cera de osso para ambos os buracos para selá-los usando o final de madeira de uma ponta do aplicador de algodão estéril. </li><li> Sutura no couro cabeludo. </li><li> Aplicar o bloqueio do nervo para a área ferida. </li><li> Retire do mouse de stereotax e coloque em gaiola aquecida em bloco de calor para se recuperar por 45 min. </li><li> Monitorar os sinais de dor, incluindo: diminuição da atividade global ou agitação, diminuição do consumo de alimentos e água ou aumento da vocalização. A buprenorfina (0,05-0,2 mg / kg), pode ser administrada duas vezes por dia durante uma semana, se necessário para a dor. As alternativas aceitáveis ​​incluem: meperidina (20-60 mg / kg), pentazocina (10 mg / kg), nalbufina (4-8 mg / kg). </li></ol> 3. Condicionado Lugar de Preferência: Acquisition, extinção e Reintegração <ol><li> Camundongos lugar para a câmara central de um lugar aparelho preferência de três câmaras (Med Associates Inc., St. Albans, VT, EUA) para um período de habituação 60 seg com portas guilhotina fechada. </li><li> Depois de habituação, portas guilhotina abertas e permitir que os ratos livre exploração de todas as três câmaras para 1,200 seg, com o tempo gasto em cada câmara a ser gravado usando o software MedPC IV (Med Associates, Inc.). </li><li> Atribuir ratos para câmaras de condicionamento com base em seu desempenho durante o pré-teste. Usando um desenho, a administração da droga par inclinado durante os três dias seguintes, com o compartimento que era menos preferido durante o pré-teste de linha de base. </li><li> Durante os próximos 3 dias condicionado, injetar camundongos com a cocaína (10 mg / kg, ip) durante a sessão da manhã e confiná-los em sua câmara atribuído para 1200 sec imediatamente após a injeção. Voltar ratos para suas gaiolas para casa depois da sessão. Depois de quatro horas, injetar camundongos com solução salina (0,01 ml / gde peso corporal) e confinam à câmara oposta para a sessão de 1,200 seg tarde. </li><li> Para testar a aquisição, ratinhos lugar na câmara central com portas de guilhotina fechado por um período de habituação de 60 seg. Portas de guilhotina abertas e permitem a livre exploração para 1200 sec, com o tempo gasto em todas as câmaras gravadas por software MedPC IV (Med Associates, Inc.). Calcular preferência subtraindo a quantidade de tempo gasto na câmara de solução salina-emparelhado a partir da quantidade de tempo gasto na câmara de cocaína-emparelhado. </li><li> Para extinguir a preferência de lugar, expor os ratos a duas sessões de 20 min de exploração livre diários no mesmo aparelho, separados por pelo menos 2 horas, começando 24 horas após o teste para a aquisição. O tempo gasto em todas as câmaras devem voltar a ser gravado e a magnitude do CPP inicial deve ser comparado com aquele de cada sessão de extinção. </li><li> Determinar se os ratos têm extinta a preferência induzida por drogas, avaliando se a preferência por um parcâmara cular em dois dias consecutivos de extinção é significativamente menor em comparação com a pontuação preferência de aquisição. </li><li> Restabelecer ratinhos com uma dose preparatória de cocaína (5 ou 10 mg / kg, ip) e re-exposição ao aparelho permitindo a exploração livre e registar o tempo gasto em cada compartimento. </li><li> Administrar solução eutanásia Euthasol (150 mg / kg). Transcardially perfundir ratos com paraformaldeído 4% (PFA) e efectuar um exame histológico para validar a colocação microinjeção correta. </li></ol>

<ol>
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Regionalmente e ablação gene específico temporalmente via microinjeção estereotáxica de vetores virais combinados com CPP, que inclui as fases de reintegração extinção e permite uma investigação das contribuições específicas de genes de três fases distintas de comportamento viciante-like. Enquanto knockout condicional que é alcançado utilizando o sistema Cre-LoxP tradicional prevê espaço-temporalmente ablação gene restrito, stereotaxically microinjecting Cre-recombinase em áreas cerebrais distinta...

A cocaína é um psicoestimulante altamente reforço. Após a exposição repetida, várias adaptações celulares e moleculares ocorrem em circuitos cerebrais de recompensa relevante que são acreditados para resultar em compulsivo de drogas comportamento de busca, fazendo com altas taxas de recaída que representam um sério problema clínico 1. A cocaína exerce esses efeitos comportamentais de longa duração por regulação da expressão gênica. Para estudar as adaptações que surgem do uso crônico da cocaína, os modelos pré-clínicos de roedores têm sido amplamente utilizados. Um tal modelo é a preferência paradigma local condicionado (CPP). Este modelo envolve o desenvolvimento de uma associação entre um ambiente aprendido anteriormente neutro e as propriedades gratificantes de cocaína. Depois de vários pares de cocaína com uma câmara particular, os animais estão autorizados a explorar livremente os ambientes cocaína pareado e não-pareado cocaína e se eles preferem o compartimento de droga pareado, eles dizem ter adquirido uma plac induzida pela cocaínae de preferência. Além disso, na sequência de um período de treino de extinção, este paradigma podem ser usadas para estudar a reintegração específica do contexto do comportamento de procura de cocaína. Em comparação com outros modelos comportamentais de comportamento aditivo semelhante, tais como a sensibilização psicomotora, que é usada para estudar a longo prazo induzida por cocaína comportamental e plasticidade molecular 2,3 e auto-administração (SA), que é pensado para mimetizar mais precisamente viciante -como o comportamento encontrado em seres humanos, o paradigma CPP é um procedimento simples para estudar cocaína aprendizagem contextual 4. Protocolos CPP pode ser convenientemente estendido para incluir as fases reintegração extinção e, à semelhança do SA, que permitem a investigação dos mecanismos subjacentes a ânsia da droga e recaída 5-7 e que são acreditados para recapitular aspectos do que ocorre em humanos droga comportamento de busca e drogas - e cue-induced recaída 8-10. Um mecanismo que está na base dainduzida por cocaína plasticidade comportamental, que é característica de cada uma das fases distintas do CPP, incluindo a aquisição, extinção, e reposição, é a activação de assinaturas únicas de expressão do gene em diferentes regiões do cérebro. Para testar quais genes directamente dentro das regiões do cérebro relevantes para mediar a recompensa cocaína induziu alterações comportamentais, é útil ser capaz de manipular selectivamente de forma regional específica. Uma maneira de conseguir isso é microinject vetores virais recombinantes adenoassociados (rAAV) que expressam Cre recombinase usando cirurgia estereotáxica, em áreas distintas do cérebro de ratos que têm genes alvo ladeado por sites LoxP (ratos floxed). Este método permite um controlo temporal e regionais muito preciso sobre quando e onde os genes são ablação, em ambos os neurónios que dividem e que não se dividem, sem induzir respostas imunes 11-13. Este nível de controle representa uma vantagem importante sobre a tecnologia tradicional Cre-LoxP de reprodução camundongos floxed with ratinhos que expressam a recombinase Cre sob o controle de um promotor de gene endógeno, em que a temporização e distribuição de deleção do gene pode ser mais fortemente regulado. Além disso, o vetor viral mediada por nocaute gene evita potenciais efeitos de compensação de desenvolvimento que podem ocorrer usando estratégias knockout tradicionais. Em adição à cocaína CPP, que é aqui descrito, a microinjecção de rAAV-Cre no cérebro de ratinhos floxed para avaliar o papel de genes específicos pode ser aplicado para ubiquamente paradigmas comportamentais que envolvem fases distintas, incluindo a auto-administração e sensibilização psicomotora. Por exemplo, o nosso laboratório tem utilizado a tecnologia de rAAV-Cre para estudar o papel do Ca V 1.2 canais de Ca 2 + do tipo-L de cocaína psicomotora sensibilização 14. Especificamente, rAAV-Cre foi microinjectado no núcleo accumbens (NAC) de ratinhos com o gene que codifica a Ca v 1.2 floxed, para demonstrar que o Ca v </sub&gt; 1.2 atuando nesta região medeia a fase de sensibilização psicomotora 14,15 expressão. No entanto, a estratégia de rAAV-Cre não pode ser utilizado se os ratos com um gene de interesse floxed não existem, como foi a nossa experiência quando se avalia o papel de Ca V 1.3 canais de Ca 2 + do tipo-L de sensibilização psicomotora. Assim, uma limitação da utilização de rAAV-Cre apresenta-se se os ratos condicionados não existem para um determinado gene de interesse. No entanto, rAAVs que expressam siRNA podem ser usadas para genes alvo knockdown, como fizemos para examinar o papel do CA V 1.3 canais 14,15. Microinjecting rAAV-Cre em regiões cerebrais distintas de camundongos floxed e, em seguida, testá-las no paradigma do CPP permite a investigação sobre os genes específicos que medeiam as várias fases do comportamento viciante-como e onde eles estão atuando. O uso deste paradigma tem ajudado na nossa compreensão de como a administração repetida de cocaína em essência seqüestra o brains circuito de recompensa causando alterações inadequadas em vias de transdução de sinal molecular e expressão de genes que levam ao estado viciado 1,16,17.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Conditioned place preference activity chambers</td>
			<td>Med Associates, Inc., St. Albans, VT, USA</td>
			<td>MED-CPP-MS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment system for mouse</td>
			<td>David Kopf Instruments, Tujunga, CA, USA</td>
			<td>model 900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringes</td>
			<td>Hamilton Company, Reno, Nevada, USA</td>
			<td>7634-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-Cre-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7004</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

stereotaxic microinjection of viral vectors expressing cre recombinase to study the role of target genes in cocaine conditioned place preference

Microinjecting virais (rAAV) vetores recombinantes adenoassociados expressando Cre recombinase em regiões do cérebro do rato distintas seletivamente genes knockout de interesse permite maior controle temporal e regionalmente específica de deleção do gene, em comparação com os métodos existentes. Enquanto a deleção condicional também pode ser conseguida por acoplamento ratinhos que expressa recombinase Cre sob o controle de promotores de genes específicos com ratinhos que transportam um gene floxed, microinjecção estereotáxico permite a determinação das áreas discretas do cérebro em pontos de interesse tempo experimentador-determinados. No contexto de preferência lugar cocaína condicionado, e outros paradigmas comportamentais da cocaína tais como a auto-administração ou sensibilização psicomotora, que pode envolver a retirada de restabelecimento fases, extinção e / ou, esta técnica é particularmente útil para explorar a contribuição única de genes-alvo para estes distintos fases de modelos comportamentais de plasticidade induzida pela cocaína. Especificamente,esta técnica permite a ablação seletiva de genes-alvo durante as fases distintas de um comportamento para testar a sua contribuição para o comportamento ao longo do tempo. Em última instância, esse entendimento permite terapêuticas mais específicas, que são mais capazes de lidar com os fatores de risco mais potentes que se apresentam durante cada fase do comportamento viciante.

CPP Depois de realizar CPP em ratos microinjetados, deve-se verificar que o grupo de controle de injecção (rAAV-GFP) camundongos normalmente adquirido preferência para a câmara de drogas pareado (Figura 1A, 1B). Ratos são considerados como tendo adquirido preferência por uma câmara especial quando preferência cocaína (tempo gasto na câmara de cocaína-emparelhado menos tempo gasto na câmara de solução salina-emparelhado), é significativamente superior em comparaç...

Watch this Scientific Journal Video about Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference at JoVE.com

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Este artigo descreve como microinject vetores virais em cérebro de camundongos e teste em um paradigma de preferência local condicionado, que inclui uma fase de aquisição, extinção e reintegração.

Microinjeção estereotáxica de vetores virais Expressando Cre Recombinase para estudar o papel de genes alvo em cocaína condicionado Lugar de Preferência

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

stereotaxic-microinjection-viral-vectors-expressing-cre-recombinase

Research

JoVE Journal

Behavior

18.5K visualizações.  Weill Cornell Graduate School of Biomedical Sciences. Este artigo descreve como microinject vetores virais em cérebro de camundongos e teste em um paradigma de preferência local condicionado, que inclui uma fase de aquisição, extinção e reintegração.

Vídeo: Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

Injeção estereotáxica de microRNA-expressando Lentiviruses ao mouse Região CA1 do hipocampo e avaliação do resultado Behavioral

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Comportamento

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

Um procedimento para estudar o efeito da restrição alimentar prolongada em heroína Buscando em ratos abstinentes

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Preferência de Gordura: Um Modelo Novel de comportamento alimentar em ratos

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Um procedimento para Observar Renovação induzida por Contexto de pavloviano condicionado Comportamento Álcool-seeking em Ratos

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Roedor Cérebro microinjeção para estudar substratos moleculares do comportamento motivado

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

Avaliação da sensibilização comportamental induzida por cocaína e preferência por lugar condicionado em Ratos

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

Coloque e Aprendizagem Response no Open-campo Torre Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

Reintegração de busca de drogas em ratos utilizando o paradigma de preferência lugar condicionado

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

Um protocolo de preferência condicionada lugar para medir a incubação do desejo em ratos

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

Assessing the Coherence of Parents&#039; Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Avaliando a coerência das narrativas curtas dos pais em relação ao seu filho usando o procedimento de amostra de fala de cinco minutos

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...