Os autores agradecem Gerrits Dr. Peter para os desenhos usados ​​no vídeo.

Nota: técnicas de anti-séptico deve ser empregado durante todo o procedimento. Todos os instrumentos e materiais (cotonetes, gazes, etc) que serão utilizados durante a cirurgia deve ser esterilizado em autoclave. A máscara cirúrgica, gorro cabelo e luvas estéreis devem ser usados. A área de trabalho e do aparelho estereotáxico deve ser cuidadosamente limpos e desinfetados com uma solução de etanol 70%. 1. Procedimentos pré-cirúrgicos <ol><li> Configurar o aparelho estereotáxico e todos os materiais necessários. Pré-aquecer a almofada de aquecimento. </li><li> Coloque a cânula em seu apoio e verificar se ele é reto. </li><li> Ligue o sistema de gás - mistura de ar ambiente e oxigênio (30-35% do fluxo total deve ser de oxigênio). </li><li> Pesar o rato e administrar o anestésico. Estamos usando uma mistura de cetamina (37,5 mg / kg) e dexmedetomidina (0,25 mg / kg) administrado por via subcutânea. Para protocolos de anestesia diferente, consulte Flecknell 4 e Hellebrekers et al. 7. </li><li> Após o rato perdeu a consciência, raspar a área que vai da cabeça ouvidos apenas entre os olhos com um barbeador elétrico. </li><li> Coloque o rato sobre a almofada de aquecimento, com o nariz na frente do tubo de ar. Use um oxímetro para garantir que o rato tem um nível de oxigenação adequada do sangue (não deve cair &lt;90%). Por favor, siga as instruções do fabricante para a utilização correcta do equipamento. </li><li> Aplicar creme para os olhos (Duratears Z, Alcon) em ambas as córneas para evitar a desidratação. </li><li> Verificar os reflexos do rato (cauda reflexo ou dedo do pé pinch-reflex, como demonstrado no Walantus et al. 8) para garantir que ele seja adequadamente anestesiados. Se o rato continua a mostrar reflexos fortes, a suplementação de anestesia pode ser necessária. </li><li> Se nenhum reflexo toe pinch é mostrado, coloque o rato no aparelho estereotáxico, ajustar as barras de ouvido para que ele mostra a leitura igual em ambos os lados, e coloque novamente o tubo de ar na frente do animalfixando-a com a barra de nariz. Verifique novamente se o rato mostra um nível de oxigenação do sangue de 90% ou mais. Se não, ajuste ou a tubulação, aproximando-a do nariz, ou aumentar o fluxo de oxigênio. Monitorar o nível de oxigenação do sangue e da freqüência cardíaca durante a cirurgia. </li><li> Monitorar continuamente a temperatura do rato com um termômetro retal (preferencialmente ligado a uma almofada de aquecimento) e registre os valores no início e no final da cirurgia. Ajuste a almofada de aquecimento ou usar um cobertor para manter uma temperatura corporal de 37,5 a 38,5 ° C. </li></ol> 2. Cirurgia <ol><li> Injetar o analgésico. Estamos usando uma administração peri-operatória única de carprofeno (4,0-5,0 mg / kg, por via subcutânea). Para diferentes protocolos analgésicos, ver Hellebrekers et al. 7. </li><li> Limpar a área raspada de tele pele do centro para o perímetro cabelos três vezes com um desinfectante (por exemplo, clorexidina 0,5%) e localmente injetar uma mistura de lidocaína (20 mg / ml) e adrenalina (5 mg / ml) para anestesia local e vasoconstrição (para evitar sangramento excessivo). </li><li> Faça uma incisão ântero-posterior de cerca de 2,5 cm na linha média do couro cabeludo, passando de entre os olhos até a parte de trás das orelhas. Use grampos 06/04 bulldog para beliscar fora da pele e manter a incisão aberta. Remover qualquer tecido conjuntivo com uma espátula e / ou cotonetes e limpe a área para expor a superfície do crânio. </li><li> Verifique se a cabeça é o nível: Primeiro, encontre Lambda e coloque a cânula guia exatamente sobre esse local, tocando o crânio. Registro a coordenada dorso-ventral. Em seguida, coloque a cânula guia exatamente sobre bregma, tocando o crânio, e registrar sua coordenada dorso-ventral. Estas duas coordenadas devem ser idênticos. Se a diferença for&gt; 0,3 mm, ajuste a barra de nariz para corrigir isso. </li><li> Make dois pequenos furos para a fixação dos parafusos de caveira usando uma broca de mão esterilizados (um milímetro de aproximadamente 5 para o local anterior cânula em um dos hemisférios e os outros 5 mm posterior ao local da cânula no hemisfério contralateral). Coloque dois parafusos estéril nestes buracos até que eles estão bem ancoradas, sem estar completamente inserida dentro do crânio. </li><li> Com a cânula guia colocado exatamente no bregma, gravar as coordenadas ântero-posterior e lateral. O local correto da guia colocação cânula para cada região do cérebro pode ser calculado somando ou subtraindo bregma, com a ajuda de um atlas estereotáxico 9-11. </li><li> Posição da cânula guia no seu local correto, ligeiramente tocando o crânio. Registro a coordenada dorso-ventral. Para a colocação de cânula bilateral, encontrar próxima do local da cânula no outro hemisfério, e novamente recorde a coordenada dorso-ventral. Ambas as coordenadas devem ser idênticos (ou diferir &lt;0,3 mm). </li><li>Marque os locais cânula no crânio com um lápis estéril e, com o perfurador de mão, fazer os furos rebarba, verificar o tamanho ea localização correta com a ajuda da cânula guia. Uma vez que os buracos são feitos, use uma agulha estéril para perfurar o meninges suavemente para permitir a inserção desobstruída da cânula. </li><li> Coloque a cânula no orifício primeiro e abaixá-lo com cuidado até que ele atinja a cota final ventral. Prepare o cimento dental e generosamente aplicá-la ao redor da cânula e um ou dois parafusos para fixar a cânula. Aguarde até que o cimento secar por completo. Depois, remova cuidadosamente o apoio cânula girando para cima dorso-ventral bar. </li><li> Coloque a cânula segundo para o apoio e vá até o local da cânula no outro hemisfério. Coloque a cânula no orifício e repita o passo anterior. Cobrir os parafusos e uma grande superfície das cânulas com o cimento, e antes que o cimento secar, retirar qualquer excesso da pele. </li><li> Injetar quente (~ 37 ° C) solução salina estéril (~ 10 ml / kg, sc) para garantir a reidratação. </li><li> Depois que o cimento esteja completamente seca, retire o apoio cânula e coloque um alfinete estéril em cada cânula para evitar obstrução. </li><li> Limpar a área da ferida com solução salina estéril e sutura da frente e as costas da ferida. </li><li> Remover o animal do aparelho estereotáxico, substituindo a tubulação de gás na frente de seu nariz. Continuar a acompanhar o nível de saturação de oxigênio e temperatura corporal. </li><li> Se um anestésico injetável com dexmedetomidina é usada, injetam sua atipamezol antagonista (0,25 mg / kg, sc) e aguarde até que o animal acorda (aproximadamente 5 minutos). </li><li> Coloque o rato em uma gaiola de recuperação. Para evitar a hipotermia, coloque a gaiola em uma incubadora a 28 ° C ou em uma almofada de aquecimento em um lugar onde você pode observar o animal por pelo menos uma hora, antes de retornar à sala de viveiro. </li></ol> 3. Cuidados pós-cirúrgicos <ol><li> Durante<html> os primeiros 4 dias após a cirurgia, monitor de recuperação do rato, mantendo os registros diários de peso e outras observações a respeito da condição do animal em laboratório ou diários de bordo &quot;diários bem-estar animal&quot;. </li><li> Animais que mostram sinais evidentes de infecção doença, do ferimento, perda de peso corporal ou outros sinais de redução de bem-estar devem ser submetidos a cuidados especiais: por exemplo, uma dose extra de analgésicos para minimizar a dor pós-operatória, uma mistura de alimentos em pó e água além de ração padrão para estimular o apetite do rato, e / ou uma injecção subcutânea de solução salina para apoiar a reidratação. </li><li> Se o rato não mostra qualquer melhora após essas intervenções, ou a perda de peso corporal é&gt; 15% (em comparação à cirurgia pré-peso), o sacrifício do animal com uma overdose de anestésico (humana ponto final). </li><li> Ratos normalmente precisam de recuperar, pelo menos, sete dias antes do início dos experimentos comportamentais. </li></ol> 4. Resultados representante &gt; jove_content &quot;Para determinar se a várias modificações ao nosso procedimento cirúrgico, particularmente com relação à suplementação de oxigênio e do monitoramento contínuo dos níveis sanguíneos de oxigenação, freqüência cardíaca e temperatura corporal, maior a sobrevida do animal e melhorou sua condição geral após a cirurgia, foram comparados os não-sobrevivência taxa de 20 grupos de animais (composto de 20 ratos cada) que foram submetidos à cirurgia depois que implementou essas modificações com a taxa de sobrevivência não de 24 coortes (20 ratos cada) operado com o protocolo padrão. Como é mostrado na Figura 1a , a taxa de sobrevivência não foi significativamente reduzida na amostra de coortes que foi operado com o protocolo modificado (P &lt;0,05; Mann-Whitney, duas caudas). Além disso, como é mostrado na Figura 1b, perda de peso pós-cirúrgica de ratos operados com o protocolo modificado também foi significativamente reduzida quando comparada com a de ratos operados com o procedimento normal (dia pós-operatório 1: P &lt;0,05; dia pós-operatório 2: P &lt;0,01; dia pós-operatório 3: P = 0,17; Student t-tests). <img alt="Figura 1" src="/files/ftp_upload/3528/3528fig1.jpg" /> Figura 1. Efeito de modificações cirúrgicas sobre a não-sobrevivência taxa e pós-cirúrgico de perda de peso. (A.) taxa de não-sobrevivência de ratos operados com o protocolo modificado em comparação com a de ratos operados com o protocolo padrão. A taxa de não-sobrevivência (média ± interquartil faixas) foi calculado como o percentual de ratos, por coorte de 20 ratos, que não sobreviveu a cirurgia. * P &lt;0,05, Mann-Whitney bicaudal (n = 20 coortes para o protocolo modificado e 24 coortes para o protocolo padrão). (B) A perda de peso (média ± EPM em percentagem do peso pré-cirurgia), durante o primeiro (Pod1), segundo (POD2) e terceira (POD3) dia pós-operatório. * P &lt;0,05, ** P &lt;0,01, teste t de Student (n = 60 por grupo).

<ol>
	<li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li><li>Richardson, C. A., Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+and+post-operative+analgesia+following+experimental+surgery+in+laboratory+rodents:+are+we+making+progress?.">Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.</a> Altern. Lab Anim. 33, 119-127 (2005).</li><li>Stokes, E. L., Flecknell, P. A., Richardson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reported+analgesic+and+anaesthetic+administration+to+rodents+undergoing+experimental+surgical+procedures.">Reported analgesic and anaesthetic administration to rodents undergoing experimental surgical procedures.</a> Lab Anim. 43, 149-154 (2009).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia - A Practical Introduction for Research Workers and Technicians. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIRECTIVE+2010/63/EU+OF+THE+EUROPEAN+PARLIAMENT+AND+OF+THE+COUNCIL+of+22+September+2010+on+the+protection+of+animals+used+for+scientific+purposes.">DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes.</a> Official Journal of the European Union. , (2010).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+Stereotaxic+Surgery+in+Rodents.">Survivable Stereotaxic Surgery in Rodents.</a> J. Vis. Exp. (20), e880-e880 (2008).</li><li>Hellebrekers, L. J., Booij, L. H. D. J., Flecknell, P. A., Van Zutphen, L. F. M., Baumans, V., Beynen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia,+analgesia+and+euthanasia.">Anaesthesia, analgesia and euthanasia.</a> Principles of Laboratory Animal Science. , 277-311 (2001).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J. Vis. Exp. (6), e236-e236 (2007).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2007).</li><li>Swanson, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain Maps: Structure of the Rat Brain. , (1992).</li></ol>

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O principal objectivo desta apresentação de vídeo é familiarizar os neurocientistas comportamentais com os princípios básicos da cirurgia estereotáxica. Pesquisadores que já estão realizando a cirurgia estereotáxica pode também beneficiar deste vídeo e considerar alguns dos refinamentos processuais para o uso em seu próprio laboratório. Um conhecimento cada vez maior sobre a otimização de técnicas cirúrgicas 1-3, o desenvolvimento de novos anestésicos e analgésicos para uso em medicina hum...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td></tr><tr><td> Álcool a 70% </td><td> VWR </td></tr><tr><td> Antisedan (atipamezole) </td><td> Orion </td></tr><tr><td> Sulfato de atropina 0,5 </td><td> Pharmachemie BV </td></tr><tr><td> Bulldog pinça hemostática </td><td> Aesculap </td></tr><tr><td> Cânulas </td><td> Component Supply Co. </td></tr><tr><td> Clorexidina 0,5% </td><td> AppepPharma </td></tr><tr><td> Limpeza em pó </td><td> Alconox </td></tr><tr><td> Varas de algodão </td><td> Hartmann BV </td></tr><tr><td> Dexdomitor (dexmedetomidina) </td><td> Orion </td></tr><tr><td> Broca </td><td> Dremel 8000 </td></tr><tr><td> Duratears Z </td><td> Alcon </td></tr><tr><td> Durogrip Naaldvoerder conversar 130 milímetros </td><td> Aesculap </td></tr><tr><td> Durotip Fijne schaar ret.sp / st 110 milímetros </td><td> Aesculap </td></tr><tr><td> Gaze </td><td> Medicomp (5x5) </td></tr><tr><td> Almofada de aquecimento </td><td> Harvard Apparatus </td></tr><tr><td> Pinos de insetos (estiletes) </td><td> Entosphinx (Czech Republik) </td></tr><tr><td> Ketamina 10% (ketamina) </td><td> Alfasan </td></tr><tr><td> Micro-Halsted ponta reta </td><td> Aesculap vet- </td></tr><tr><td> Molinea </td><td> Hartmann BV </td></tr><tr><td> NaCl 0,9% </td><td> Baxter </td></tr><tr><td> Agulhas (25G) </td><td> Terumo </td></tr><tr><td> Oxímetro </td><td> Edan Instruments, Inc. </td></tr><tr><td> Pentobarbital </td><td> Farmácia da UMCG </td></tr><tr><td> Rimadyl </td><td> Pfizer </td></tr><tr><td> Lâmina de bisturi n º 23 </td><td> Swann Morton </td></tr><tr><td> NR Scalpelholder. 4 133 milímetros </td><td> Aesculap </td></tr><tr><td> Chave de fenda </td><td> Loja Hardware convencionais </td></tr><tr><td> Simplex rápida (cimento dental) </td><td> Kemdent </td></tr><tr><td> Parafusos crânio </td><td> Component Supply Co. </td></tr><tr><td> Espátula </td><td> VWR </td></tr><tr><td> Spongestan especiais </td><td> Johnson &amp; Johnson </td></tr><tr><td> Stereotacts </td><td> Kopf Instruments </td></tr><tr><td> Pinça cirúrgica 100 milímetros </td><td> Aesculap </td></tr><tr><td> Material de sutura Safil 5 / 0 </td><td> Aesculap </td></tr><tr><td> Seringa de 10 ml </td><td> Omnifix </td></tr><tr><td> Seringa de 1ml </td><td> Terumo </td></tr><tr><td> 5ml da seringa </td><td> Omnifix </td></tr><tr><td> Xilocaína (lidocaína / adrenalina) </td><td> Astra Zeneca </td></tr></tbody></table>

rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience

Cirurgia estereotáxica para implantação de cânulas em regiões específicas do cérebro tem por muitas décadas foi uma técnica muito sucesso experimental para investigar os efeitos do neurotransmissor localmente manipulados e vias de sinalização em vigília, os animais se comportando. Além disso, a implantação estereotáxica de eletrodos para a estimulação eletrofisiológica e estudos de gravação tem sido fundamental para a nossa compreensão atual de neuroplasticidade e redes cerebrais no comportamento dos animais. Conhecimento cada vez maior sobre a otimização de técnicas cirúrgicas em roedores 1-4, conscientização pública sobre questões de bem-estar animal e legislação rigorosa (por exemplo, de 2010 a Directiva da União Europeia sobre o uso de animais de laboratório 5) nos levou a aperfeiçoar esses procedimentos cirúrgicos, particularmente com respeito a implementação de novos procedimentos para a suplementação de oxigênio e da monitorização contínua da oxigenação do sangue e os níveis de frequência cardíaca durante a cirurgia, bem comointrodução de um protocolo padronizado para atendimento pós-cirúrgico. Nossas observações indicam que essas modificações resultaram em uma taxa de aumento da sobrevida e uma melhora na condição geral dos animais após a cirurgia (por exemplo, perda de peso menor e um animal mais ativo). Esta apresentação de vídeo vai mostrar os procedimentos gerais envolvidos neste tipo de cirurgia estereotáxica com especial atenção para o nosso várias modificações. Vamos ilustrar esses procedimentos cirúrgicos em ratos, mas também é possível realizar este tipo de cirurgia em ratos ou outros animais de laboratório pequenos usando adaptadores especiais para o aparelho estereotáxico 6.

Watch this Scientific Journal Video about Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience at JoVE.com

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Cirurgia estereotáxica em roedores permite a administração alvo de drogas ou estimulação elétrica e gravações em despertos, comportando-se animais. Neste vídeo de apresentação iremos demonstrar recentes refinamentos processuais para este procedimento de longa data que a taxa de sobrevivência melhorou com sucesso e perda de peso reduzido pós-cirúrgico.

Cirurgia estereotáxica de roedores e animais Melhorias Resultado do Bem-Estar de Neurociência Comportamental

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

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Research

JoVE Journal

Neuroscience

39.0K visualizações.  University of Groningen. Cirurgia estereotáxica em roedores permite a administração alvo de drogas ou estimulação elétrica e gravações em despertos, comportando-se animais. Neste vídeo de apresentação iremos demonstrar recentes refinamentos processuais para este procedimento de longa data que a taxa de sobrevivência melhorou com sucesso e perda de peso reduzido pós-cirúrgico.

Vídeo: Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Entrega da droga sistêmica e local para o tratamento de doenças do sistema nervoso central em modelos de roedores

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Neurociência

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Combinando Behavioral Endocrinologia e Economia Experimental: Decisão da testosterona e Social Fazendo

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Um método geral para a avaliação de Incubação de Craving Sacarose em Ratos

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

Fixação de Perfusão todo animal para Roedores

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Cirurgia estereotáxica para lesão excitotóxica de áreas específicas do cérebro do rato adulto

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Um leve, sistema de fones baseado para manipular feedback auditivo em Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirurgia estereotáxica para manipulação genética em células estriadas de cérebro de rato Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.