Esta pesquisa foi apoiada pela Grants-in-Aid para a Investigação Científica da Sociedade Japonesa para a Promoção da Ciência (JSPS), Grant-in-Aid do Ministério da Educação, Cultura, Desporto, Ciência e Tecnologia do Japão, Grants-in- ajuda do BIRD e do Japão CREST Ciências e Tecnologia, Grant-in-Aid da Neuroinformática Japan Center (NIJC), RIKEN e por Grant-in-Aid para a Investigação Científica da Prioridade Brain Research Areas-Integrativa (Shien) - de MEXT em Japan. Agradecemos a O&#39;hara &amp; Co. e Mariko Hayashi por sua assistência na criação deste filme.

Protocolo <ol><li> O aparelho utilizado para o teste de labirinto em cruz elevado é na configuração de um + e compreende dois braços abertos (25 x 5 x 0,5 cm) em frente a si e perpendiculares a dois braços fechados (25 x 5 x 16 cm) com um centro de plataforma (5 x 5 x 0,5 cm). Os braços abertos têm uma pequena parede (0,5 cm) para diminuir o número de quedas, enquanto os braços fechados e uma parede (16 cm) de altura para colocar o braço. Todo o aparelho é de 50 cm acima do piso (Ohara &amp; Co., Tokyo) e é colocado em um tanque vazio circular (100 cm de diâmetro, 35 cm de altura, normalmente utilizada para a tarefa de labirinto aquático de Morris) para proteger os ratos que caem ou tentativa de fuga durante o experimento. O aparelho é feito de materiais plásticos. A plataforma é branco e as paredes são transparentes. Há uma variação de materiais e cores dos aparelhos de labirinto elevado. </li><li> Os ratos são alojados com uma luz de 12 h / ciclo escuro (luzes acesas às 07:00), como descrito anteriormente (Takao &amp; Miyakawa, 2006a). Testes comportamentais é realizada 09h00 - 18:00. Todos os camundongos experimental são transferidos para a sala de testes de comportamento min 30 antes de começar o primeiro teste para habituar à condição da sala de testes de comportamento. A ordem dos ensaios é contrabalançada em genótipos. Há dois fins de julgamento de teste usando um animal prática. Uma delas é para se certificar de que está tudo bem com o sistema de gravação. Outra é manter a condição de teste mais uniforme possível. Ou seja, o mouse primeiro em toda a sessão seria condição experiência um pouco diferente com os outros (isto é, sem ruídos, antes feita por operações de teste e sem odor pistas de ensaios anteriores), sem um tal prática julgamento. Os animais são mantidos de acordo com as diretrizes do Comitê de Pesquisa Animal da Universidade de Kyoto. </li><li> A sala de testes de comportamento (170 x 210 x 200 cm, Ohara &amp; Co., Tóquio) é à prova de som e do nível de iluminação é mantida a 100 lux. Um rato é colocado na área central do labirinto com a cabeça voltada para um braço fechado. O teste de labirinto em cruz elevado é gravado com uma câmera de vídeo conectada a um computador, que é controlado por um dispositivo remoto. O número de entradas (uma entrada é definida como o centro de massa do mouse entra o braço) em cada braço e do tempo gasto nos braços abertos são gravados e estas medidas servem como um índice de ansiedade-como o comportamento. </li><li> Os ratos são autorizados a circular livremente sobre o labirinto por 10 min. Cada rato recebe um julgamento em nossa bateria de testes. O aplicativo utilizado para a aquisição e análise de dados comportamentais (Image EP) é baseado no programa público de domínio J Imagem (desenvolvido por Wayne Rasband no Instituto Nacional de Saúde Mental e disponível em <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ ij /</a> ), que foi modificado por Tsuyoshi Miyakawa (disponível através de O&#39;Hara &amp; Co., Tóquio, Japão). </li><li> A distância percorrida, o número de entradas em cada braço, o tempo gasto em cada braço, ea porcentagem de entradas nos braços abertos são calculados pelo programa PE Image. </li><li> Depois de cada julgamento, todas as armas e na área central são limpos com água hipocloroso super, que é um agente eficiente remoção de odor e tem odor relativamente fraco de si mesmo em comparação com outras soluções de limpeza, para evitar uma polarização com base em pistas olfactivas. Assim, podemos realizar os testes sob condições controladas sobre pistas olfativas. </li></ol>

<ol>
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Todos os procedimentos foram aprovados pelo Comitê de Uso Animal e Cuidados da Universidade de Kyoto.

Embora o genoma do rato foi seqüenciado, as funções da maioria dos genes não são conhecidas. Técnicas de modificação genética permitem exclusão ou outra manipulação de um gene específico em camundongos (Austin et al, 2004;.. Aiba et al, 2007). A influência de um determinado gene em um comportamento específico pode ser determinada através da realização de análises comportamentais dos camundongos mutantes (Takao e Miyakawa, 2006b;. Takao et al, 2007). O teste de labirinto em cruz elevado é um dos testes mais populares de todos os modelos animais atualmente disponíveis de ansiedade (Rodgers e Dalvi, 1997; Crawley, 2007). Este teste para a ansiedade-como o comportamento tem sido utilizado para triagem e fenotipagem transgênicos e knockout camundongos (Crawley, 1999) e para a descoberta de drogas (Hogg et al 1996;. Crawley, 2007). O teste de labirinto em cruz elevado tem uma forte validade preditiva para a triagem de drogas ansiolíticas (Rodgers e Dalvi, 1997; Mechiel Korte e De Boer, 2003; Crawley, 2007); ansiolíticos especificamente aumentar, e as drogas ansiogênicas diminuem especificamente, o número de entradas os braços abertos eo tempo gasto lá. A pontuação total de entradas e distância total são considerados um índice de atividade geral. Pontuação total de entradas é também um índice de ansiedade, e as porcentagens de entradas e tempo gasto em cada braço constituem o índice de ansiedade primária (Rodgers e Dalvi, 1997, Mechiel Korte e De Boer, 2003). Os braços abertos e fechados são considerados para evocar a mesma unidade exploratório, portanto, evitar os braços abertos é considerado como um resultado da indução de níveis mais elevados de medo (Rodgers e Dalvi, 1997). Pensa-se que a aversão de camundongos para explorar os braços abertos do labirinto é causada pelo medo de espaços abertos e elevados. Em 1984, Handley e Mithani informou sobre os trabalhos preliminares com o teste de labirinto elevado X (plus) descrito acima. O aparelho de teste original foi elevada 70 centímetros acima do chão, e composta por dois fechados e dois braços abertos, cada um dos quais medido 45 centímetros de comprimento por 10 cm de largura. Em seus estudos iniciais, eles relataram a relação das inscrições aberto / total do braço (Handley e Mithani, 1984). Posteriormente, outros índices foram desenvolvidos, que incluiu o número de entradas nos braços fechados e abertos eo tempo gasto nos braços abertos e fechados para ratos (Pellow et al, 1985; Pellow e File, 1986) e camundongos (Lister, 1987) . Alterações no teste de labirinto em cruz elevado incluiu alongamento de ambos os braços abertos (50 x 10 cm) e os braços fechados (50 x 10 x 40 cm) de altura com paredes em volta e um teto aberto nos braços fechados, e todo o labirinto foi elevada a uma altura de 50 cm (Pellow et al, 1985; Pellow e Arquivo, 1986). Atualmente, em nosso laboratório, o aparelho de teste de labirinto em cruz elevado é configurado na forma de um +, com dois braços abertos (25 x 5 cm, com uma muito ligeira, de 0,5 cm, parede) em frente a si e perpendiculares a dois braços fechados (25 x 5 x 16 cm), e é levantado 50 cm acima do chão (Miyakawa et al, 1996; Manabe et al, 2000; Miyakawa et al, 2001; Seeger et al, 2004; Morishima et al, 2005; Miyamoto et al, 2005; Arrow et al, 2006; Hattori et al, 2007; Niemann et al, 2007; Sano et al, 2008; Horii et al, 2008; Fukuda et al, 2008; Ikeda et al; 2008). O mouse é colocado no centro do + (5 cm x 5 cm) e é permitido explorar o labirinto livremente. Apesar de 5 min de gravação é comum, o comportamento é registrado por 10 min em nosso protocolo para aumentar a oportunidade de detectar o fenótipo. Os braços abertos e fechados elevados induzem um conflito de exploração (Mechiel Korte e De Boer, 2003; Crawley, 2007). As medidas do teste de labirinto em cruz elevado são registrados por um observador durante o experimento. Em nosso laboratório, o teste é gravado com uma câmera de vídeo conectada a um computador e os dados comportamentais (Image EP) são adquiridos e analisados ​​utilizando o programa PE Image. Número de entradas para os braços abertos versus número de entradas nos braços total, eo tempo gasto nos braços abertos versus braços fechados, fornecem as medidas de ansiedade-como o comportamento. Foram avaliadas mais de 90 linhagens de ratos mutantes geneticamente modificados usando o protocolo mostrado no filme e tem um grande conjunto de dados brutos para mais de 5000 ratos (incluindo do tipo selvagem e camundongos mutantes). Em nossa bateria de testes, do tipo selvagem littermates são normalmente utilizados como controle. Como uma tensão de fundo, os camundongos C57BL/6J são amplamente utilizados. Foram coletados os dados de camundongos C57BL/6J nos testes comportamentais. Os valores obtidos de camundongos C57BL/6J em nosso teste de labirinto em cruz elevado são as seguintes (n = 914, média SEM), a distância total percorrida: 1.547,55 14,27 centímetros; duração do tempo gasto: 56,49 2,42 s (braços abertos), 384,02 3,13 s (braços fechados), 161,90 2,17 s (centro de labirinto), relação do tempo gasto nos braços abertos: 9,19% 0,36, proporção de tempo gasto nos braços fechados: 63,82 0,52%; número de entradas: 7.64 0.21 (braços abertos), 24,32 0,28 (braços fechados); entradas% aberta braço: 21,9 0,05% fechado braços entradas: 78,1 0,05. Camundongos C57BL/6J gastar menos tempo de braços abertos que nos braços fechados (p &lt;0,0001, n = 914, T pareado test). Isto indica que os camundongos C57BL/6J tendem a evitar os braços abertos, e que o tempo gasto nos braços abertos é um índice válido de ansiedade-como o comportamento. Além disso, a ordem dos ensaios é contrabalançada em genótipos, porque o número de teste afeta o tempo gasto nos braços abertos ea plataforma central. Ou seja, os índices de todo o aumento durante o 3 º e 4 ratos em comparação com o primeiro camundongos (p = 0,0089, n = 476) (dados não publicados). Com nosso protocolo, a análise de mais de 1.661 camundongos mostraram que a ordem seqüencial dos ratos testados em uma gaiola não afeta significativamente o tempo permanecer aberto braço (dados não publicados). Ou seja, os desempenhos dos ratos primeira tirada da gaiola, não são significativamente diferentes das do segundo, o terceiro ou os ratos passado. Embora o teste de labirinto em cruz elevado eo teste de transição claro / escuro são ambos usados ​​para avaliar a ansiedade-como o comportamento, os resultados nem sempre são consistentes entre si (Holmes et al, 2000; Tujimura et al, 2008; Nakajima et al, no prelo ) Por exemplo, prosencéfalo específicos calcineurina knockout-ratos gastar uma diminuição da quantidade de tempo na câmara de luz no teste de transição claro / escuro, mas um aumento da quantidade de tempo nos braços abertos no teste de labirinto em cruz elevado (Miyakawa et al. , 2003). Análise fatorial da nossa bateria de testes comportamentais indica que o teste de labirinto em cruz elevado eo teste de transição claro / escuro avaliar diferentes aspectos da ansiedade-como o comportamento, como o brilhante espaço ansiedade no teste de transição claro / escuro e abrir espaço de ansiedade como comportamento no teste de campo aberto (Takao e Miyakawa, 2006b; Yamasaki et al, 2006). Assim, tanto o teste de transição claro / escuro eo teste de labirinto em cruz elevado estão incluídos na nossa bateria de testes comportamentais. Crabbe e colegas relataram que as variáveis ​​não controladas e experimentos caracterizando mutantes podem produzir resultados que são específicos para um laboratório particular (Crabbe et al., 1999). As diferenças que existem entre processuais laboratórios tornam difícil replicar ou comparar os resultados entre eles. Elaboração de documentação visual do protocolo irá promover uma melhor compreensão dos procedimentos experimentais, permitindo a padronização dos protocolos utilizados em laboratórios e para as comparações dos fenótipos comportamentais de várias linhagens de ratos mutantes avaliados com estes testes. Nós já publicou um protocolo de filme do teste de transição claro / escuro (Takao e Miyakawa, 2006a). Da mesma forma, os filmes de outros protocolos, como para o teste de campo aberto, o teste de natação forçada porsolt, e testes de condicionamento do medo que usamos em nossa bateria de testes comportamentais estão actualmente a ser feitos para publicação como artigos de periódicos futuro vídeo.

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elevated plus maze for mice

Embora o genoma do rato é agora completamente seqüenciado, as funções da maioria dos genes expressos no cérebro não são conhecidos. A influência de um determinado gene em um comportamento específico pode ser determinado pela análise comportamental de ratos mutantes. Se um gene alvo é expressa no cérebro, fenótipo comportamental dos camundongos mutantes poderiam elucidar o mecanismo genético de comportamentos normais. O teste de labirinto em cruz elevado é um dos testes mais utilizados para medir a ansiedade-como o comportamento. O teste se baseia na aversão natural de ratos para áreas abertas e elevados, bem como sobre o seu comportamento espontâneo naturais exploratório em ambientes novos. O aparelho consiste de braços abertos e os braços fechados, cruzou no meio perpendicularmente uns aos outros, e uma área central. Ratos têm acesso a todas as armas e estão autorizados a circular livremente entre eles. O número de entradas nos braços abertos eo tempo gasto nos braços abertos são usados ​​como índices de ansiedade induzida espaço aberto em camundongos. Infelizmente, as diferenças que existem entre processuais laboratórios torná-lo difícil de duplicar e comparar os resultados entre laboratórios. Aqui, apresentamos um filme detalhado demonstrando nosso protocolo para o teste de labirinto em cruz elevado. Em nosso laboratório, temos avaliados mais de 90 linhagens de camundongos mutantes usando o protocolo mostrado no filme. Estes dados serão divulgados como parte de um banco de dados público que estamos construindo. Visualização do protocolo irá promover uma melhor compreensão dos detalhes de todo o procedimento experimental, permitindo a padronização dos protocolos utilizados em laboratórios diferentes e as comparações dos fenótipos comportamentais de várias linhagens de ratos mutantes avaliados através deste teste.

Watch this Scientific Journal Video about Elevated Plus Maze for Mice at JoVE.com

Elevated Plus Maze for Mice

O teste de labirinto em cruz elevado é um dos testes mais utilizados para medir a ansiedade-como o comportamento em camundongos. Aqui, apresentamos um filme mostrando os procedimentos detalhados para a realização do ensaio.

Labirinto elevado para Ratos

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Research

JoVE Journal

Biology

90.2K visualizações.  Graduate School of Medicine, Kyoto University. O teste de labirinto em cruz elevado é um dos testes mais utilizados para medir a ansiedade-como o comportamento em camundongos. Aqui, apresentamos um filme mostrando os procedimentos detalhados para a realização do ensaio.

Vídeo: Elevated Plus Maze for Mice

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Claro / escuro Teste de transição para Ratos

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biologia

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Morris Experiment Maze Água

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Reação em Cadeia da Polimerase: Protocolo Basic Plus Estratégias de solução de problemas e otimização

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Métodos de coleta de leite para Ratos e Reeves Muntjac cervos

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Injeções intravenosas em Neonatal Ratos

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subclonagem Além disso Inserção (SPI) - Um Método recombineering Novel para a construção rápida de Gene Segmentação Vetores

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

Uma estratégia para validar o papel da mediada por calejado Plasmodesmal Gating na Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

Diferença de potencial nasal para quantificar o transporte do íon Trans-epitelial em ratos

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

Um método de perfusão antegrada para isolamento cardiomiocócito de camundongos

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

Terminal H-reflex Measurements in Mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

Medições do reflexo H terminal em camundongos

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...