Esta pesquisa foi suportada por concessões do Medical Research Council, as Ciências Naturais e Pesquisa de Engenharia Council of Canada e da Rede canadense Stroke. GAM também foi apoiado pela Heritage Foundation Alberta para a investigação médica.

Degrau da escada a pé aparelho de teste O degrau da escada horizontal andando aparelho de teste consistiu de paredes laterais feitas de Plexiglas clara e degraus de metal (3 mm de diâmetro), que pode ser inserido para criar um piso com uma distância mínima de 1 cm entre os degraus (ver Fig. 1;. Metz e Whishaw , 2003). As paredes laterais foram 1 m de comprimento e 19 cm de altura medidos a partir da altura dos degraus. A escada foi elevada 30 centímetros acima do solo com uma gaiola de partida em neutro e um refúgio (gaiola) no final. Porque os animais foram habituados durante o treinamento, a elevação do aparelho era improvável que cause ansiedade. A largura da pista foi ajustado para o tamanho do animal, de modo que foi cerca de 1 cm mais largo que um animal para evitar que o animal de se virar. A dificuldade da tarefa foi modificada pela variação da posição dos degraus de metal. Um padrão regular dos degraus permitidos os animais para aprender sobre o padrão de várias sessões de formação e de antecipar a posição dos degraus (Fig. 1, Padrão A). Um padrão irregular que foi mudado de julgamento a julgamento impediu o animal de aprendizagem o padrão (Fig. 1, Padrão B). Para o regime regular, os degraus foram espaçadas em intervalos de 2 cm. Para o padrão irregular, a distância dos degraus variados sistematicamente 1-5 cm. Cinco modelos de padrões de degrau irregular foram utilizadas, de modo que os mesmos padrões foram aplicados a todos os animais para padronizar a dificuldade do teste e melhorar a comparabilidade dos resultados (ver resultados). Gravação em vídeo Uma câmera (Canovision, Canon Inc.) foi posicionado em um ângulo ventral, para cargos de todos os quatro membros podem ser gravadas simultaneamente. A velocidade do obturador foi fixado em 500 - 2000 s. O gravações de vídeo foram analisados ​​usando-frame por frame-análise de 30 m / seg. Treinamento comportamental e análise do teste Os animais foram treinados para atravessar a escada de uma gaiola neutro para alcançar sua gaiola em casa, assim que a gaiola com os irmãos desde que o reforço positivo para caminhar. Todos os animais atravessaram a escada na mesma direção. Sem novo reforço foi dado para motivar os animais para cruzar a escada. Todos os animais foram treinados e testados cinco vezes por sessão. Pontuação culpa pé A avaliação qualitativa da colocação forelimb e hindlimb foi realizada utilizando um sistema de pontuação pé culpa, conforme descrito anteriormente (Metz e Whishaw, 2003). Análise foi feita por inspeção das gravações de vídeo quadro a quadro. Apenas etapas consecutivas de cada membro foram analisados. Portanto, o último passo antes de uma interrupção de marcha, como uma parada ou uma falha do pé, eo primeiro passo depois de uma interrupção não foram marcados. O ciclo de pisar última realizada no final da escada também foi excluído do gol. Colocação do membro foi marcado em termos de posicionamento do membro em uma protrusão degrau e membro entre os degraus, quando uma falta ocorreu. Os tipos de pé ou pata colocação nos degraus foram avaliados utilizando uma escala de 7-categoria (ver fig. 2). Pé ou pata colocação no degrau foi classificado de acordo com sua posição e erros que ocorreram na precisão de posicionamento. (0) Total de miss. 0 pontos foram dadas quando o membro completamente perdido um degrau, ou seja, não tocá-lo, e uma queda ocorreu. A queda foi definido como um membro profundamente caindo no meio degraus e postura corporal e equilíbrio foram perturbados. (1) deslizamento profundo. O membro foi inicialmente colocado em uma linha, em seguida, escorregou quando o peso-rolamento e causou uma queda. (2) deslizamento leve. O membro foi colocado em um degrau, escorregou quando a sustentação de peso, mas não resultou em uma queda, nem interromper o ciclo da marcha. Neste caso, o animal era capaz de manter o equilíbrio e continuar uma marcha coordenada. (3) de substituição. O membro foi colocado em um degrau, mas antes que fosse rolamento de peso foi rapidamente levantado e colocado em um outro degrau. (4) Correção. O membro apontado para um degrau, mas foi então colocado em outro degrau, sem tocar o primeiro. Alternativamente, uma pontuação de 4 foi gravado se um membro foi colocado em um degrau e foi rapidamente reposicionado, permanecendo no mesmo degrau. (5) colocação parcial. O membro foi colocado em uma linha com um pulso ou dígitos do membro anterior ou calcanhar ou dedos dos membros posteriores. (6) Colocação correta. A porção média da palma da mão de um membro foi colocado no degrau, com o apoio de peso total. Quando os erros ocorreram diferentes ao mesmo tempo, a mais baixa das pontuações foi gravado. Por exemplo, se um pé foi o primeiro colocado em um degrau e depois colocado em um outro na mesma etapa (pontuação 3), e, em seguida, escorregou e caiu no meio degraus (escore 1), uma pontuação de 1 foi gravada. When uma queda ocorreu, apenas o membro de iniciar o erro foi avaliado e nenhum dos outros membros foi marcado até que o animal havia reposicionado todos os membros. Pontuações de erro de cinco ensaios foram calculados para análise. Análise de precisão pé colocação (número de erros) O número de erros em cada cruzamento foi contado. Erros foram determinados com base no sistema de pontuação pé culpa. Um erro foi definida como a colocação de cada membro que recebeu uma pontuação de pontos 0, 1 ou 2, ou seja, um erro representa qualquer tipo de deslize pé ou perca total. O número de erros eo número de passos foi registrado para cada membro separadamente. A partir destes dados, o número médio de erros por passo foi calculado com uma média de cinco ensaios. O segundo parâmetro quantitativo analisado foi o tempo médio necessário para atravessar todo o comprimento da tarefa escada. Medição do tempo começou depois que o animal foi colocado na escada e começou a caminhar a todo o comprimento do mesmo. O tempo gasto em um animal uma parada não foi incluído na medição. Forepaw pontuação dígitos A pontuação forepaw dígito foi registrado quando a pata foi colocado corretamente com a sua porção média em uma linha (ou seja, 6 pontos no sistema de pontuação pé culpa). O grau em que os dígitos da pata dianteira poderia ser flexionado em torno de um degrau foi classificado em uma escala de três pontos (Fig. 2). A pontuação foi aplicado para a posição com o apoio de peso máximo, ou seja, o membro anterior foi posicionado verticalmente sobre um degrau. As pontuações foram dadas como segue: (0) Dígitos fechada em um ângulo de 90 graus aproximados. (1) Dígitos fechada em um ângulo de aproximadamente 45. (2) dígitos completamente flexionados em torno da linha. Dezenas de cinco etapas foram calculados e utilizados para análise. As legendas das figuras <img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="figura 1" /> Fig. 1. O degrau da escada qualificados andando aparelho de teste na visão frontolateral, com as medidas específicas do aparelho indicado. Dois padrões degrau diferentes foram utilizadas no presente estudo: Padrão A, arranjo degrau regular, Padrão B, arranjo degrau irregular. O arranjo degrau irregular variou entre ensaios consecutivos. <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="figura 2" /> Fig. 2. Fotografias Representante ilustrando as três categorias do escore de dígitos. Observe o ângulo dos dígitos em relação ao membro anterior, quando em apoio de peso total.

<ol>
	<li>Chu, C. J., Jones, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+structural+plasticity+in+cortex+heterotopic+to+focal+sensorimotor+cortical+damage.">Experience-dependent structural plasticity in cortex heterotopic to focal sensorimotor cortical damage.</a> Exp Neurol. 166, 403-414 (2000).</li><li>Emerick, A. J., Kartje, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+recovery+and+anatomical+plasticity+in+adult+rats+after+cortical+lesion+and+treatment+with+monoclonal+antibody+IN-1.">Behavioral recovery and anatomical plasticity in adult rats after cortical lesion and treatment with monoclonal antibody IN-1.</a> Behav Brain Res. 152, 315-325 (2004).</li><li>Faraji, J., Metz, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+bilateral+striatal+lesions+have+additive+effects+on+single+skilled+limb+use+in+rats.">Sequential bilateral striatal lesions have additive effects on single skilled limb use in rats.</a> Behav Brain Res. 177, 195-204 (2007).</li><li>Farr, T. D., Liu, L., Colwell, K. L., Whishaw, I. Q., Metz, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+alteration+in+stepping+pattern+after+unilateral+motor+cortex+injury:+a+new+test+strategy+for+analysis+of+skilled+limb+movements+in+neurological+mouse+models.">Bilateral alteration in stepping pattern after unilateral motor cortex injury: a new test strategy for analysis of skilled limb movements in neurological mouse models.</a> J Neurosci Methods. 153, 104-113 (2006).</li><li>Kleim, J. A., Barbay, S., Nudo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reorganization+of+the+rat+motor+cortex+following+motor+skill+learning.">Functional reorganization of the rat motor cortex following motor skill learning.</a> J Neurophysiol. 80, 3321-3325 (1998).</li><li>Merkler, D., Metz, G. A., Raineteau, O., Dietz, V., Schwab, M. E., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+recovery+in+spinal+cord-injured+rats+treated+with+an+antibody+neutralizing+the+myelin-associated+neurite+growth+inhibitor+Nogo-A.">Locomotor recovery in spinal cord-injured rats treated with an antibody neutralizing the myelin-associated neurite growth inhibitor Nogo-A.</a> J Neurosci. 21, 3665-3673 (2001).</li><li>McVea, D. A., Pearson, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepping+of+the+forelegs+over+obstacles+establishes+long-lasting+memories+in+cats.">Stepping of the forelegs over obstacles establishes long-lasting memories in cats.</a> Curr Biol. 17, R621-R623 (2007).</li><li>Metz, G. A. S., Dietz, V., Schwab, M. E., Meent, H. v. a. n. d. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+unilateral+pyramidal+tract+section+on+hindlimb+motor+performance+in+the+rat.">The effects of unilateral pyramidal tract section on hindlimb motor performance in the rat.</a> Behav Brain Res. 95, 37-46 (1998).</li><li>Metz, G. A. S., Dietz, V., Schwab, M. E., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+testing+of+motor+function+in+spinal+cord+injured+rats.">Efficient testing of motor function in spinal cord injured rats.</a> Brain Res. 883, 165-177 (2000).</li><li>Metz, G. A., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+rotation+intensity+in+unilateral+dopamine-depleted+rats+is+not+correlated+with+end+point+or+qualitative+measures+of+forelimb+or+hindlimb+motor+performance.">Drug-induced rotation intensity in unilateral dopamine-depleted rats is not correlated with end point or qualitative measures of forelimb or hindlimb motor performance.</a> Neuroscience. 111, 325-336 (2002).</li><li>Miklyaeva, E. I., Castaneda, E., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+deficits+in+unilateral+dopamine-depleted+rats:+impairments+in+movement+and+posture+and+compensatory+adjustments.">Skilled reaching deficits in unilateral dopamine-depleted rats: impairments in movement and posture and compensatory adjustments.</a> J Neurosci. 14, 7148-7158 (1994).</li><li>Muir, G. D., Whishaw, I. Q., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Locomotion in The Behavior of the laboratory rat. , 150-161 (2005).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground+reaction+forces+in+locomoting+hemi-parkinsonian+rats:+a+definitive+test+for+impairments+and+compensations.">Ground reaction forces in locomoting hemi-parkinsonian rats: a definitive test for impairments and compensations.</a> Exp Brain Res. 126, 307-314 (1999).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+nucleus+lesions+impair+overground+locomotion+in+rats:+a+kinetic+analysis.">Red nucleus lesions impair overground locomotion in rats: a kinetic analysis.</a> Eur J Neurosci. 12, 1113-1122 (2000).</li><li>Riek-Burchardt, M., Henrich-Noack, P., Metz, G. A., Reymann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+chronic+sensorimotor+impairments+in+the+ladder+rung+walking+task+in+rats+with+endothelin-1-induced+mild+focal+ischemia.">Detection of chronic sensorimotor impairments in the ladder rung walking task in rats with endothelin-1-induced mild focal ischemia.</a> J Neurosci Methods. 137, 227-233 (2004).</li><li>Ploughman, M., Attwood, Z., White, N., Dor&#233;, J. J., Corbett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endurance+exercise+facilitates+relearning+of+forelimb+motor+skill+after+focal+ischemia.">Endurance exercise facilitates relearning of forelimb motor skill after focal ischemia.</a> Eur J Neurosci. 25, 3453-3460 (2007).</li><li>Thallmair, M., Metz, G. A. S., Z'Graggen, W. J., Kartje, G. L., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+parallels+enhanced+plasticity+of+the+lesioned+adult+CNS+by+antibodies+to+neurite+growth+inhibitors.">Functional recovery parallels enhanced plasticity of the lesioned adult CNS by antibodies to neurite growth inhibitors.</a> Nature Neurosci. 1, 124-131 (1998).</li><li>Meyenburg, J. v. o. n., Brosamle, C., Metz, G. A., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+sprouting+of+chronically+injured+corticospinal+tract+fibers+in+adult+rats+promoted+by+NT-3+and+the+mAb+IN-1,+which+neutralizes+myelin-associated+neurite+growth+inhibitors.">Regeneration and sprouting of chronically injured corticospinal tract fibers in adult rats promoted by NT-3 and the mAb IN-1, which neutralizes myelin-associated neurite growth inhibitors.</a> Exp Neurol. 154, 583-594 (1998).</li><li>Whishaw, I. Q., Coles, B. L., Pellis, S. M., Miklyaeva, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+and+compensation+in+mouth+and+limb+use+in+free+feeding+after+unilateral+dopamine+depletions+in+a+rat+analog+of+human+Parkinson's+disease.">Impairments and compensation in mouth and limb use in free feeding after unilateral dopamine depletions in a rat analog of human Parkinson's disease.</a> Behav Brain Res. 84, 167-177 (1997a).</li><li>Whishaw, I. Q., Woodward, N. C., Miklyaeva, E., Pellis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+limb+use+by+control+rats+and+unilateral+DA-depleted+rats+in+the+Montoya+staircase+test:+movements,+impairments+and+compensatory+strategies.">Analysis of limb use by control rats and unilateral DA-depleted rats in the Montoya staircase test: movements, impairments and compensatory strategies.</a> Behav Brain Res. 89, 167-177 (1997b).</li><li>Whishaw, I. Q., Gorny, B., Sarna, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paw+and+limb+use+in+skilled+and+spontaneous+reaching+after+pyramidal+tract,+red+nucleus+and+combined+lesions+in+the+rat:+behavioral+and+anatomical+dissociations.">Paw and limb use in skilled and spontaneous reaching after pyramidal tract, red nucleus and combined lesions in the rat: behavioral and anatomical dissociations.</a> Behav Brain Res. 93, 167-183 (1998).</li><li>Z'Graggen, W. J., Metz, G. A. S., Kartje, G. L., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+and+enhanced+ccrtico-fugal+plasticity+in+the+adult+rat+after+unilateral+pyramidal+tract+section+and+blockade+of+myelin-associated+neurite+growth+inhibitors.">Functional recovery and enhanced ccrtico-fugal plasticity in the adult rat after unilateral pyramidal tract section and blockade of myelin-associated neurite growth inhibitors.</a> J Neurosci. 18, 4744-4757 (1998).</li></ol>

O presente estudo apresenta a escada degrau a pé tarefa como um novo teste para avaliar a pé qualificados, colocação dos membros e dos membros da coordenação. O degrau da escada a pé tarefa permite discriminação entre distúrbios sutis da função motora através da combinação de análise qualitativa e quantitativa de andar qualificados. O procedimento de análise de vídeo pode ser usado para uma conta de mais descritivo da deficiência, ea escala de classificação simples descritas neste documento revela o...

the ladder rung walking task: a scoring system and its practical application.

Progresso no desenvolvimento de modelos animais para / acidente vascular cerebral, lesão medular e outras doenças neurodegenerativas requer testes de alta sensibilidade para elaborar aspectos distintos da função motora e até mesmo para determinar a perda sutil da capacidade de movimento. Para melhorar a eficácia e resolução de testes, os testes devem permitir que medidas qualitativas e quantitativas da função motora e ser sensíveis a mudanças no desempenho durante os períodos de recuperação. O presente estudo descreve uma nova tarefa para avaliar a pé qualificados no rato para medir ambos os membros anteriores e função dos membros posteriores, ao mesmo tempo. Animais são obrigados a caminhar ao longo de uma escada horizontal em que o espaçamento dos degraus é variável e é periodicamente alterada. Alterações no espaçamento degrau evitar que os animais de aprender a localização absoluta e relativa dos degraus e assim minimizar a capacidade dos animais para compensar deficiências através da aprendizagem. Além disso, alterar o espaçamento entre os degraus permite que o teste para ser usado repetidas vezes em estudos de longo prazo. Métodos são descritos para a descrição quantitativa e qualitativa do desempenho do frente-e hindlimb, incluindo membros colocação, pisar, coordenação. Além disso, o uso de estratégias compensatórias é indicada por erros ou passos de compensação em resposta a outro membro do extravio.

Watch this Scientific Journal Video about The Ladder Rung Walking Task: A Scoring System and its Practical Application. at JoVE.com

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

O degrau da escada a pé tarefa é um novo teste para avaliar a pé qualificados e medir tanto forelimb e hindlimb colocação, pisando, e inter-membro da coordenação.

O Ladder Rung Walking Tarefa: um sistema de pontuação e sua aplicação prática.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

the-ladder-rung-walking-task-scoring-system-its-practical

Research

JoVE Journal

Biology

25.8K visualizações.  University of Lethbridge. O degrau da escada a pé tarefa é um novo teste para avaliar a pé qualificados e medir tanto forelimb e hindlimb colocação, pisando, e inter-membro da coordenação.

Vídeo: The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Uso de Rotorod como um método de análise qualitativa de Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Biologia

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Transplante de lente em Zebrafish e sua Aplicação na Análise de Mutantes Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualização de Caenorhabditis elegans Estruturas cutícula Usando o Dye lipofílicos Vital, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Um Guia Prático para Phylogenetics para não especialistas

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Reconstrução de 3-Dimensional Volume Histologia e sua Aplicação para o Estudo de Rato glândulas mamárias

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imagiologia reorganização espacial de uma via de sinalização MAPK Utilizar o sistema de expressão transitória do Tabaco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Agrobacterium-Transformação Genética Mediada, Produção Transgênica e Sua Aplicação para o Estudo do Desenvolvimento Reprodutivo Masculino no Arroz

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.