这项研究得到了补助金，由英国医学研究理事会，自然科学和工程研究理事会，加拿大和加拿大中风网络。自由亚齐运动也支持由阿尔伯塔省文物基金会的医学研究。

梯级步行测试仪器水平梯级步行测试仪器，包括明确的有机玻璃和金属梯级（3毫米直径），这可能是插入创建一个1厘米之间的梯级的最小距离（见图1的地板的侧墙;。梅斯和Whishaw ，2003）。侧墙1米长，19厘米高的梯级的高度来衡量。阶梯高架地面30厘米以上，用中性启动笼和一个避难所（家笼）结束。由于动物习惯于在训练期间，仪器的海拔是不太可能导致焦虑。巷的宽度调整为动物的大小，所以，这是比动物，以防止动物转身更广泛的约1厘米。 修改任务的难度是由不同的金属梯级的位置。一个规律的梯级让动物过几次培训班学习的模式，并预测梯级的位置（图1，模式A）。一个不规则的格局被改变了从审判审判，防止动物的学习模式（图1，模式B）。经常安排，梯级间距为2厘米的间隔。梯级距离为不规则的图案，不同的系统从1至5厘米。分别用五种不规则的梯级模式的模板，使相同的模式适用于所有动物的标准化测试的难度，提高结果的可比性（见结果）。 录像一个摄像头（Canovision，佳能公司）是定位在一个轻微的腹角，所以，四肢的位置，可以同时记录。快门速度设置为500 - 2000秒。 videorecordings被使用于F /秒30帧帧分析分析。 行为训练和测试分析动物训练交叉从一个中立的笼梯，以达到他们的家庭笼，所以窝在家笼步行提供积极的强化。所有的动物都在同一方向越过阶梯。没有进一步强化了激励的动物跨越的阶梯。所有的动物都进行了培训和测试，每节5次。 足故障评分脚故障如前所述（梅斯和Whishaw，2003年）的计分制，采用定性评价的前肢和后肢的位置。通过检查帧一帧的录像分析。只有连续的步骤，每一个肢体进行了分析。因此，步态中断，如停止或脚故障，中断后的第一步前的最后一步，没有进球。加强循环的阶梯年底完成最后得分也被排除在外。肢体的位置是在肢体上放置一个小姐发生梯级之间的梯级和肢体突取得的。 步行或爪子梯级布局的类型被评为使用7类规模（见图2）。梯级足步行或安置被评为根据自己的立场和错误的贴装精度，在发生。 （0）共错过。0分，当患肢完全错过了一个梯级，即没有去碰它，并出现下降。被定义为一个肢体深之间的梯级和身体姿势和平衡不安的下降。 （1）深层滑动。肢体最初被放在一个梯级上，然后下滑时负重，并引起了下降。 （2）轻微的滑动 。患肢放置在梯级上，脱下时负重，但并不会导致在秋季也没有中断的步态周期。在这种情况下，动物能够保持平衡，并继续以协调的步态。 （3）更换。肢体被放置在梯级上，但它是负重前很快就被解除，放在另一个梯级。 （4）校正。肢体旨在为一个梯级，但不接触的第一个，然后放在另一个梯级。另一方面，录得4分如果一个肢体上放置一个梯级，并很快重新定位，而在同一梯级。 （5）部分的位置。肢体被放置在一个梯级，无论是手腕或前肢或后肢脚跟或脚趾的数字。 （6）正确的位置。midportion肢体手掌放在梯级上的分量十足支持。 当不同的错误发生在同一时间，分数最低的记录。举例来说，如果一只脚先放在梯级上，然后放置在另外一个相同的步骤（3分），然后脚下一滑，倒在梯级（1分）之间，1分录得。瓦途中发生跌倒，只有启动错误的肢体被评为和其他肢体没有得分，直到动物已重新定位，所有的肢体。五项试验的错误分数的平均值进行分析。 足的贴装精度分析（错误数） 在每个路口的错误的数量计算。脚故障评分系统的基础上确定了错误。每个肢体的位置，获得了0，1或2分的得分是错误的定义，即一个错误，代表任何一种足部滑移或总错过。录得分别为每一个肢体的错误和一些步骤。从这些数据中，每一步的错误的平均数量计算，平均五项试验。第二个定量参数分析是跨越整个长度的阶梯任务所需的平均时间。时间测量开始后，动物放在梯子上，开始走在它的整个长度。动物在停止花费的时间是不包括在测量中。 前爪两位数的评分前爪两位数的得分录得的爪子放在梯级midportion（即得分在脚下故障评分系统6）正确。被评为三点规模的前爪的数字，可以围绕一个梯级弯曲的程度（图2）。比分是应用最大重量的支持的立场，即前肢垂直定位在一个梯级。分数如下： （0）数字封闭在一个约90度角。 （1）数字封闭在一个约45度角。 （2）数字完全弯曲周围的梯级。 五个步骤的分数的平均值，用于分析。 图例<img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="图1" /> 图1。走在额侧查看具体测量仪器，测试仪器的熟练的梯级表示。在本研究中使用两种不同的梯级模式：模式，定期梯级安排，模式B，不规则的梯级安排。不规​​则的梯级安排不同之间的连续试验。 <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="图2" /> 图2，代表照片说明两位数得分的三类。请注意数字的角度与前肢在全重量的支持。

<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>

本研究介绍了梯级步行作为一种新的测试，以评估熟练的行走，患肢安置和肢体协调的任务。行走任务的梯级允许之间微妙的干扰，通过定性和定量相结合的熟练步行分析的运动功能歧视。视频分析程序，可用于描述一个障碍，并抓一个梯级时，本文中所描述的简单的评量表显示，在肢体安置和数字屈曲任何错误。计数客流量错误的定性分析和交叉设备所需的时间。这些措施让脚的贴装精度和数?...

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

/中风，脊髓损伤和其他神经退行性疾病的动物模型的发展进步需要高灵敏度的测试，来阐述运动功能的不同方面，并确定哪怕是细微的运动能力丧失。为了提高疗效和分辨率的测试，测试应允许定性和定量的运动功能和敏感，在性能上的变化，在恢复期间。本研究介绍了一种新的任务，以评估在大鼠熟练的走在同一时间来衡量两个前肢和后肢功能。动物需要沿着水平梯梯级间距是可变的，并定期改变。梯级间距的变化，防止学习的梯级绝对和相对位置的动物，以便尽量减少动物的能力，通过学习障碍补偿。此外，改变的梯级之间的间距可以用于长期研究反复测试。前后肢性能的定量和定性的描述，包括肢体配售，加强合作协调的方法进行了介绍。此外，使用补偿性的战略失误或其他肢体的错位的补偿性步骤表示。

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.

梯级行走任务，是一个新的测试，以评估熟练的步行和配售，加强和肢体间协调措施，前肢和后肢。

梯级步行任务：一个评分系统，其实际应用。

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 次观看.  University of Lethbridge. 梯级行走任务，是一个新的测试，以评估熟练的步行和配售，加强和肢体间协调措施，前肢和后肢。

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

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).
Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.
The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.

This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

归纳和慢性复发性实验性自身免疫性脑脊髓炎的临床评分

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

科研

生物学

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.

走在大鼠作为定性分析方法使用Rotorod

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

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.

在斑马鱼和镜头移植在眼突变分析中的应用

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.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

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;

实用指南系统发育的非专家

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.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

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.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

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.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

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.