이 연구는 의학 연구위원회, 자연 과학 및 캐나다의 공학 연구위원회에서 보조금, 그리고 캐나다 획 네트워크에 의해 지원되었다. GAM은 또한 의료 연구를 위해 앨버타 헤리티지 재단에 의해 지원되었다.

시험 장치 도보 레더 안울렸으면 . 메츠와 Whishaw, 테스트 장치를 걸어 수평 래더 안울렸으면는 rungs 사이에 1cm의 최소 거리 (그림 참조 1과 바닥을 만들 삽입 수 명확 플렉시 및 금속 rungs (3mm 직경)로 만들어진 측면 벽 구성되어 , 2003). 측면 벽 높은 rungs의 높이에서 측정 1m 길이 19cm했다. 사다리는 마지막에 30cm 중립 시작 케이지 및 피난 (홈 케이지)와 지상 고가했다. 동물 훈련 동안 길들여지지 않았기 때문에, 장치의 상승이 불안을 일으킬 가능성이 희박했다. 골목의 너비가 주변을 공전의 동물을 방지하기 위해 동물보다 약 1cm 폭되었습니다 있도록 동물의 크기로 조정했다. 작업의 어려움은 금속 rungs의 위치를​​ 변화하여 바뀌었습니다. rungs의 일반적인 패턴은 동물들이 여러 교육 세션을 통해 패턴을 학습하고 rungs의 위치 (그림 1, 패턴)을 예상 수있었습니다. 재판에서 재판으로 변경되었습니다 불규칙한 패턴은 패턴 (그림 1, 패턴 B)를 학습에서 동물을하지 못했습니다. 일반 배열은 rungs는 2cm 간격으로 간격했다. 불규칙 패턴, rungs의 거리는 1 ~ 5cm로 체계적으로 다양. 불규칙 렁 패턴의 다섯 가지 템플릿은 동일한 패턴 (결과 참조) 테스트의 어려움을 표준화하고 결과 comparability을 향상시키기 위해 모든 동물에 적용되었다 있도록 사용되었습니다. Videorecording 카메라 (Canovision, 캐논 주식 회사)은 약간의 복부 각도로 위치를했습니다, 그래서 모든 사지의 위치를​​ 동시에 기록 수 있습니다. 셔터 속도는 500으로 설정되었습니다 - 2000 s을 (를) videorecordings 30 F / 초에서 프레임별로 프레임 분석을 사용하여 분석했다. 행동 훈련 및 시험 분석 동물들의 홈 케이지에 도달하는 중립적인 케이지에서 사다리를 십자가에 훈련했다, littermates와 홈 케이지는 산책에 대한 긍정적인 강화를 제공하므로. 모든 동물은 같은 방향으로 사다리를 넘어. 더 이상의 보강은 사다리를 건너 동물 의욕을주지 않았다. 모든 동물은 훈련 세션 당 다섯 번 테스트를했다. 풋 폴트 점수 forelimb 및 hindlimb 배치의 질적 평가로 (메츠와 Whishaw, 2003) 앞에서 설명한 발 결함 채점 시스템을 사용하여 수행되었습니다. 분석은 비디오 레코딩을 프레임별로 프레임의 검사에 의해 만들어졌습니다. 각 사지의 단 연속 단계를 분석했다. 따라서, 이러한 중지 또는 도보 결함으로 게이트 중단, 그리고 중단 후 첫 번째 단계는 전에 마지막 단계는 득점되지 않았습니다. 사다리의 끝에 공연 마지막 스텝 사이클도 채점에서 제외되었다. 림브 게재는 미스가 발생 rungs 사이 안울렸으면 위험한 돌출부에 사지 배치의 관점에서 득점했다. rungs 도보 또는 발 배치의 종류 (그림을 참조하십시오. 2) 7 카테고리 척도를 사용하여 평가되었다. 안울렸으면 도보 또는 포 게재는 자신의 위치와 배치 정확도에서 발생한 오류에 따라 평가되었다. (0) 총 싶어요. 0 점은 사지가 완전히 안울렸으면를 놓쳤을 때 주어진, 즉 그것을 만지지 않았 구요, 그리고 가을가 발생했습니다. 가을은 깊은 사이에 rungs 및 신체 자세와 균형을 교란했다 낙하 분수령으로 정의되었다. (1) 딥 슬립합니다. 분수령이 처음 안울렸으면에 배치했다, 그때 때 무게 베어링을 미끄러져서 가을을 일으켰습니다. (2) 약간 전표가. 분수령이 울렸으면에 배치 되었음 때 체중 베어링을 미끄러하지만 가을에 그 결과이나 보행주기를 방해하지 않았다. 이 경우, 동물은 균형을 유지하고 조정 게이트를 계속 수있었습니다. (3) 교환. 갈기갈기는 안울렸으면에 배치했지만, 그것이 체중 베어링 전에 그것은 신속하게 해제하고 다른 안울렸으면에 배치되었다. (4) 보정. 갈기갈기 한 살에 대한 목표지만, 그 첫 번째을 건드리지 않고도 다른 안울렸으면에 배치되었다. 사지가 울렸으면에 게재 경우 또는 4의 점수가 기록되었고 같은 안울렸으면에 남아있는 동안 신속하게 재배치했다. (5) 부분 배치가. 갈기갈기는 forelimb 또는 발뒤꿈치 또는 hindlimb의 발가락 중 손목이나 숫자와 안울렸으면에 배치되었다. (6) 올바른 배치가. 분수령의 손바닥 midportion는 전체 체중 지원 안울렸으면에 배치되었다. 다른 오류가 동시에 발생하면, 점수의 가장 낮은이 기록되었다. 예를 들어, 발을 처음 안울렸으면에 배치하고 같은 단계 (점수 3)에서 다른 하나를 배치하고, 1의 점수를 미끄러져서 rungs (점수 1) 사이에 떨어진 경우이 기록되었다. 뭐가을이 발생 EN에만 오류를 시작 분수령이 평가되었으며 동물이 모두 손발을 재설정했다 때까지 다른 사지의 아무도는 득점 않았다. 다섯 재판의 오류 점수는 분석을 위해 평균했다. 풋 배치 정확도 분석 (오류 번호) 각 교차점에서 오류의 개수가 계산되었다. 오류가 발 오류 채점 방식에 따라 결정됩니다. 오류가 0, 1 또는 2 포인트의 점수를받은 각 사지 배치로 정의 되었음 오류가 발 전표 또는 합계를 놓치지 어떠한 종류를 나타냅니다 즉. 오류와 단계의 수를은 별도로 각 사지에 대한 기록했습니다. 이러한 데이터에서 단계마다 오류의 의미 번호는 계산 다섯 실험에 대한 평균. 분석 두 번째 매개 변수는 양적 사다리 작업의 전체 길이를 교차하는 데 필요한 평균 시간이었습니다. 시간 측정은 동물이 사다리에 배치 후에 시작하고의 전체 길이를 산책했다. 동물 중지에 보낸 시간은 측정에 포함되지 않았습니다. 앞발 자리 점수 앞발 자리 점수는 앞발이 울렸으면에 미치는 midportion (발 오류 점수 시스템에 즉 점수 6) 올바르게 배치했을 때 기록했다. 앞발의 자리가 울렸으면 주위 근육이 수축 될 수 정도는 (그림 2) 3 점 만점으로 평가했다. 점수는 forelimb가 울렸으면에 수직으로 위치했다 즉, 최대 체중 지원 위치에 적용했다. 점수는 다음과 같이 주어진되었습니다 (0) 자리 대략 90도 각도로 마감했다. (1) 자리 약 45도 각도로 마감했다. (2) 자리 완전히 안울렸으면 주변 근육이 수축. 다섯 단계의 점수는 평균과 분석에 사용되었다. 그림 전설 <img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="그림 1" /> 그림 1. 장치의 특정 측정, frontolateral보기에서 테스트 장치를 도보로 숙련된 래더 안울렸으면는 지적했다. 두 개의 다른 렁 패턴은 현재의 연구에 사용되었습니다 패턴, 일반 렁 배치, 패턴 B, 불규칙한 렁 배치. 불규칙 렁 배열은 연속 실험 사이에 다양. <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="그림 2" /> 그림. 2. 대표 사진은 자리 점수의 세 범주를 설명. forelimb 때 전체 체중 지원 관련 자릿수의 각도를합니다.

<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>

현재의 연구는 숙련된 산책, 사지 배치 및 사지 ​​공동 성직을 평가하는 새로운 시험으로 작업을 걸어 래더 안울렸으면을 소개합니다. 작업을 산책 래더 안울렸으면은 숙련된 걷는 정성 및 정량 분석​​을 결합하여 운동 기능의 미묘한 교란 사이에 차별을 수 있습니다. 비디오 분석 절차는 장애인의 좀 더 자세한 설명 계정에 대한 사용과 안울렸으면 쥐고 때이 문서에서 설명하는 간단한 평가...

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

/ 뇌졸중, 척수 부상, 그리고 다른 neurodegenerative 질병에 대한 동물 모델의 개발 진행 모터 기능의 독특한 측면을 정교하고 운동 능력도 미묘한 손실을 결정하기 위해 고감도의 테스트를 필요로합니다. 테스트 효능과 해상도를 향상시키기 위해, 테스트 모터 기능의 질적 양적 및 조치를 허가 및 복구 기간 동안 성능의 변화에​​ 민감하여야한다. 현재의 연구는 동시에 forelimb과 hindlimb 기능을 모두 측정하는 쥐의 숙련된 산책을 평가하는 새로운 작업을 설명합니다. 동물은 rungs의 간격이 변수이고 정기적으로 변경되는 수평 사다리를 따라 도보로 필요합니다. 렁 간격의 변화는 rungs의 절대 및 상대 위치를 학습에서 동물을 방지하고 있으므로 학습을 통해 장애에 대한 보상하기 위해 동물의 능력을 최소화합니다. 또한, rungs 사이의 간격을 변경하면 테스트가 장기적인 연구에서 반복적으로 사용할 수 있습니다. 방법은 사지 배치, 스테핑, 공동 성직을 포함하여 이물 및 hindlimb 성능을 모두 양적 및 질적 모두 설명에 대해 설명되어 있습니다. 또한, 보상 전략의 사용은 잘못하면 다른 사지의 misplacement에 대한 응답으로 보상 단계로 표시됩니다.

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.

작업을 산책 래더 안울렸으면는 숙련된 산책을 평가하고 forelimb과 hindlimb, 배치 스테핑 및 간 사지 공동 성직을 모두 측정할 수있는 새로운 테스트입니다.

작업 워킹 레더 렁 : 채점 시스템과 실용적인 프로그램입니다.

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 조회수.  University of Lethbridge. 작업을 산책 래더 안울렸으면는 숙련된 산책을 평가하고 forelimb과 hindlimb, 배치 스테핑 및 간 사지 공동 성직을 모두 측정할 수있는 새로운 테스트입니다.

비디오: 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.

쥐에서 도보의 질적 분석 방법으로 Rotorod의 사용

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

연구

생물학

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.

렌즈 Zebrafish의 이식과 눈 돌연변이의 분석에의 응용

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.

의 시각화 Caenorhabditis Elegans</emLipophilic 바이탈 염료, DiI를 사용하기> 표피 구조

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;

비전문가를위한 계통 발생에 대한 실용 가이드

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.

드로소필라의 온도 선호도 행동과 Circadian 리듬의 설계 및 분석

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 차원 조직학 볼륨 및 마우스 유방 땀샘을 연구에의 응용의 재구성

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.

담배 과도 식 시스템을 사용하여 MAPK 신호 전달 통로의 공간 개편 이미징

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-매개 유전 변환, 트랜스 제닉 생산, 그리고 쌀에서 남성 생식 개발의 연구에 대 한 그것의 응용 프로그램

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.