Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

이 프로토콜에 사용되는 모든 재료는 표 1을 참조하십시오. 실험은 템플 대학에서 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1 동물 <ol><li> 야생형 애벌레 바다 칠성 장어 (Petromyzon 스 marinus L.)를 얻어, 10 - 메인에서 델라웨어 강 (펜실베니아) 또는 스트림 지류에서 미시간 호수를 공급 스트림에서 - (7 세 4)의 길이는 14 센티미터. </li><li> 16 ° C에서 50갤런 담수 탱크 (100) 동물 - 실험실에서, 50 그룹의 유충을 유지한다. 선 자갈 인치와 탱크 애벌레에서 굴을하기 위해, 물은 숯 여과하고 그들 칠성 장어하기 전에 염소 및 기타 불순물을 제거하기 위해 최소 48 시간 동안 공기 돌을 조절한다. 사용 일까지, 자신의 암성 재활용 동물, 먹이를 할 필요는 없다. </li></ol> 2 완전 척수코드 절개 <ol><li> 척수 수술하기 전에 다음과 같은 조성으로 심고 링거 용액 제조 : 110 mM의 염화나트륨, 2.1 mM의 KCl을 2.6 mM의 염화칼슘, 1.8 mM의 MgCl2를, 10 mM 트리스 완충액을; pH가 7.4. </li><li> 척수 절개를 수행하려면 작은 용기에 유충을 배치 얼음 차가운 링거 용액 1 % tricaine 메탄으로 마취합니다. </li><li> 마취 대신 일단 반 실 가드 (실리콘) 가득 배양 접시에있는 동물과 하나가 꼬리에, 동물의 주둥이에 하나씩 배치 (수술 위치에있는 동물을 잡아 4 곤충 핀 (0.15 mm 직경)를 사용 그리고이 다섯 번째 아가미의 수준에서). 거의 링거 용액으로 가기까지 배양 접시를 채 웁니다. 실체 현미경을 이용하여 수술을 수행하는 동안 얼음에 유충으로 요리를 넣습니다. </li><li> 척수 절개에게 등쪽을 수행하려면, 정중선, 5 번째의 레벨 메스 (# 11)와 세로 절개아가미는 척수를 시각화합니다. 초기에 피부가 위로 향하게 척수 이상에서 알 수있는 때까지 근육 조직이 매우 부드럽게 절단 메스 블레이드의 선단을 삽입함으로써 개방된다. 패션이 곤충 핀에서 후크와 척수 절개를 수행하는 동안 몸의 벽이 열려 유지하는 데 사용할. </li><li> 완전히 가위 (8 Castroviejo #)를 이용하여 제 5 아가미의 수준에서 척수를 쪼개다 척수에 수직으로 위를 삽입한다. 척수가 완전히 한 깨끗한 잘라 가로로해야합니다. 척수가 완전히 가로로되어 있음을 확인하기 위해 실체 현미경 척수 절단 단부를 시각화. 그리고, 한 쌍의 집게 (# 5)를 사용하여 절개 후 척수의 절단 단부를 재조정. </li><li> 척수 절개 미세 집게 한 쌍의 지느러미 몸 벽에 상처를 닫고 상처 t 수 있도록 1 시간 동안 얼음에 유충을 넣어 수행 한 후오 건조. 얼음 및 유충 사이 필터 종이 조각을 넣고 심고 링거 용액에 담가. 낮은 온도는 건조 따라서 감염을 방지 할 수 있도록 공기에 상처를 허용 동시에 살아있는 동물을 유지한다. </li><li> 그런 다음 24 시간 동안 얼음 냉수 작은 탱크 유충 (1 조 당 한 유충)를 전송합니다. </li><li> 행동면 부상의 사이트에 더 이동 꼬리가 없음을 확인하기 위해 척추 절개 후 병변 애벌레 일일를 검사합니다. 유충 (5 번째 아가미의 수준에서) 부상의 사이트에만 머리의 주동이를 이동할 수 있습니다 경우 척수 절개가 완료된 것으로 간주됩니다. 이 경우는 현재 23 유생 (실온에서 담수와 탱크 (1 조 당 한 유충)으로 전송 될 수있다 ° C)는 그들이 선택한 시점에 복구 할 수 있습니다. </li><li> 회복 기간 중 매일 2-3일 물을 변경합니다. </li></ol> 3 뇌 해부 및 준비현장 하이브리드 화에 대한 뇌 <ol><li> 부상 후 원하는 시점에서 애벌레 바다 칠성 장어와 장소를 다시 마취 상기와 같이 뇌 해부를 수행하기 위해 링거 용액과 페트리 접시에 그들을 핀. </li><li> 뇌를 해부하려면 먼저 콧 구멍과 뇌의 장기 사이의 메스 횡 절개를합니다. 여기에서 한 쌍의 집게로 잡고 Castroviejo 위의 쌍을 이용하여 뇌를 둘러싸고있는 조직을 절단하기 시작한다. </li><li> 뇌가 노출되면, 한 쌍의 집게를 이용하여 맥락총를 제거하고 뇌 신경을 동물의 머리를 확산하고 노출시키는 후크를 사용한다. 이어서, Castroviejo 가위와 척수 횡 절단. 뇌를 해부 두개골 신경을 잘라 집게의 또 다른 쌍을 사용하는 동안 집게 한 쌍의 척수를 잡습니다. </li><li> 절개 후, 실리콘의 작은 조각에 뇌를 넣고이 기능을 삽입하여 위치를 잡고척수 후각 전구 및 하나의 요법 핀입니다. 그런 다음, 후방 및 가위로 지느러미 중간 선을 따라 뇌의 cerebrotectal commissures을 절감하고 측면 날개의 판을 편향과 4 곤충 핀 (그림 2)와 실리콘에 평평하게 고정. </li><li> 회에 3 시간 동안 방 temparature로 고정 인산염 완충 생리 식염수에 4 % 파라 포름 알데히드 (산도 7.4)를 포함하는 튜브에 뇌와 실리콘의 조각을 전송합니다. </li><li> 전술 한 바와 같이 고정 후 현장 하이브리드 화에 wholemount을 위해 뇌를 사용 (스웨인 외., 1994). 제어 또는 허위 조작 동물의 뇌 부상 동물의 뇌와 병렬로 항상 실행해야합니다. wholemounted 애벌레 뇌 때문에 평평하고 얇은 모양과 인해 뇌가 상당히 투명하게 수초 (10)의 부재에 현장 하이브리드 화에 의해 연구 될 수있다. </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, 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=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, 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=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, 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=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, 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=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, 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=A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

여기에서 우리는 애벌레 바다 칠성 장어의 완전 척수 절개 후방 뇌 해부를 수행하기 위해 상세한 프로토콜을 제시한다. 이 절차는 원위치 혼성화에 전체 실장 뇌에 의한 척수 손상 후 척수 식별 돌출 뉴런 간의 유전자 발현의 차이를 분석한다. 절차에서 중요한 단계는 부드럽게 한 쌍의 유충의 주둥이를 터치하여 24 시간 후 stereomicrocope 하에서 척수의 절단 단부를 관찰함으로써 제어되고 확인 될 ?...

포유 동물에서 척수 손상 (SCI)는 부상 축삭은 외상 영역을 통해 재생과 적절한 대상에 다시 연결하지 않기 때문에 부상의 사이트 아래 기능의 영구적 인 손실로 이어지는 치명적인 조건이다. 포유류는 달리, 칠성 장어는 완전한 척수 손상 후 운동력을 복구. 1 흥미롭게 칠성 장어 때문에 그들의 큰 크기 2,3 (도 1)의 뇌 제제 마운트 전체 36 척수에서 개별적으로 식별되어 뉴런 돌출 세트를 가지고 . 이러한 척추-돌출 뉴런은 모두 높은 수준의 완전한 척수 절개하여 axotomized된다. 우리 그룹 등의 이전의 연구는 다른 일반적으로 손상 부위를 통해 자신의 축삭을 재생하는 동안, 심지어 이러한 뉴런의 일부는 매우 낮은 재생 능력을 보여 SCI 후 기능 회복의 존재 (그들이 &quot;나쁜 재생기를&quot;으로 간주된다) 것으로 나타났다 (그들이 간주된다 &quot;g짐 우드 재생기 &quot;). 2,3이 특성은 칠성 장어에게 차례를 시도 뉴런의 고유 재생 능력의 차이로 이어질 것입니다 좋고 나쁜 재생기 척추 - 투사 신경 세포 간의 유전자 발현의 차이를 연구하는 재미있는 척추 동물 모델을 만드는 같은 외부 환경에서 축삭을 재생. 1 우리가 이전에 보여 주었다이 모델을 사용하여 척추 - 투사 각각 netrin와 RGM의 억제 작용을 매개 UNC5 4,5 및 neogenin, 6 등의 축삭지도 분자 수용체의 낮은 재생 능력 쇼 식 뉴런. 또한,이 방법을 사용하여 우리의 그룹은 단지 좋은 재생기가 손상 후 및 재생 공정 동안에 neurofilaments의 표현의 회복을 보여주는 것으로 나타났다. 최근 부시 모건 7은 나쁜 재생기가 increas를 표시하는 면역에 의해 보여 주었다&quot;나쁜 재생기&quot;척추 - 투사 신경 세포가 서서히 완전한 척수 절개의 5,7,8 후 사망 사실을 저자가 관련되어 부상 후 시누 클레인의 에드 표현. 그래서 완전한 척수 손상의 칠성 장어 모델은 척수 투사 신경 세포 axotomy 후 &quot;나쁜 재생기를&quot;무엇이 이해하는 매우 유용한 모델로 등장했다. 우리는 현장 하이브리드 화에 wholemount 수행 손상 후 원하는 시점에서 완전한 척수 절개 수술 프로토콜과 후방 뇌 해부를 수행하는 우리의 연구를 수행합니다. 본 방법론 기사에서 우리는 애벌레 칠성 장어의 완전 척수 손상 수술의 적절한 성능에 대한 자세한 프로토콜을 제시, 동물의 후속 유지 보수 및 현장 하이브리드 화에 wholemount에 대한 최종 뇌의 해부 및 뇌의 준비. 자세한 프로토콜 퍼가기합니다유생 칠성 장어의 뇌에서 원위치 혼성화 wholemount을 rform는 이전에보고 된 바있다. 9는 또한, 척수 손상 및 뇌 해부 본 프로토콜은 또한 다음, 면역 조직 화학 또는 다른 조직 학적 방법에 대한 뇌를 처리하는 데 사용될 수있다.

<table><tbody><tr><td>Tricaine methane sulfonate</td><td>Spectrum</td><td>TR108</td><td>Benzocaine saturated solution in PBS for sacrifice</td></tr><tr><td>Scalpel #3</td><td>Fine Science Tools (FST)</td><td>10003-12</td><td></td></tr><tr><td>Blades for scalpel: #11</td><td>Fine Science Tools&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>Castroviejo scissors #8</td><td>Fine Science Tools&amp;nbsp;</td><td>15002-08</td><td></td></tr><tr><td>Forceps #4 &amp;amp; #5</td><td>Dumont, Switzerland</td><td>Roboz RS4955</td><td>#4 for dissection of Spinal cord; #5 for stripping menninges</td></tr><tr><td>Dissecting Microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Sylgard</td><td>Dow Corning Co.</td><td>184</td><td></td></tr><tr><td>Insect pins 0.15, 0.20 mm</td><td>Austerlitz</td><td>No catalogue #</td><td>0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td></tr><tr><td>7 ml HDPE Scintillation Tubes with Caps</td><td>Fisher Scientific</td><td>03-337-1</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Science (EMS)</td><td>19210</td><td>Dilute to 4% in PBS</td></tr></tbody></table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

완전 척수 손상 후 처음 바다 칠성 장어는 절개의 수준 이하로 마비된다. 그러나, 몇 주 후에 운동력을 복구하고,이 propriospinal 축삭 및 뇌간으로부터 돌출-척수 축삭의 재생 단거리 (몇 mm)을 동반한다. 36 대 식별 척추 - 투사 신경 세포 중 일부는 좋은 재생기가 있고 다른 사람은 나쁜 재생기입니다. 이 신경 세포는 쉽게 wholemount CNS 준비에 식별 할 수 있습니다. 선호하는 신경 세포 고유의 메커니즘을 이해하거나 척추 동물의 중추 신경계에 손상 후 축삭 재생을 억제하기 위해, 우리는 좋고 나쁜 재생기 간의 유전자 발현의 차이를 결정하는 방법과 표현은 척수 절개에 의해 영향을 받는다. 이 논문은 미세한 비전 아래 완전 척수 transections을 생산하고, BR을 준비하고, 신선한 물 탱크에서 주택 애벌레에 대한 기술과 최근 변화 성인 바다 칠성 장어를 보여줍니다아인 및 현장 하이브리드에 대한 척수 wholemounts. 간단히, 동물은 16로 유지됩니다 &#160; C를 °와 칠성 장어의 벨소리에 1 %의 벤조 카인에 마취. 척수 등쪽 접근 방식을 통해 홍채 절제술 가위로 가로로하고, 동물은 23 ° C에서 신선한 물 탱크에 복구 할 수 있습니다. 현장 하이브리드의 경우, 동물 reanesthetized하고 뇌와 코드가 지느러미 접근 방식을 통해 제거했다.

이 방법을 사용할 때 얻을 수있는 결과의 예를 들어, 식별 척수-돌출 제어의 신경 세포와 이주의 neogenin 성적 증명서의 발현을 보여주는 wholemounted 뇌의 대표 이미지는 병변 애벌레 바다 칠성 장어는 그림 2에 나타내었다 게시 할 수 있습니다. 독자는 완전한 척수 절개 및 자세한 사항은 바다 칠성 장어의 신경 세포를 돌출 식별 척수의 재생 능력 후 neogenin의 표현 사이의 관계를보고 이?...

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

칠성 장어는 완전 척수 손상 후 운동을 복구 할 수 있습니다. 그러나 일부 척추 - 투사 뉴런 좋은 재생기하고 다른 사람은 없습니다. 이 문서는 완전한 척수 transections 생산 및 현장 하이브리드에 대한 wholemount 뇌와 척수를 준비, 주택 바다 칠성 장어 유생 (최근 변화 성인)에 대한 기술을 보여줍니다.

이후 Wholemount을위한 완벽한 척수 손상 및 뇌 해부 프로토콜<em&gt; 제자리에서</em&gt; 애벌레 바다 장어의 하이브리드

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

complete-spinal-cord-injury-brain-dissection-protocol-for-subsequent

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K 조회수.  University of Edinburgh.  칠성 장어는 완전 척수 손상 후 운동을 복구 할 수 있습니다. 그러나 일부 척추 - 투사 뉴런 좋은 재생기하고 다른 사람은 없습니다. 이 문서는 완전한 척수 transections 생산 및 현장 하이브리드에 대한 wholemount 뇌와 척수를 준비, 주택 바다 칠성 장어 유생 (최근 변화 성인)에 대한 기술을 보여줍니다. 

비디오: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

마우스 배아 척수의 전체 마운트 준비 분기 Axonal 공부 DRG 뉴런의 DiI - 라벨링

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

연구

신경과학

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

오픈 책 준비에 DiI 주입하여 개발 신경 튜브와 Phenotypes의 평가에 miRNA 기반 Plasmids의 비켜 Electroporation에

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

해부 및 제브라 피쉬 배아의 측면 장착 : 척수 개발의 분석

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

활용 칙 신경 튜브에서 뉴런의 유전 정의된 그룹 Axonal 경로를 벗기기 비켜에서 Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

생물학

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Zebrafish의 성인 Telencephalon의 자상 부상 : 척추 동물의 뇌 신경 발생과 재생을 조사하는 방법

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

장어 Reticulospinal 축삭의 급성 해리는 개별 기능 시냅스 터미널의 릴리스 얼굴 막에서 녹음을 활성화하는

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish의<em&gt; 시츄</em&gt; 척수 척추 감각과 운동 뉴런 전기 생리 녹음 준비

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

외상 성 부상에 의해 영향을 하는 뇌 영역의 정위 적 아틀라스 유도 레이저 캡처 기정

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

제브라피쉬 유충의 척수 재생 연구를위한 제어 된 반자동 레이저 유도 부상

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

이후 Wholemount을위한 완벽한 척수 손상 및 뇌 해부 프로토콜

요약

더 많은 비디오 탐색

이 비디오의 챕터