이 작품은 Fondation 뿌린 라 공들인 Médicale (FRM), ALS 연구 (ALS 협회), NIH NINDS 교부금 NS05462와 NS034382, 그리고 ANR 부여 HyperMND에 대한 밀튼 Safenowitz 박사 동지의 재정 지원 덕분에 가능했다되었습니다.

1. 단계 하나 사전 마취 약물 : 마취의 유도 전에 10-15 분, 각각 타액의 분비 및 부종을 방지하기 위해 아트로핀 (0.20 밀리그램 / kg)와 methylprenidsolone (0.05 MG) 하위 cutaneously를 삽입. 2. 2 단계 마취의 유도 : pentobarbital 나트륨 (70 밀리그램 / kg) 또는 케타민 / xylazine (10 MG / kg, 각각 100 밀리그램 / kg 이상) 간 peritoneally의 혼합물을 주입. 마우스가 더 발가락 핀치 반사를 얻을 수 없습니다 때까지 아래 가자. 마취가 너무 빛이 발생하는 경우 복용량의 1 / 4 보충. 3. 세 번째 단계 * 참고 사항 :이 수술은 터미널 절차입니다. 마취의 수술 비행기에 도달 할 때 발생하기 쉬운 위치에 제기 따뜻한 담요 위에 마우스를 전송합니다. <ol><li> 100 ML / 분 주위에 흐름에서 순수 O 2를 제공 마스크와 마우스의 주둥이를 다룹니다. </li><li&gt;을 머리 깎기를 사용하여 꼬리의 기지로 목의 목덜미에서 등쪽 지역을 면도. </li><li> 또한 오른쪽 hindlimb 면도. </li><li> 위로 향한 위치로 마우스를 뒤집하지만 장소에 산소 마스크를 남겨하는데주의를 기울여야. </li></ol> 4. 4 단계 사지 주변에 고정하고 작업 표면의 네 모서리에 고정 봉합과 장소에 마우스를 고정합니다. 5. 5 단계 마우스 코어 온도를 모니터링하기 위해 온도 프로브를 삽입합니다. 36 ° C와 38 ° C. 사이의 핵심 온도를 유지하기 위해 가열 담요 / 램프 전력을 조정 6. 기관 절개술 및 인공 환기 <ol><li> 무딘 가위를 사용하여 기관을 통해 상처를 만들고 양쪽에 떨어져 피부를 당긴다. </li><li> 무딘 집게를 사용하여 기관을 다루는이 얇은 근육 (Sternohyoid)을 노출로 침샘 때문에 찢어. </li><li> 무딘 집게를 사용, 두 musc을 분리기관을 표시하는 방법으로 자신의 중간 분리를 따라 레. </li><li> 뒤몽 7 포셉을 사용하여 기관에서 4.0 실크 봉합의 두 길이를 끼 웁니다. </li><li> 이 연골 성의 고리 사이에있는 기관에서 횡단 컷을하지만 섹션의 호흡 관을 완전히하는데주의를 기울여야. </li><li> 기관 아래 tracheal 튜브를 넣고 그 위에 봉합을 매는하여 삽입 개방의 양쪽에 고정하십시오. tracheal 튜브는 마우스 환풍기 (SAR-830​​/AP, CWE 주식회사)과 capnograph (μcapstar, CWE 주식회사)에 연결되어 있습니다. 환풍기는 순수 O 2의 소스로, 준수 백을 통해 연결되어 있습니다. 마우스가 인공 환기 싸우고되지 않고 최종 갯벌 PCO 2는 4와 5 % 사이 안정되도록 환풍기 (호흡 속도 100-150 BPM, 최종 갯벌 볼륨 170-310 μl)의 매개 변수를 조정합니다. </li></ol> 7. 정맥 선의 위치 <ol><li> 무딘 절개 기법 사용, 경정맥에 노출목의 한쪽면에 정맥. 이 단계에서, 경정맥 정맥은 두 가지 주요 줄기, 앞쪽과 뒤쪽 얼굴 정맥으로 나눕니다. </li><li> 두 번 한이 줄기 각각에 대해 설정 한 다음 단계를 수행합니다 : <ol><li> 뒤몽 4 ~ 5 포셉 사용하여 주변 결합하는 조직에서 정맥을 신중하게 구분합니다. </li><li> 뒤몽 7 포셉을 사용하여 정맥에서 6.0 실크 봉합의 두 길이를 끼 웁니다. 정맥의 길이를 따라 멀리 가능한 한 그들을 구분합니다. </li><li> 정맥 (심장에 가장 가까운 쪽)의 근위 측면에 작은 혈관 클립을 배치하고, 정맥의 말초 측면을 묶을. </li><li> 아주 좋은 홍채 가위 사용을 완전히 섹션에 정맥 큰 신경 안 복용, 정맥에 작은 가로 절개를합니다. </li><li> 최대 선박 클립에, 개방에 미리 기입 1Fr 카테 테르 (premicath, Vygon)를 삽입합니다. </li><li> 뒤몽 4 포셉에 함께 정맥과 카테터를 잡고, 조심스럽게 선박 클립을 제거 후, ​​카테터 몇 가지 mor을 밀어정맥에 전자 mm. </li><li> 삽입의 양쪽에 모두 봉합으로 매는하여 카테터를 고정합니다. </li></ol></li><li> 카테터 중 하나의 anesthetics 때마다 필요한 (보통 매 10-30 분). 보충 용량을 주입하는 주사기 또는 주사기 펌프에 연결되어 IV 투여는 중 6 밀리그램 / pentobarbital 나트륨 또는 1250 40 μg / kg / 케타민 / xylazine 12 분 kg. 다른 카테터는 NaHCO 3 (1 %)과 plasmion (14 %)를 포함하는 4 % 포도당 솔루션의 속도가 느린 정맥 주입 (50 μl / 시간)에 주사기 펌프에 연결되어 있습니다. </li></ol> 8. 니들과 봉합사로 목 피부를 닫고 쉽상 위치에 마우스로 돌아 가기 9. 뒷다리 - 사지 근육과 신경의 해부 <ol><li> 가위를 사용하여 아킬레스 힘줄의의 허벅지의 상단에서 절개를합니다. 혈관을 손상하지 돌보는, 기본 근육에서 피부를 분리합니다. 필요에 따라이 부식. </li><lI&gt; 허벅지를 실행하는 흰 선으로 나타납니다 팔뚝 Femoris의 앞쪽 가장자리를 식별합니다. 가위를 사용 조심스럽게 무릎에서 라인을 따라 엉덩이 뼈에있는 모든 방법을 엽니 다. 근육을 분리합니다. 좌골 신경은 이제 팔뚝 Femoris 아래에서 볼 수 있습니다. </li><li> 조심스럽게 어깨를 Femoris을 해부. 출혈을 방지하기 위해 필요에 따라 / 끈을 부식. 팔뚝 Femoris는 완전히 제거하거나 좌골 신경과 삼두근 Surae 근육을 노출 reclined 할 수 있습니다. </li><li> Sural 신경을 해부. </li><li> 8 / 0 실크 스레드를 사용하여 일반 Peroneal 신경의 말초 부분을 묶고, 매듭을 말초 컷. 가능한 한 유행에 가까이, 신경 끝까지를 해부. </li><li> 일반 Peroneal와 Sural 신경 사이에 정강 신경을 확인합니다. 정강 신경의 서로 다른 지점 사이에, (지금의 정강 신경로 칭함) 더 깊은 지점에서 삼두근의 Surae을 innervating 지점을 식별합니다. </li><li> 8 / 0 실크를 사용하여나무 가지가 삼두근의 Surae을 innervating 그대로 유지하면서 목록, 정강 신경의 모든 가지를 함께 묶어. 매듭에 distally 정강 신경을 잘라까지 가능한 신경을 해부. </li><li> 다음 단계로 진행하면서 dessication을 방지하기 위해 식염수에 imbibed 거즈로 전체 hindlimb을 다룹니다. </li></ol> 10. Laminectomy <ol><li> 가위를 사용하여 백본을 따라 절개를합니다. 기본 근육에서 피부를 분리합니다. </li><li> 피하 근육을 분리 할 수​​있는 척추의 각 측면에 두 절개를합니다. 다음 각 측면에있는 척추의 측면에 부착 근육의 각 힘줄을 잘라. </li><li> 무딘 집게와 고급 퀴렛 사용하여 명확하게 각각의 척추 뼈를 식별 할 수 spinous 프로세스 주위 척추의 등쪽에 근육의 나머지 부분을 제거합니다. </li><li> 척추 T13와 L1을 식별합니다. T13는 늑골이 첨부 할 마지막 척추 뼈이다. 척추 열 w병 커닝햄 척추 척추 겸자 (주 Stoelting)를 사용하여 고정합니다. T13와 L1의 각 측면에있는 척추 클램프를 놓습니다. 척수를 압축하지 않도록주의하지만, 종 방향 축에 긴장이 약간 넣어. 척추 잘 집게로 살짝 아래로 눌러 고정되어 있는지 확인합니다. </li><li> 고급 rongeurs를 사용함으로써 척수를 노출 T13와 L1을 통해 다음 laminae을 척추 프로세스를 제거합니다. </li><li> 탈수를 방지하기 위해 식염수에 imbibed면이나 spongel의 작은 비트로 노출 된 척수를 다룹니다. </li><li> 척수를 둘러싸고있는 다시 상단에 맞춤 제작 플라스틱 욕조를 배치합니다. 4 / 0 실크 스레드를 사용하여 장소에서 목욕을 고정합니다. 욕조가 편의점 - 캐스트 밀봉 (WPI)와 함께 밀폐하여 방수인지 확인하십시오. </li><li> 편의점 - 캐스트가 건조되면, 척수를 덮고있는면을 제거하고 미네랄 오일과 함께 목욕탕을 입력합니다. </li><li> 뒤몽 5 포셉 매우 훌륭한 홍채 가위를 사용하여 부드럽게 경질의 친구를 당겨R은 척수를 둘러싸고 있으며, 두 방향으로까지 가능한 엽니 다. 척추의 각면에 경질를 접으십시오. </li><li> 이 척추 클램프에 의해 중지 유지하기로 마우스가 그렇게 거짓말을하는 제기 플랫폼을 낮 춥니 다. </li><li> 동물의 뒤쪽 지역을 지원하는 엉치 지역의 spinous 프로세스를 클램프 다른 척추 클램프를 사용하십시오. </li><li> 세 번째 클램프가 오른쪽 hindlimb과 발목을 고정하는 데 사용됩니다, 무릎에서 90 ° 각도로 근육이 수축. </li></ol> 11. 아킬레스의 힘줄의 해부 <ol><li> 다리는 무릎과 발목에서 90 °에서 구부러으로 hindlimb 면적 거즈를 제거하고 조직을 주변의 무료 아킬레스의 힘줄 해부. 많은뿐만 아니라 조직을 주변에서 가능한 삼두근의 Surae으로 해부. </li><li> calcaneus에 가까운 Plantaris 근육의 힘줄을 잘라 다음 완전히 힘줄을 제거하려면 다시 잘라. </li><li> 스레드 바늘 사용, 일을 통해 6 / 0 실크 실을 첨부전자 아킬레스의 힘줄과 힘줄 주위에 트리플 매듭을합니다. </li><li> 매듭에 가까운 힘 변환기를 배치, 힘줄의 말초 부분을 잘라 내고 실크 스레드를 사용하여 힘 변환기에 첨부합니다. </li><li> 삼두근의 Surae 근육의 근막 아래 스테인레스 스틸 와이어의 두 길이를 넣습니다. 이 와이어는 EMG 기록에 대한 세포 AC 앰프에 연결되어 있습니다. </li><li> 이 양극성 훅 전극에 공통 Peroneal 신경과 정강 신경을 배치합니다. </li><li> 양극은 주변 근육을 만지면으로 훅 전극의 음극에 삼두근의 Surae 신경을 놓으십시오. </li><li> 절연 장치에 대한 모든 자극 전극을 연결합니다. </li><li> 코드 dorsum 잠재력을 기록하는 세포 AC 증폭기에 연결된 척추의 등 표면에 볼 전극을 배치합니다. 다시 근육과 연락 자세 / AgCl 참조 전극을 배치합니다. </li></ol> 12. 단계 천 삼두근의 Surae 신경을 자극최대 트 진폭이 관찰 될 때까지 낮은 주파수 (&lt;1 Hz에서)에서 증가 강도의 50 μsec 사각형 펄스를 사용합니다. 천천히 트 진폭이 최대에 도달 할 때까지 트 응답의 진폭을 모니터링하는 동안 근육을​​ 향상시킬 수있는 힘 변환기를 이동합니다. 13. Motoneurons의 세포 레코딩 에이 시점에서, 표준 electrophysiological 기술은 세포 내 전극을 준비 척수에 신경에 침투 motoneuron로 식별하는 데 사용됩니다. <ol><li> 피펫 풀러 (P-97 Micropipette 풀러, 셔터 악기)를 사용하여 ~ 1 μm 팁에 유리 micropipette를 당겨. KCl 3M 솔루션 (전극 MΩ 10-20의 저항)으로 전극을 입력합니다. </li><li> micropositioner 사용하여 척추에 세포 증폭기 (Axoclamp 2B, 축삭 인스트루먼트)에 연결된 micropipette를 운전합니다. 전자의 자극에 의​​해 elicited 지역 필드 잠재력을 모니터링이크 신경 삼두근의 Surae의 모터 풀을 찾습니다. </li><li> microelectrode의 저항을 모니터링하면서 putative motoneurons을주의 깊게 접근한다. 막에 대한 밀어 때, 저항은 증가합니다. 침투 가끔 세포 증폭기의 &quot;버즈&quot;기능을 사용하여 용이하게 할 수 있습니다. </li></ol> 14. 안락사 절차 실험의 끝에서 동물은 pentobarbital의 과다 복용 목 베기 다음 (210 밀리그램 / kg IV)에 의해 euthanized 있습니다.

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+minimal+neuronal+networks+involved+in+flexor-extensor+alternation+in+the+mammalian+spinal+cord.">Identification of minimal neuronal networks involved in flexor-extensor alternation in the mammalian spinal cord.</a> Neuron. 71, 1071-1084 (2011).</li><li>Rabe, N., Gezelius, H., Vallstedt, A., Memic, F., Kullander, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrin-1-dependent+spinal+interneuron+subtypes+are+required+for+the+formation+of+left-right+alternating+locomotor+circuitry.">Netrin-1-dependent spinal interneuron subtypes are required for the formation of left-right alternating locomotor circuitry.</a> J. Neurosci. 29, 15642-15649 (2009).</li><li>Gurney, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science. 264, 1772-1775 (1994).</li><li>Cifuentes-Diaz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofilament+accumulation+at+the+motor+endplate+and+lack+of+axonal+sprouting+in+a+spinal+muscular+atrophy+mouse.">Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse.</a> Hum. Mol. Genet. 11, 1439-1447 (2002).</li><li>Simpson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+(12+hours)+intravenous+anesthesia+in+the+rat.">Prolonged (12 hours) intravenous anesthesia in the rat.</a> Laboratory animal science. 47, 519-523 (1997).</li><li>Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+Unit+Types+-+Functional+Specializations+in+Motor+Control.">Motor Unit Types - Functional Specializations in Motor Control.</a> Trends Neurosci. 3, 255-258 (1980).</li><li>Kerkut, G. A., Bagust, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+mammalian+spinal+cord.">The isolated mammalian spinal cord.</a> Prog. Neurobiol. 46, 1-48 (1995).</li><li>Carp, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+protocol+for+recording+from+spinal+motoneurons+of+adult+rats.">An in vitro protocol for recording from spinal motoneurons of adult rats.</a> Journal of Neurophysiology. 100, 474-481 (2008).</li><li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Spinal+Cord+Slice+Preparation+for+Recording+from+Lumbar+Motoneurons+of+the+Adult+Mouse.">An In Vitro Spinal Cord Slice Preparation for Recording from Lumbar Motoneurons of the Adult Mouse.</a> Journal of Neurophysiology. , (2011).</li><li>Husch, A., Cramer, N., Harris-Warrick, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+duration+perforated+patch+recordings+from+spinal+interneurons+of+adult+mice.">Long duration perforated patch recordings from spinal interneurons of adult mice.</a> Journal of Neurophysiology. , (2011).</li><li>Manuel, M., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha,+beta+and+gamma+motoneurons:+functional+diversity+in+the+motor+system's+final+pathway.">Alpha, beta and gamma motoneurons: functional diversity in the motor system's final pathway.</a> J. Integr. Neurosci. 10, 243-276 (2011).</li><li>Nakanishi, S. T., Whelan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decerebrate+adult+mouse+model+for+examining+the+sensorimotor+control+of+locomotion.">A decerebrate adult mouse model for examining the sensorimotor control of locomotion.</a> Journal of Neurophysiology. 107, 500-515 (2012).</li><li>Meehan, C. F., Grondahl, L., Nielsen, J. B., Hultborn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fictive+locomotion+in+the+adult+decerebrate+and+spinal+mouse+in+vivo.">Fictive locomotion in the adult decerebrate and spinal mouse in vivo.</a> The Journal of Physiology. 590, 289-300 (2012).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기에 설명 된 준비가 성인 마우스, 허리 motoneuron과 축삭에 의해 innervated 근육 섬유에 의해 생성 된 힘의 측정을 동시에 세포 내 기록에서 허용하는 첫 번째입니다. 때문에 동물의 작은 크기이 준비에 필요한 수술 기술 얻기 위해 도전 할 수 있습니다. 이러한 기술을 마스터하고 나면 단, 전체 수술은 세 시간 수행 할 수 있으며, 동물 레코딩을위한 수술 절차의 종료 후 7 더 ?...

A correction was made to <a href="http://www.jove.com/video/4312">Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo</a>. There was an error in the name of one author, Marin Manuel. The author's name has been corrected to:
Marin Manuel 
 
instead of: 
 
Manuel Marin

A correction to author list has been made for article <a href="http://www.jove.com/video/4312">Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo</a>.

Erratum: Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In Vivo

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 아트로핀 황산 </td><td> Aguettant </td><td></td><td></td></tr><tr><td> Methylprenidsolone </td><td> 화이자 </td><td> Solu - Medrol </td><td></td></tr><tr><td> 나트륨 pentobarbitone </td><td> 사노피 - Aventis </td><td> Pentobarbital </td><td></td></tr><tr><td> 케타민 </td><td></td><td></td><td></td></tr><tr><td> Xylazine </td><td></td><td></td><td></td></tr><tr><td> 포도당 </td><td></td><td></td><td></td></tr><tr><td> 혈장 증량제 </td><td> 로저 Bellon </td><td> Plasmagel </td><td></td></tr><tr><td> 무딘 가위 </td><td> FST </td><td> 14079-10 </td><td></td></tr><tr><td> 무딘 고급 가위 </td><td> FST </td><td> 15025-10 </td><td></td></tr><tr><td> Vannas 봄 가위 </td><td> FST </td><td> 15002-08 </td><td></td></tr><tr><td> 좋은 포셉 톱니 모양의 </td><td> FST </td><td> 11370-32 </td><td></td></tr><tr><td> 좋은 포셉 톱니 모양의 </td><td> FST </td><td> 11370-31 </td><td></td></tr><tr><td> 커닝햄 척수 어댑터 </td><td> Stoelting (주) </td><td></td><td></td></tr><tr><td> 편의점 - 캐스트 밀봉 </td><td> WPI </td><td> # 편의점 - CAST </td><td></td></tr><tr><td> 송풍기 </td><td> CWE 주식회사 </td><td> SAR-830​​/AP </td><td></td></tr><tr><td> Capnograph </td><td> CWE 주식회사 </td><td> μcapstar </td><td></td></tr><tr><td> 난방 담요 </td><td> 하버드 장치 </td><td> 507221F </td><td></td></tr><tr><td> 세포 증폭기 </td><td> 축삭 Instrume국세청 </td><td> 2B를 Axoclamp </td><td></td></tr><tr><td> 피펫 풀러 </td><td> 셔터 악기 </td><td> P-97 </td><td></td></tr><tr><td> KCl </td><td> 시그마 - 알드리치 </td><td> P9333-500G </td><td></td></tr></tbody></table>

simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo

가 (1) 쉽게 식별 할 목표를 (근육 섬유)를 갖는와하는 것은 따라서 매우 잘 알려진 기능을 갖는 고유의 특성을 가진 중추 신경계의 신경 세포이기 때문에 척추 motoneuron 긴 신경 기능을 연구하기위한 좋은 모델 시스템 왔습니다 (근육 수축을 조절하는), (2) 많은 척추와 내림차순 네트워크의 수렴 대상 &quot;최종 공통 경로&quot;의 따라서 이름 존재하며, (3)이 가능 날카로운 세포 전극과 함께 침투 할 수있는 대형 소마를 갖는 . 또한, 생체에서 공부했을 때, 그것은 동시에 motoneurons과 근육 목표에 의해 개발 된 힘의 전기 활동을 기록 할 수 있습니다. 생체에 motoneurons의 세포 녹음을 수행하는 것은 동시에 공부 할 수있는 독특한 위치에 experimentalist 넣어, &quot;모터 유닛&quot;(motoneuron, 그 축삭에 지정된 이름의 모든 격실, 그리고가 1 innervates 근육 섬유) : motoneuron에 impinging 입력은 motoneuron의 electrophysiological 속성 및 motoneurons의 생리적 기능에서 이러한 특성의 영향은 그 모터 장치에 의해 발생 된 힘을 즉. 준비가 마비 될 수 있으며 따라서 세포 내 기록에 대한 기계적 안정성이 감소되기 때문에 그러나,이 방법은 매우 어려운 것입니다. 따라서, 실험의이 종류는 고양이와 쥐의 달성되었습니다. 이 정상과 유전자 변형 생쥐에서 유사한 실험을 수행 할 수 있었던 경우에는 척추 모터 시스템의 연구는 강력한 도약을 수 있습니다. 기술적 인 이유로, 마우스의 척수 네트워크의 연구는 대부분 motoneurons과 척추 네트워크 유치 체외 준비에 신생아로 제한되었습니다 motoneurons는 목표에서 분리하고, 슬라이스, 미주리에서 공부했을 때 아르toneurons들은 입력의 대부분에서 구분됩니다. 최근까지 몇 그룹은 우리가 성인 마우스에 생체에 5,6를 motoneurons 매우 안정적인 레코딩을 얻을 수 새로운 준비를 출판 저희 팀을 포함, 생체 2-4 motoneurons의 세포 레코딩을 수행하기 위해 관리했습니다. 그러나, 이러한 녹음 즉, 이러한 motoneurons의 힘 출력을 기록 할 수있는 가능성이없는, 마비 동물에서 얻은되었습니다. 여기 우리가 motoneurons의 electrophysiological 속성 및 모터 유닛에 의해 개발 된 힘의 동시 녹음을 얻을 수있었습니다있는이 원래 준비의 확장을 제시한다. 그것은 우리가 자신의 힘 프로필에 따라 motoneurons의 다른 유형을 식별함으로써 자신의 기능을 공개 할 수 있으므로 이것은 중요한 성과입니다. 유전자 모델은 척추 segmental 회로 7-9 방해가, 또는 인간의 disea를 reproducting와 커플 링SE 10,11, 우리는이 기술이 척추 모터 시스템의 연구를위한 필수 도구가 될 것으로 기대합니다.

그림 1은 침투 후 삼두근의 Surae 그룹에서 motoneuron를 식별하는 방법을 보여줍니다. 낮은 자극 강도에서 만 monosynaptic EPSP는 (그림 1A) 관찰 할 수 있습니다. 높은 강도에서 EPSP는 &quot;orthodromic &#39;스파이크를 (그림 1B) 게재 할 수있을만큼 큰 수 있습니다. 더 높은 자극 강도에서 모든 - 또는 - 없음 antidromic 스파이크는 monosynaptic EPSP (그림 1C)보다 짧은 ?...

Watch this Scientific Journal Video about Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo at JoVE.com

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

이 새로운 방법은 하나의 성인 마우스 motoneuron과 근육 섬유에 의해 생성 된 힘의 측정의 동시 세포 녹음을 할 수 있습니다. 정상과 유전자 변형 동물의 모터 장치의 전기 및 기계적 성질의 결합 조사 신경 근육 시스템의 연구를위한 돌파구이다.

성인 마우스에서의 모터 단위 제작 룸버 Motoneuron과 포스의 동시 세포 녹화<em&gt; 생체에</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

simultaneous-intracellular-recording-lumbar-motoneuron-force-produced

Research

JoVE Journal

Neuroscience

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

14.6K 조회수.  Northwestern University Feinberg School of Medicine. 이 새로운 방법은 하나의 성인 마우스 motoneuron과 근육 섬유에 의해 생성 된 힘의 측정의 동시 세포 녹음을 할 수 있습니다. 정상과 유전자 변형 동물의 모터 장치의 전기 및 기계적 성질의 결합 조사 신경 근육 시스템의 연구를위한 돌파구이다.

비디오: Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

연구

신경과학

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

문화 접시에있는 기능성 모터 단위 : 척수의 Explants와 근육 세포의 공동 문화

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

성인 마우스 Forebrain의 급성 슬라이스에 Neuroblast 마이그레이션의 시간 경과 영상

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

메뚜기에 신경 활동을 특성화하기 위해 다중 단위 기록 방법 (<em&gt; Schistocerca 아메리카나</em&gt;) 강한 회로

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

동시에 두 광자<em&gt; 생체</em&gt; 마우스 Retrosplenial 코어 텍스에서 시냅틱 입력의 이미징 및 시냅스 대상

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

의 기록 세포 내 전압 응답에 대한 전기 생리 방법<em&gt; 초파리</em&gt; 빛 자극에 광수와의 interneurons<em&gt; 생체</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.