원고에 대한 토론과 논평을 해주신 Ying Xiong 박사님께 감사드립니다. 이 연구는 중국 국립과학재단(NSFC)이 C. M.(31500839)에 제공한 보조금으로 지원됩니다.

1. 유충 해부
알림: 해부 용액 혈림프 유사 식염수(HL3.1)18 및 고정 용액 4% 파라포름알데히드(PFA)19,20은 온도가 너무 낮을 때 미세소관이 해중합되기 때문에 실온에서 사용됩니다.
<ol><li>길고 뭉툭한 집게로 방황하는 3번째 인스타 유충을 골라냅니다. HL3.1로 세척하고 실체현미경 아래의 해부 접시에 놓습니다. 참고: 방황하는 3번째 인스타 유충은 분지된 전방 나선으로 식별되며 약 96시간(25°C)21에서 튜브 주위를 기어 다닙니다.</li>
	<li>등쪽이나 배쪽이 위로 향하도록 유충을 배치합니다. 두 개의 긴 기관으로 등쪽을 식별하고 복부 치아 벨트로 복부를 식별합니다.<ol><li>관찰할 조직에 따라 등쪽 또는 복부 절개를 선택하십시오. 이렇게 하면 특정 관심 영역을 보다 정확하고 자세하게 볼 수 있습니다. NMJ 및 근육 세포 관찰을 위해 유충 등쪽과 복부 부위를 각각 절개합니다.</li>
	</ol></li>
	<li>입 고리와 꼬리를 고정합니다. 핀을 조정하여 유충을 확장된 상태로 유지합니다. HL3.1 한 방울을 유충에 떨어뜨려 유충이 마르지 않도록 합니다. 알림: Ca2+ -free HL 3.1은 해부 중 근육 수축을 최소화하는 데 사용할 수 있습니다.</li>
	<li>해부 가위를 사용하여 뒤쪽 끝 가까이에 작은 가로 절단을 하되 뒤쪽 끝을 자르지 마십시오. 그런 다음 복부 정중선을 따라 앞쪽 끝을 향해 자릅니다.</li>
	<li>유충의 네 모서리에 4 개의 곤충 핀을 삽입하십시오. 유충이 모든 방향으로 최대한 늘어나도록 곤충 핀을 다시 조정하십시오.</li>
	<li>집게를 사용하여 근육을 손상시키지 않으면서 내부 장기를 제거합니다.</li>
</ol>2. 고정
<ol><li>100μL의 PFA(4%)를 추가하여 사체가 해부 접시에 고정되어 있는 동안 사체를 40분 동안 담그십시오. 주의 : PFA는 위험하므로 피부와의 직접적인 접촉이나 흡입을 피하기 위해 효과적인 보호 조치를 취합니다.</li>
	<li>핀을 분리하고 샘플을 2mL 마이크로 원심분리 튜브로 옮깁니다. 0.2% Triton X-100(0.2% PBST)을 함유한 1x 인산염 완충 식염수로 PFA를 헹구어 15rpm의 탈색 셰이커에서 10분 동안 2mL 미세 원심분리기 튜브를 채웁니다. 세탁 과정을 5회 반복합니다.</li>
</ol>3. 면역세포화학
<ol><li>사체를 차단제(0.2% PBST에 5% 염소 혈청)에 담그고 실온에서 40분 동안 차단합니다.</li>
	<li>차단제를 제거하고 4°C에서 밤새 0.2% PBST로 희석한 200μL의 1차 항체(예: anti-α-tubulin, 1:1000; anti-Futsch, 1:50)로 교체합니다. 단클론 항α-튜불린(anti--tubulin)으로 면역염색을 통해 근육 미세소관을 시각화합니다. Anti-Futsch는 NMJ의 미세소관 형태를 간접적으로 반영할 수 있습니다.</li>
	<li>1차 항체를 배양한 후 0.2% PBST로 유충을 10분 동안 세척하고 5회 반복합니다.</li>
	<li>0.2% PBST로 희석한 200μL의 2차 항체(예: goat anti-Mouse-488, 1:1000)로 유충을 어두운 곳에서 실온에서 1.5시간 동안 배양합니다.</li>
	<li>다음으로, 어두운 곳에서 미세소관을 염색할 때 3605:1000 농도의 TO-PRO(R) 3 요오드화물(T20,22)과 같은 핵 염료를 배양 튜브에 추가합니다.</li>
	<li>2차 항체와 핵 염료를 0.2% PBST로 10분 동안 헹굽니다. 어둠 속에서 5회 반복합니다.</li>
</ol>4. 장착
<ol><li>유충 사체를 유리 슬라이드에 0.2% PBST로 놓고 실체현미경으로 조정합니다. 유충 사체의 내부 표면이 위를 향하고 모든 유충 사체가 원하는 대로 배열되었는지 확인하십시오.</li>
	<li>물티슈로 과도한 PBST 용액을 흡수하고 페이드 방지 장착 매체를 부드럽게 추가합니다.</li>
	<li>슬라이드에 커버슬립을 놓아 해부된 유충을 천천히 부드럽게 덮어 기포가 생기지 않도록 합니다.</li>
	<li>커버슬립 주위에 손톱 광택제를 바릅니다. 형광 감쇠를 줄이기 위해 슬라이드를 어두운 공간에 놓으십시오.</li>
</ol>5. 이미지 취득
<ol><li>이미지를 획득하려면 레이저 스캐닝 컨포칼 현미경을 사용하여 60x 오일 이멀젼 대물렌즈(개구수 1.42) 또는 이와 유사한 것을 선택하고 실험에 따라 레이저 출력과 파장을 조정합니다.</li>
	<li>NMJ를 식별하려면 세그먼트 A4의 근육 3에서 이미지를 캡처합니다( 그림 1F의 위치 참조). 488nm 레이저를 선택하여 α-tubulin 또는 Futsch를 활성화하고 543nm를 선택하여 HRP 이미징 트랙을 활성화합니다. 파라미터를 800픽셀 x 800픽셀의 프레임 크기, 2.0의 디지털 줌, 0.8μm의 이미징 간격(NMJ)으로 조정합니다(그림 2).</li>
	<li>근육의 미세소관을 식별하려면 기관 가지가 더 적기 때문에 세그먼트 A3-A5( 그림 1G의 위치 참조)에서 근육 2의 이미지를 캡처합니다. α-튜불린을 활성화하기 위해 488nm 레이저를 선택하고 T3605 이미징 트랙을 활성화하기 위해 635nm 레이저를 선택하십시오. 파라미터를 1024픽셀 x 1024픽셀의 프레임 크기, 3.0의 디지털 줌, 근육 세포의 이미징 간격 0.4μm로 조정합니다(그림 3).</li>
</ol>

Observation Microtubule networks in Drosophila Neuromuscular junctions and muscle cells(초파리 신경근 접합부 및 근육 세포의 미세소관 네트워크 관찰)

<ol>
	<li>Halpain, S., Dehmelt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAP1+family+of+microtubule-associated+proteins.">The MAP1 family of microtubule-associated proteins.</a> Genome biology. 7 (6), 224 (2006).</li><li>S&#225;nchez, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+microtubule-associated+protein+2+(MAP2)+and+its+relevance+for+the+regulation+of+the+neuronal+cytoskeleton+function.">Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function.</a> Progress in neurobiology. 61 (2), 133-168 (2000).</li><li>Dehmelt, L., Halpain, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAP2/Tau+family+of+microtubule-associated+proteins.">The MAP2/Tau family of microtubule-associated proteins.</a> Genome biology. 6 (1), 204 (2005).</li><li>Roll-Mecak, A., McNally, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-severing+enzymes.">Microtubule-severing enzymes.</a> Current opinion in cell biology. 22 (1), 96-103 (2010).</li><li>Walczak, C. E., Gayek, S., Ohi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-depolymerizing+kinesins.">Microtubule-depolymerizing kinesins.</a> Annual review of cell and developmental biology. 29, 417-441 (2013).</li><li>Bramblett, G. T., 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=Abnormal+tau+phosphorylation+at+Ser396+in+Alzheimer's+disease+recapitulates+development+and+contributes+to+reduced+microtubule+binding.">Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding.</a> Neuron. 10 (6), 1089-1099 (1993).</li><li>Van Vactor, D., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+morphogenesis,+active+zone+organization+and+structural+plasticity+in+Drosophila.">Presynaptic morphogenesis, active zone organization and structural plasticity in Drosophila.</a> Current opinion in neurobiology. 43, 119-129 (2017).</li><li>Hummel, T., Krukkert, K., Roos, J., Davis, G., Kl&#228;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Futsch/22C10+is+a+MAP1B-like+protein+required+for+dendritic+and+axonal+development.">Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development.</a> Neuron. 26 (2), 357-370 (2000).</li><li>Roos, J., Hummel, T., Ng, N., Kl&#228;mbt, C., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Futsch+regulates+synaptic+microtubule+organization+and+is+necessary+for+synaptic+growth.">Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth.</a> Neuron. 26 (2), 371-382 (2000).</li><li>Packard, M., 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=The+Drosophila+Wnt,+wingless,+provides+an+essential+signal+for+pre-+and+postsynaptic+differentiation.">The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation.</a> Cell. 111 (3), 319-330 (2002).</li><li>Dent, E. W., Callaway, J. L., Szebenyi, G., Baas, P. W., Kalil, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+and+movement+of+microtubules+in+axonal+growth+cones+and+developing+interstitial+branches.">Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 19 (20), 8894-8908 (1999).</li><li>Tanaka, E. M., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+behavior+in+the+growth+cones+of+living+neurons+during+axon+elongation.">Microtubule behavior in the growth cones of living neurons during axon elongation.</a> The Journal of cell biology. 115 (2), 345-363 (1991).</li><li>Ahmad, F. J., Yu, W., McNally, F. J., Baas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+katanin+in+severing+microtubules+in+the+neuron.">An essential role for katanin in severing microtubules in the neuron.</a> The Journal of cell biology. 145 (2), 305-315 (1999).</li><li>Hu, Z., 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=Fidgetin+regulates+cultured+astrocyte+migration+by+severing+tyrosinated+microtubules+at+the+leading+edge.">Fidgetin regulates cultured astrocyte migration by severing tyrosinated microtubules at the leading edge.</a> Molecular biology of the cell. 28 (4), 545-553 (2017).</li><li>Feizabadi, M. S., Castillon, V. 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+effect+of+Tau+and+taxol+on+polymerization+of+MCF7+microtubules+in+vitro.">The effect of Tau and taxol on polymerization of MCF7 microtubules in vitro.</a> International journal of molecular sciences. 23 (2), 677 (2022).</li><li>Velasco, C. D., 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=Microtubule+depolymerization+contributes+to+spontaneous+neurotransmitter+release+in+vitro.">Microtubule depolymerization contributes to spontaneous neurotransmitter release in vitro.</a> Communications biology. 6 (1), 488 (2023).</li><li>H&#246;&#246;g, J. L., 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=Electron+tomography+reveals+a+flared+morphology+on+growing+microtubule+ends.">Electron tomography reveals a flared morphology on growing microtubule ends.</a> Journal of cell science. 124, 693-698 (2011).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> Journal of neurogenetics. 18 (2), 377-402 (2004).</li><li>Broadie, K. S., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+embryonic+neuromuscular+synapse+of+Drosophila+melanogaster.">Development of the embryonic neuromuscular synapse of Drosophila melanogaster.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 13 (1), 144-166 (1993).</li><li>Xiong, Y., 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=HDAC6+mutations+rescue+human+tau-induced+microtubule+defects+in+Drosophila.">HDAC6 mutations rescue human tau-induced microtubule defects in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (12), 4604-4609 (2013).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Mao, C. X., 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=Microtubule-severing+protein+Katanin+regulates+neuromuscular+junction+development+and+dendritic+elaboration+in+Drosophila.">Microtubule-severing protein Katanin regulates neuromuscular junction development and dendritic elaboration in Drosophila.</a> Development. 141 (5), 1064-1074 (2014).</li><li>Jin, 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=Drosophila+Tubulin-specific+chaperone+E+functions+at+neuromuscular+synapses+and+is+required+for+microtubule+network+formation.">Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation.</a> Development. 136 (9), 1571-1581 (2009).</li><li>Sarthi, J., Elefant, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dTip60+HAT+activity+controls+synaptic+bouton+expansion+at+the+Drosophila+neuromuscular+junction.">dTip60 HAT activity controls synaptic bouton expansion at the Drosophila neuromuscular junction.</a> PloS one. 6 (10), 26202 (2011).</li><li>Weingarten, M. D., Lockwood, A. H., Hwo, S. Y., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+factor+essential+for+microtubule+assembly.">A protein factor essential for microtubule assembly.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (5), 1858-1862 (1975).</li><li>Kadavath, H., 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=Tau+stabilizes+microtubules+by+binding+at+the+interface+between+tubulin+heterodimers.">Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (24), 7501-7506 (2015).</li><li>Trotta, N., Orso, G., Rossetto, M. G., Daga, A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hereditary+spastic+paraplegia+gene,+spastin,+regulates+microtubule+stability+to+modulate+synaptic+structure+and+function.">The hereditary spastic paraplegia gene, spastin, regulates microtubule stability to modulate synaptic structure and function.</a> Current biology: CB. 14 (13), 1135-1147 (2004).</li></ol>

저자는 공개할 것이 없습니다.

여기에서는 초파리 유충 신경근 접합부 및 근육 세포의 해부 및 면역염색을 위한 프로토콜이 설명되어 있습니다. 고려해야 할 몇 가지 필수 사항이 있습니다. 첫째, 관찰된 근육의 부상을 피하는 것은 해부 과정에서 매우 중요합니다. 집게와 근육 사이의 직접적인 접촉을 방지하기 위해 내부 장기를 제거하기 전에 필렛을 고정하는 것이 좋습니다. 근육 손상이나 유충 표피로부터의 분리를 ?...

미세소관(MT)은 세포골격의 구조적 구성 요소 중 하나로서 세포 분열, 세포 성장 및 운동성, 세포 내 수송, 세포 모양 유지 등 다양한 생물학적 과정에서 중요한 역할을 합니다. 미세소관의 역학 및 기능은 MAP1, MAP2, Tau, Katanin 및 Kinesin 1,2,3,4,5와 같은 다른 단백질과의 상호 작용에 의해 조절됩니다.
뉴런에서 미세소관은 축삭돌기와 수상돌기의 발달과 유지에 필수적입니다. 미세소관의 이상은 기능 장애와 심지어 뉴런의 죽음으로 이어집니다. 예를 들어, 알츠하이머 환자의 뇌에서 타우 단백질 과인산화는 미세소관 네트워크의 안정성을 감소시켜 신경학적 불규칙성을 유발한다6. 따라서 미세소관 네트워크를 검사하는 것은 신경 발달과 신경 질환의 발병 기전을 이해하는 데 기여할 것입니다.
신경근 접합부(NMJ)는 운동 뉴런 축삭 말단과 근육 섬유 사이에 형성된 말초 시냅스로, 시냅스 구조와 기능을 연구하기 위한 우수하고 강력한 모델 시스템이다7. Futsch는 포유류에서 발견되는 미세소관 결합 단백질 MAP1B와 상동적인 초파리의 단백질입니다8. 그것은 뉴런에서만 발현되며 NMJ의 시냅스 버튼 8,9의 발달에 중요한 역할을합니다. 야생형에서 NMJ 공정의 중심을 따라 흐르는 사상 다발은 anti-Futsch를 사용한 면역염색을 통해 시각화됩니다. NMJ의 말단에 도달하면, 이 다발은 미세소관으로 구성된 고리를 형성하거나 사상 구조를 잃게 되어, 확산 및 반점 모양(10)을 형성할 수 있다. 미세소관 고리는 일시 중지된 성장 원뿔과 관련이 있으며, 이는 미세소관 배열이 안정적임을 시사한다11. 따라서 Futsch 염색을 통해 NMJ에서 안정적인 미세소관 발달을 간접적으로 확인할 수 있습니다. 초파리 유충의 근육 세포의 크기가 크기 때문에 미세소관 네트워크를 명확하게 시각화할 수 있습니다. 미세소관 네트워크의 안정성에 영향을 미치는 요인은 미세소관의 밀도와 모양을 분석하여 찾을 수 있습니다. 동시에 근육 세포의 미세소관 네트워크 상태를 NMJ 결과와 교차 검증하여 보다 포괄적인 결론을 얻을 수 있습니다.
미세소관의 네트워크와 역학을 조사하기 위해 많은 프로토콜이 사용되었습니다. 그러나, 이러한 연구들은 종종 시험관 내 연구(in vitro studies)12,13,14,15,16에 초점을 맞추었다. 대안적으로, 일부 생체내 실험은 세포골격(17)을 검출하기 위해 전자 현미경을 이용하였다. 형광 표지된 항체 또는 화학 염료가 단백질 또는 DNA에 특이적으로 결합하는 것에 따르면, 여기에 제시된 방법을 사용하면 생체 내 개별 뉴런 수준에서 NMJ의 미세소관 네트워크를 검출할 수 있으며, 결과는 근육 세포의 관찰에 의해 확증됩니다. 이 프로토콜은 Drosophila melanogaster에서 사용할 수 있는 강력한 유전 도구와 결합할 때 간단하고 안정적이며 반복 가능하여 생체 내 신경계에서 미세소관 네트워크 조절 단백질의 역할에 대한 다양한 표현형 검사 및 유전자 스크리닝을 가능하게 합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor Plus 405 phalloidin</td>
			<td>invitrogen</td>
			<td>A30104</td>
			<td>dilute 1:200</td>
		</tr>
		<tr>
			<td>Enhanced Antifade Mounting Medium</td>
			<td>Beyotime</td>
			<td>P0128M</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW confocal microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>Thermo Fisher</td>
			<td>A-11001</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Laser confocal microscope LSM 710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>66vision</td>
			<td>54138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Futsch antibody</td>
			<td>Developmental Studies Hybridoma Bank&#160;&#160;</td>
			<td>22C10</td>
			<td>dilute 1:50</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5168</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Wako</td>
			<td>168-20955</td>
			<td>Final concentration: 4% in PB Buffer</td>
		</tr>
		<tr>
			<td>Stainless Steel Minutien Pins</td>
			<td>Entomoravia</td>
			<td></td>
			<td>0.1mm Diam</td>
		</tr>
		<tr>
			<td>Stereomicroscope SMZ161</td>
			<td>Motic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereoscopic fluorescence microscope BX41</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-conjugated goat anti-HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>dilute 1:100</td>
		</tr>
		<tr>
			<td>TO-PRO(R) 3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Transfer decoloring shaker TS-8</td>
			<td>Kylin-Bell lab instruments</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>BioFroxx</td>
			<td>1139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>dumont</td>
			<td>500342</td>
			<td></td>
		</tr>
	</tbody>
</table>

using drosophila larval neuromuscular junction and muscle cells to visualize microtubule network

미세소관 네트워크는 신경계의 필수 구성 요소입니다. 많은 미세소관 조절 단백질의 돌연변이는 신경퇴행성 질환에 대한 미세소관 관련 단백질 타우(Tau), 미세소관 절단 단백질 스파스틴(Spastin) 및 카타닌 60(Katanin 60)과 같은 신경 발달 장애 및 신경 발달 질환과 관련이 있습니다. 뉴런에서 미세소관 네트워크를 검출하는 것은 신경 장애의 발병 기전을 밝히는 데 유리합니다. 그러나 뉴런의 크기가 작고 축삭 미세소관 다발이 조밀하게 배열되어 있어 미세소관 네트워크를 시각화하는 것이 어렵습니다. 이 연구에서는 유충 신경근 접합부와 근육 세포의 해부 방법과 α-튜불린 및 미세소관 관련 단백질 Futsch의 면역염색을 통해 Drosophila melanogaster의 미세소관 네트워크를 시각화하는 방법을 설명합니다. 신경근 접합부는 시냅스 전후 미세소관을 모두 관찰할 수 있게 해주며, 초파리 유충의 근육 세포는 크기가 커서 미세소관 네트워크를 명확하게 시각화할 수 있습니다. 여기에서는 초파리 멜라노가스터(Drosophila melanogaster)에서 카타닌 60을 돌연변이시키고 과발현시킨 다음 신경근 접합부와 근육 세포의 미세소관 네트워크를 조사하여 신경 발달에서 카타닌 60의 조절 역할을 정확하게 밝힙니다. 그러므로, Drosophila melanogaster의 강력한 유전 도구와 결합해, 이 의정서는 매우 신경계에 있는 microtubule 네트워크 통제 단백질의 역할을 위한 유전자 검열 그리고 microtubule 역동성 분석을 촉진합니다.

우리는 신경근 접합부(NMJ)와 근육 세포 모두에서 미세소관 네트워크를 시각화하기 위한 단계별 절차를 시연했습니다. 개략도(그림 1A-E)에 따라 해부한 후 면역염색을 수행하고 레이저 컨포칼 현미경 또는 입체 형광 현미경(그림 1F,G)으로 이미지를 관찰 및 수집합니다.
NMJ의 시냅스 전후 미세소관 조직 모두...

Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

저자 스포트라이트: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

여기에서는 신경근 접합부와 근육 세포의 미세소관 네트워크를 시각화하기 위한 자세한 프로토콜을 제시합니다. Drosophila melanogaster의 강력한 유전 도구와 결합된 이 프로토콜은 신경계에서 미세소관 네트워크 조절 단백질의 역할에 대한 유전자 스크리닝 및 미세소관 역학 분석을 크게 용이하게 합니다.

초파리 유충 신경근 접합부 및 근육 세포를 사용하여 미세소관 네트워크 시각화

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

현재 미세소관 역학은 EB1과 같은 미세소관 결합 단백질을 통해 간접적으로 관찰됩니다. 알파벳 튜불린 헤테로이디머는 세포질에 풍부하기 때문에 직접 접히기 때문에 인접한 튜불린 헤테로다이머는 생체 내 미세소관 네트워크의 역학이 부족합니다. 당사의 프로토콜은 신경근 시냅스와 근육 세포의 미세소관을 시각화합니다.Drosophila melanogaster에서 사용할 수 있는 강력한 유전 도구와 결합하면 다양한 표현형 검사 및 생체 내 신경계에서 미세소관 네트워크 조절 단백질의 역할에 대한 유전자 스크리닝이 가능합니다. 우리의 연구는 세포골격과 신경 발달 사이의 관계와 신경 질환에 대한 영향에 중점을 둡니다. 우리는 세포골격 조절 단백질이 뉴런에 미치는 영향을 강조함으로써 신경 발달의 메커니즘과 신경 질환에 대한 단형성을 발견하기를 희망합니다.

using-drosophila-larval-neuromuscular-junction-muscle-cells-to

Research

JoVE Journal

Neuroscience

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

1.4K 조회수.  Hubei University. 현재 미세소관 역학은 EB1과 같은 미세소관 결합 단백질을 통해 간접적으로 관찰됩니다. 알파벳 튜불린 헤테로이디머는 세포질에 풍부하기 때문에 직접 접히기 때문에 인접한 튜불린 헤테로다이머는 생체 내 미세소관 네트워크의 역학이 부족합니다. 당사의 프로토콜은 신경근 시냅스와 근육 세포의 미세소관을 시각화합니다.Drosophila melanogaster에서 사용할 수 있는 강력?...

비디오: 저자 스포트라이트: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

연구

신경과학

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

전신 탈염 신경 근육 정션 분석을위한 트랜스 대 복부 근육의 해부

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

상기 표현형의 특성을 : 정량이 중요한 이유<em&gt; 초파리</em&gt; 애벌레 신경 근육 학 분기점

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

선형 아가로 오스 채널을 사용하여 공부하기<em&gt; 초파리</em&gt; 애벌레 크롤링 동작

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

높은 처리량 및 다중 파라 메트릭 정량화를위한 ​​두 알고리즘<em&gt; 초파리</em&gt; 신경 근육 접합 형태학

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

확인된 모터 뉴런을 위한 신경근육 정션에서 변경을 검출하기 위하여 Drosophila 성인 다리의 해부 및 면역 조직 화학

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...