نشكر روسباش مايكل له الإرشاد والدعم خلال المرحلة الأولى من تطوير هذا الأسلوب. تم تمويل هذا العمل من قبل JST المعزوفة البرنامج، مؤسسة العلوم الوطنية السويسرية (31003A_149893 و 31003A_169548)، ومجلس البحوث الأوروبي (ERC-الجنيه الاسترليني-311194)، ومؤسسة نوفارتيس "البحوث" الطبية البيولوجية الطبية (13A39) وجامعة جنيف .

<ol>
	<li>Sheeba, V., Kaneko, M., Sharma, V. K., Holmes, T. C. <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+circadian+pacemaker+circuit:+Pas+De+Deux+or+Tarantella?.">The Drosophila circadian pacemaker circuit: Pas De Deux or Tarantella?.</a> Crit Rev Biochem Mol Biol. 43 (1), 37-61 (2008).</li><li>Granados-Fuentes, D., Herzog, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clock+shop:+coupled+circadian+oscillators.">The clock shop: coupled circadian oscillators.</a> Exp Neurol. 243, 21-27 (2013).</li><li>Zhang, Y., Emery, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15+-+Molecular+and+neural+control+of+insects+circadian+rhythms.">Chapter 15 - Molecular and neural control of insects circadian rhythms.</a> Insect molecular biology and biochemistry. , 513-551 (2012).</li><li>Hardin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+analysis+of+circadian+timekeeping+in+Drosophila.">Molecular genetic analysis of circadian timekeeping in Drosophila.</a> Adv Genet. 74, 141-173 (2011).</li><li>Yoshii, T., Rieger, D., Helfrich-Forster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+clocks+in+the+brain:+an+update+of+the+morning+and+evening+oscillator+model+in+Drosophila.">Two clocks in the brain: an update of the morning and evening oscillator model in Drosophila.</a> Prog Brain Res. 199, 59-82 (2012).</li><li>Malpel, S., Klarsfeld, A., Rouyer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+synchronization+and+rhythmicity+in+larval+photoperception-defective+mutants+of+Drosophila.">Circadian synchronization and rhythmicity in larval photoperception-defective mutants of Drosophila.</a> J Biol Rhythms. 19 (1), 10-21 (2004).</li><li>Mazzoni, E. O., Desplan, C., Blau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+pacemaker+neurons+transmit+and+modulate+visual+information+to+control+a+rapid+behavioral+response.">Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response.</a> Neuron. 45 (2), 293-300 (2005).</li><li>Sabado, V., Vienne, L., Nunes, J. M., Rosbash, M., Nagoshi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+circadian+imaging+reveals+a+PDF-dependent+transcriptional+regulation+of+the+Drosophila+molecular+clock.">Fluorescence circadian imaging reveals a PDF-dependent transcriptional regulation of the Drosophila molecular clock.</a> Sci Rep. 7, 41560 (2017).</li><li>Hyun, 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+GPCR+Han+is+a+receptor+for+the+circadian+clock+neuropeptide+PDF.">Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF.</a> Neuron. 48 (2), 267-278 (2005).</li><li>Lear, B. 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=A+G+protein-coupled+receptor,+groom-of-PDF,+is+required+for+PDF+neuron+action+in+circadian+behavior.">A G protein-coupled receptor, groom-of-PDF, is required for PDF neuron action in circadian behavior.</a> Neuron. 48 (2), 221-227 (2005).</li><li>Mertens, I., 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=PDF+receptor+signaling+in+Drosophila+contributes+to+both+circadian+and+geotactic+behaviors.">PDF receptor signaling in Drosophila contributes to both circadian and geotactic behaviors.</a> Neuron. 48 (2), 213-219 (2005).</li><li>Ayaz, 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=Axonal+injury+and+regeneration+in+the+adult+brain+of+Drosophila.">Axonal injury and regeneration in the adult brain of Drosophila.</a> J Neurosci. 28 (23), 6010-6021 (2008).</li><li>Prithviraj, R., Trunova, S., Giniger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culturing+of+whole,+developing+Drosophila+brains.">Ex vivo culturing of whole, developing Drosophila brains.</a> J Vis Exp. (65), (2012).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Mol Biol Cell. 16 (11), 5127-5140 (2005).</li><li>Zschatzsch, 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=Regulation+of+branching+dynamics+by+axon-intrinsic+asymmetries+in+Tyrosine+Kinase+Receptor+signaling.">Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling.</a> Elife. 3, e01699 (2014).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuroblast+lineages+within+intact+larval+brains+in+Drosophila.">Live imaging of neuroblast lineages within intact larval brains in Drosophila.</a> Cold Spring Harb Protoc. 2013 (10), 970-977 (2013).</li><li>Januschke, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interphase+microtubule+aster+is+a+determinant+of+asymmetric+division+orientation+in+Drosophila+neuroblasts.">The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.</a> The Journal of Cell Biology. 188 (5), 693-706 (2010).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging.">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> J Vis Exp. (37), (2010).</li><li>Roberts, 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=Light+evokes+rapid+circadian+network+oscillator+desynchrony+followed+by+gradual+phase+retuning+of+synchrony.">Light evokes rapid circadian network oscillator desynchrony followed by gradual phase retuning of synchrony.</a> Curr Biol. 25 (7), 858-867 (2015).</li><li>Sehadova, 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=Temperature+entrainment+of+Drosophila's+circadian+clock+involves+the+gene+nocte+and+signaling+from+peripheral+sensory+tissues+to+the+brain.">Temperature entrainment of Drosophila's circadian clock involves the gene nocte and signaling from peripheral sensory tissues to the brain.</a> Neuron. 64 (2), 251-266 (2009).</li><li>Sellix, M. T., Currie, J., Menaker, M., Wijnen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence/luminescence+circadian+imaging+of+complex+tissues+at+single-cell+resolution.">Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution.</a> J Biol Rhythms. 25 (3), 228-232 (2010).</li><li>Liang, X., Holy, T. E., Taghert, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+Drosophila+circadian+pacemakers+display+nonsynchronous+Ca(2)(+)+rhythms+in+vivo.">Synchronous Drosophila circadian pacemakers display nonsynchronous Ca(2)(+) rhythms in vivo.</a> Science. 351 (6276), 976-981 (2016).</li><li>Liang, X., Holy, T. E., Taghert, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Series+of+Suppressive+Signals+within+the+Drosophila+Circadian+Neural+Circuit+Generates+Sequential+Daily+Outputs.">A Series of Suppressive Signals within the Drosophila Circadian Neural Circuit Generates Sequential Daily Outputs.</a> Neuron. , (2017).</li><li>Kuppers-Munther, B., 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=A+new+culturing+strategy+optimises+Drosophila+primary+cell+cultures+for+structural+and+functional+analyses.">A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses.</a> Dev Biol. 269 (2), 459-478 (2004).</li><li>Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Larval+Drosophila+Eye-Antennal+Discs+in+Vitro.">Differentiation of Larval Drosophila Eye-Antennal Discs in Vitro.</a> J Exp Zool. 156, 91-103 (1964).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J Neurophysiol. 94 (1), 491-500 (2005).</li><li>Hafer, N., Schedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+larval+CNS+in+Drosophila+melanogaster.">Dissection of larval CNS in Drosophila melanogaster.</a> J Vis Exp. (1), e85 (2006).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Rabinovich, D., Mayseless, O., Schuldiner, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+ex+vivo+culturing+of+Drosophila+brain+as+a+method+to+live+image+pupal+brains:+insights+into+the+cellular+mechanisms+of+neuronal+remodeling.">Long term ex vivo culturing of Drosophila brain as a method to live image pupal brains: insights into the cellular mechanisms of neuronal remodeling.</a> Front Cell Neurosci. 9, 327 (2015).</li><li>Forer, A., Pickett-Heaps, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+clots+keep+non-adhering+living+cells+in+place+on+glass+for+perfusion+or+fixation.">Fibrin clots keep non-adhering living cells in place on glass for perfusion or fixation.</a> Cell Biol Int. 29 (9), 721-730 (2005).</li><li>Forer, A., Pickett-Heaps, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochalasin+D+and+latrunculin+affect+chromosome+behaviour+during+meiosis+in+crane-fly+spermatocytes.">Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes.</a> Chromosome Res. 6 (7), 533-549 (1998).</li><li>Lukinavi&#269;ius, G., 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=SiR-Hoechst+is+a+far-red+DNA+stain+for+live-cell+nanoscopy.">SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.</a> Nature Communications. 6, 8497 (2015).</li><li>Medioni, C., Ephrussi, A., Besse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+axonal+transport+in+Drosophila+pupal+brain+explants.">Live imaging of axonal transport in Drosophila pupal brain explants.</a> Nat Protoc. 10 (4), 574-584 (2015).</li><li>Enoki, R., Ono, D., Hasan, M. T., Honma, S., Honma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+resolution+fluorescence+imaging+of+circadian+rhythms+detected+with+a+Nipkow+spinning+disk+confocal+system.">Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system.</a> J Neurosci Methods. 207 (1), 72-79 (2012).</li><li>Ozel, M. N., Langen, M., Hassan, B. A., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodial+dynamics+and+growth+cone+stabilization+in+Drosophila+visual+circuit+development.">Filopodial dynamics and growth cone stabilization in Drosophila visual circuit development.</a> Elife. 4, (2015).</li><li>Yao, Z., Macara, A. M., Lelito, K. R., Minosyan, T. Y., Shafer, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+functional+neuronal+connectivity+in+the+Drosophila+brain.">Analysis of functional neuronal connectivity in the Drosophila brain.</a> J Neurophysiol. 108 (2), 684-696 (2012).</li></ol>

الكتاب ليس لها علاقة بالكشف عن.

هنا وصفت لنا طريقة طويلة الأجل مجهرية الوقت الفاصل بين الأسفار من العقول اليرقات المستزرعة. يتوقف نجاح هذا النوع من التجارب على عدة عوامل، مثل صحة الثقافة، وطريقة لتجميد explant الدماغ، وكثافة الأسفار ونسبة الإشارة إلى الضوضاء مراسل، الزماني والمكاني القرار، و الوصول إلى explant. وهذه العوامل ?...

الساعات الإيقاعية تساعد الكائنات الحية على التكيف مع التغييرات البيئية الدورية الناتجة عن دوران الأرض ح 24. عادة وراء حلقات متداخلة ردود الفعل النسخي متعدية الآلية الجزيئية للساعات الإيقاعية نطاق الأنواع1. تتألف الدارة تنظيم ضربات القلب الإيقاعية المحتوية على مدار الساعة الخلايا العصبية يدمج معلومات الوقت من اليوم تنقلها العظة البيئية، مثل الضوء/الظلام (دينار) ودورات درجة الحرارة، تنسق الإيقاعات من عدد كبير صحيفة الفسيولوجية و العمليات السلوكية2،3. تنسيق إيقاعات الجزيئية مع الخلايا العصبية المدخلات والمخرجات من الأهمية بمكان لتشغيل الدائرة الإيقاعية لكن يظل مفهوما جزئيا فقط.
في المورفولوجية، في صميم عقارب الساعة الجزيئية، ينشط هيتيروديمير ساعة/دورة (CLK/الفتوة) النسخ من الفترة (الواحد) و زمان (تيم). تشكل مجمعا في وتيم وإدخال النواة، حيث أنها تكبح النشاط الترانسكربتي CLK/الفتوة، ونتيجة لذلك النسخ الخاصة بهم. أنظمة بوستترانسكريبشونال وبوستترانسلاشونال يسبب التأخير بين النسخ CLK/الفتوة-التوسط والقمع من جانب في/تيم، ضمان توليد سيركا ذبذبات الجزيئية 24-ح1،،من34 . حوالي 150 من الخلايا العصبية التي تحتوي على النموذج الجزيئي الساعات هذه دائرة للتحكم في سلوك الكبار الإيقاعية الذباب5. كثير أبسط لم تعمل بكامل طاقتها الإيقاعية دارة تتكون من 3 مجموعات من الخلايا العصبية على مدار الساعة-5 البطني الجانبي الخلايا العصبية (اللازم؛ 4 PDF-الإيجابي اللازم وواحد سالب PDF لنف، انظر أدناه)، 1s العصبية الظهرية 2 (DN1s) و 2s العصبية الظهرية 2 (DN2s)-موجود في اليرقات الدماغ6،7.
الدائرة الإيقاعية اليرقات بسيط يوفر نموذجا ممتازا لدراسة التفاعلات بين الاتصالات بين الخلايا العصبية والبرتقالة الجزيئية. استخدام مراسلنا الفلورسنت المطورة حديثا في--TDT، الذي يحاكي المستويات كل البروتين وموقعها سوبسيلولار، وسعينا إلى وصف ديناميات الجزيئية البرتقالة في مجموعات مختلفة على مدار الساعة العصبية فرعية في الدائرة الإيقاعية اليرقات 8. وعلاوة على ذلك، مع العلم بالدور الرئيسي لمعامل تفريق الصباغ neuropeptide (PDF) التي تنتجها اللازم 4 في تنظيم إيقاعات circadian في ال10،الخلايا العصبية المستوى9،11، أردنا النظر المباشر أثر PDF على الساعات الجزيئية. وتحقيقا لهذه الغاية، قمنا بتطوير طريقة لرصد الجينات الإيقاعية إيقاعات في الدماغ اليرقات explant على مدى عدة أيام من الوقت الفاصل بين الفحص المجهري [كنفوكل]. وكان أيضا تكييف البروتوكول لفحوصات الدوائية لاختبار تأثير PDF أو غيرها من المركبات على المستوى في--TDT. وهكذا، وتكييف يتألف من إتاحة ثقافة explant الدماغ لتطبيق المخدرات وزيادة دقة الزمانية والتصوير لمدة أقصر.
السابقين فيفو ثقافة العقول المورفولوجية لمراحل تنموية مختلفة قد حددت سابقا12،13،،من1415،16،17 ،18. بينما استخدمت هذه البروتوكولات لتصوير مختلف الظواهر البيولوجية، البعض منهم غير متوافقة مع التصوير في القرار خلية واحدة أو لا تدعم الثقافة لأكثر من عدة ساعات. تشمل أساليب بديلة لإجراء تصوير حية طويلة الأجل من الخلايا العصبية الإيقاعية في المورفولوجية تصوير الإضاءة الحيوية الجزيئية إيقاعات19،،من2021 وتصوير الأسفار مؤشر الكالسيوم مع الورقة الخفيفة الميكروسكوب22،23. على الرغم من أن التصوير بالإضاءة الحيوية يمكن تحقيق أعلى الأزمنة والفحص المجهري الورقة الخفيفة يمكن أن تكون قابلة للتكيف للتصوير في فيفو ، أنها تقتصر على القرار المكانية وتتطلب أنظمة المجهر المتخصصة.
لتلائم الطريقة الموصوفة هنا هو تصور إشارات مضيئة في ثقافة الجامعة الدماغ في القرار خلية واحدة على مدى عدة أيام. هذه طريقة سهلة ومرنة يمكن تكييفها وفقا للصورة مثقف الكبار يطير العقول والتجارب الدوائية لدراسة العديد من مشاكل مختلفة في بيولوجيا الأعصاب المورفولوجية .

<table><tbody><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td>I am not sure they are exactly the same ones we have in the lab. I chose &quot;suitable for insect cell culture&quot; whenever available</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>D-(+) Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1000</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I0516-5ML</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>BIS-TRIS</td><td>Sigma-Aldrich</td><td>B4429</td><td></td></tr><tr><td>L-(&amp;minus;)-Malic acid</td><td>Sigma-Aldrich</td><td>M7397</td><td></td></tr><tr><td>D-(+)-Trehalose dihydrate</td><td>Sigma-Aldrich</td><td>T0167</td><td></td></tr><tr><td>Succinic acid</td><td>Sigma-Aldrich</td><td>S9512</td><td></td></tr><tr><td>Fumaric acid</td><td>Sigma-Aldrich</td><td>F8509</td><td></td></tr><tr><td>&amp;alpha;-Ketoglutaric acid</td><td>Sigma-Aldrich</td><td>K1128</td><td></td></tr><tr><td>Non-heat-inactivated, Foetal Calf Serum (FCS) Mycoplasma and Virus screened</td><td>BioConcept Ltd. Amimed</td><td>2-01F30-I</td><td></td></tr><tr><td>HEPES-KOH, pH 7.4</td><td>E&amp;amp;K Scientific Products</td><td>EK-654011</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5011</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Calbiochem (Merck)</td><td>341573-1GM</td><td>CAUTION: Harmful by inhalation, in contact with skin and if swallowed. Manipulate under laminar flow</td></tr><tr><td>Thrombin from bovine plasma</td><td>Sigma-Aldrich</td><td>T9549</td><td>CAUTION: Health Hazard, use gloves</td></tr><tr><td>PDF, NH&lt;sub&gt;2&lt;/sub&gt;-NSELINSLLSLPKNMNDA-OH</td><td>Chi Scientific</td><td>custom made</td><td></td></tr><tr><td>Vaccum grease</td><td>Sigma-Aldrich</td><td>18405</td><td></td></tr><tr><td>35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated</td><td>MatTek</td><td>P35G-1.5-20-C</td><td></td></tr><tr><td>Corning Falcon Easy-Grip Tissue Culture Dishes</td><td>fisherscientific</td><td>08-772A</td><td></td></tr><tr><td>Sterile 500 mL Steritop-GP 33 mm threaded bottle top filter, 0.22 &amp;mu;m</td><td>Millipore</td><td>SCGPS05RE</td><td></td></tr><tr><td>Polytetrafluoroethylene (PTFE) film</td><td>Dupont</td><td>200A Teflon FEP Film</td><td></td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45&amp;nbsp;&amp;micro;m, PVDF, 33&amp;nbsp;mm, gamma sterilized</td><td>Millipore</td><td>SLHV033RS</td><td></td></tr><tr><td>Millex-GV Syringe Filter Unit, 0.22&amp;nbsp;&amp;micro;m, PVDF, 33&amp;nbsp;mm, gamma sterilized</td><td>Millipore</td><td>SLGV033RS</td><td></td></tr><tr><td>Three-well glass dissection dish</td><td>Any company</td><td></td><td></td></tr><tr><td>Fine forceps, size 5, Dumont</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Tandem scanner inverted TCS SP5 confocal microscope, with resonant scanner and HyD photo-multiplier detectors</td><td>Leica microsystem CMS GmbH</td><td></td><td></td></tr><tr><td>Temperature control chamber</td><td>Life Imaging Services</td><td>The CUBE &amp;amp; BOX temperature control system, custom designed</td><td></td></tr><tr><td>Stage-top humidity controller</td><td>Life Imaging Services</td><td>custom made</td><td></td></tr><tr><td>Water Immersion Micro Dispenser: dispenser, extended micro-pump MP6 series and Autoimmersion Objective Controller software</td><td>Leica microsystem CMS GmbH</td><td></td><td></td></tr><tr><td>SUM-stack creation and 3D correction drift plugin</td><td>ImageJ software</td><td></td><td></td></tr><tr><td>10x iterative deconvolution</td><td>AutoQuant and Imaris software</td><td></td><td></td></tr></tbody></table>

single-cell resolution fluorescence live imaging of drosophila circadian clocks in larval brain culture

دائرة تنظيم ضربات القلب الإيقاعية ينظم الإيقاعي النواتج السلوكية والفسيولوجية منسقة مع إشارات بيئية، مثل دورات اليوم/ليلة. ساعة جزيئية داخل كل تنظيم ضربات القلب العصبية يولد إيقاعات circadian في التعبير الجيني، التي تكمن وراء الإيقاعي وظائف الخلايا العصبية اللازمة لعمل الدائرة. التحقيق في خصائص التذبذب الجزيئية الفردية في مختلف أقسام فرعية لتنظيم ضربات القلب من الخلايا العصبية وتفاعلها مع الإشارات العصبية غلة فهم أفضل لتنظيم ضربات القلب الإيقاعية الدائرة. نقدم هنا، نهجاً الوقت الفاصل بين الفحص المجهري نيون لرصد البرتقالة الجزيئية في الخلايا العصبية على مدار الساعة لمثقف المورفولوجية اليرقات الدماغ. يسمح هذا الأسلوب تسجيل عدة أيام من إيقاعات الصحفيين الإيقاعية الفلورسنت وراثيا مرمزة في القرار خلية واحدة. يمكن أن يكون هذا الإعداد جنبا إلى جنب مع المعالجات الدوائية لتحليل استجابة في الوقت الحقيقي على مدار الساعة الجزيئية للمركبات المختلفة عن كثب. خارج إيقاعات circadian، يقدم هذا الأسلوب متعدد الأغراض في تركيبة مع التقنيات الجينية المورفولوجية القوية إمكانية دراسة العمليات العصبية أو الجزيئية المختلفة في أنسجة الدماغ حية.

هنا، نحن إظهار النتائج الممثل التسجيل طويلة الأجل لمراسل الفلورسنت الإيقاعية في السابقين فيفو ثقافة الدماغ اليرقات، ويعيش التصوير نتائج تطبيق حمام PDF في التعبير مراسل.
عدم تجول اليرقات L3 التعبير عن ساعة جزيئية مراسل في--TDT و UAS-mCD8::YFP ?...

Watch this Scientific Journal Video about Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture at JoVE.com

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

والهدف من هذا البروتوكول إنشاء ثقافة الدماغ اليرقات السابقين فيفو المورفولوجية الأمثل لرصد إيقاعات الجزيئية الإيقاعية مع المدى الطويل fluorescence الوقت الفاصل بين التصوير. وتناقش أيضا تطبيق هذا الأسلوب لفحوصات الدوائية.

Fluorescence القرار خلية واحدة تعيش تصوير ساعات الإيقاعية المورفولوجية في الدماغ اليرقات الثقافة

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

single-cell-resolution-fluorescence-live-imaging-drosophila-circadian

Research

JoVE Journal

Neuroscience

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

7.5K Views.  University of Geneva. والهدف من هذا البروتوكول إنشاء ثقافة الدماغ اليرقات السابقين فيفو المورفولوجية الأمثل لرصد إيقاعات الجزيئية الإيقاعية مع المدى الطويل fluorescence الوقت الفاصل بين التصوير. وتناقش أيضا تطبيق هذا الأسلوب لفحوصات الدوائية.

Video: Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

يعيش التصوير لل<em&gt; ذبابة الفاكهة</em&gt; العذراء العين

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Developmental Biology

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

استخدام خلايا Drosophila S2 للتصوير الحي لقسم الخلايا

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Genetics

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

التصوير المباشر ل<em&gt; ذبابة الفاكهة</em&gt; اليرقات Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

إعداد وتطوير الكبار ذبابة الفاكهة الأمخاخ والشبكية للتصوير لايف

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

في التصوير من يرقات ذبابة الفاكهة فيفو سليمة الفرعية الخلوية في القرار

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

تصوير نبتات الدماغ الحية من ذبابة الفاكهة الميلانوجاستر يرقات Instar الثالثة

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

تسليط الضوء على المؤلف: التحقيق في ديناميكيات انقسام الخلايا غير المتماثلة: بروتوكول للتصوير الحي لنباتات دماغ يرقات ذبابة الفاكهة 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

تصوير الخلايا الحية لذبابة الفاكهة الميلانوجاستر أدمغة اليرقات الثالثة

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

السابقين فيفو تصوير الكالسيوم لتصور استجابات الدماغ للإشارات الغدد الصماء في المورفولوجية

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Fluorescence القرار خلية واحدة تعيش تصوير ساعات الإيقاعية المورفولوجية في الدماغ اليرقات الثقافة

Summary

Explore More Videos

Chapters in this video

يعيش التصوير لل

استخدام خلايا Drosophila S2 للتصوير الحي لقسم الخلايا

التصوير المباشر ل

إعداد وتطوير الكبار ذبابة الفاكهة الأمخاخ والشبكية للتصوير لايف

في التصوير من يرقات ذبابة الفاكهة فيفو سليمة الفرعية الخلوية في القرار

تصوير الخلايا الحية لذبابة الفاكهة الميلانوجاستر أدمغة اليرقات الثالثة

تصوير الخلايا الحية لذبابة الفاكهة الميلانوجاستر أدمغة اليرقات الثالثة

تصوير الخلايا الحية لذبابة الفاكهة الميلانوجاستر أدمغة اليرقات الثالثة

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

السابقين فيفو تصوير الكالسيوم لتصور استجابات الدماغ للإشارات الغدد الصماء في المورفولوجية