نود أن نعترف أعضاء مختبر ماري ومختبر Godenschwege، في Yonezawa ألين وجه الخصوص، للتعليق عليها والمساعدة في هذا البروتوكول. وقد تم تمويل هذا العمل من قبل المعهد الوطني للاضطرابات العصبية والسكتة الدماغية R21NS06637 إلى منح وزير الخارجية وTAG، وكان يمول من قبل عدد AB الوطنية للعلوم جائزة مؤسسة 082925، URM: البيولوجيا التكاملية للباحثين في المستقبل.

<ol>
	<li>Koehn, F. E., Carter, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+role+of+natural+products+in+drug+discovery.">The evolving role of natural products in drug discovery.</a> Nat. Rev. Drug Discov. 4, 206-220 (2005).</li><li>Miljanich, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ziconotide:+neuronal+calcium+channel+blocker+for+treating+severe+chronic+pain.">Ziconotide: neuronal calcium channel blocker for treating severe chronic pain.</a> Curr. Med. Chem. 11, 3029-3040 (2004).</li><li>Layer, R. T., Wagstaff, J. D., White, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conantokins:+peptide+antagonists+of+NMDA+receptors.">Conantokins: peptide antagonists of NMDA receptors.</a> Curr. Med. Chem. 11, 3073-3084 (2004).</li><li>Lewis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conotoxins+as+selective+inhibitors+of+neuronal+ion+channels,+receptors+and+transporters.">Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters.</a> IUBMB Life. 56, 89-93 (2004).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Fayyazuddin, A., Zaheer, M. A., Hiesinger, P. R., Bellen, H. 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+nicotinic+acetylcholine+receptor+Dalpha7+is+required+for+an+escape+behavior+in+Drosophila.">The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila.</a> PLoS biology. 4, e63 (2006).</li><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-glutamate+as+an+excitatory+transmitter+at+the+Drosophila+larval+neuromuscular+junction.">L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction.</a> The Journal of physiology. 262, 215-236 (1976).</li><li>Usherwood, P. N., Machili, P., Leaf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-Glutamate+at+insect+excitatory+nerve-muscle+synapses.">L-Glutamate at insect excitatory nerve-muscle synapses.</a> Nature. 219, 1169-1172 (1968).</li><li>Marrus, S. B., Portman, S. L., Allen, M. J., Moffat, K. G., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+localization+of+glutamate+receptor+subunits+at+the+Drosophila+neuromuscular+junction.">Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 24, 1406-1415 (2004).</li><li>Petersen, S. A., Fetter, R. D., Noordermeer, J. N., Goodman, C. S., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+glutamate+receptors+in+Drosophila+reveals+a+retrograde+signal+regulating+presynaptic+transmitter+release.">Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release.</a> Neuron. 19, 1237-1248 (1997).</li><li>Qin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+different+subunits+are+essential+for+expressing+the+synaptic+glutamate+receptor+at+neuromuscular+junctions+of+Drosophila.">Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 3209-3218 (2005).</li><li>Schuster, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+an+invertebrate+glutamate+receptor+subunit+expressed+in+Drosophila+muscle.">Molecular cloning of an invertebrate glutamate receptor subunit expressed in Drosophila muscle.</a> Science. 254, 112-114 (1991).</li><li>Tanouye, M. A., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+outputs+of+giant+nerve+fiber+in+Drosophila.">Motor outputs of giant nerve fiber in Drosophila.</a> Journal of. 44, 405-421 (1980).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. (47), e2412 (2011).</li><li>Allen, M. J., Godenschwege, T., Zhang, B., Freeman, M. R., Waddell, 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 Neurobiology. , 215-224 (2010).</li><li>Blagburn, J. M., Alexopoulos, H., Davies, J. A., Bacon, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Null+mutation+in+shaking-B+eliminates+electrical,+but+not+chemical,+synapses+in+the+Drosophila+giant+fiber+system:+a+structural+study.">Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study.</a> J. Comp. Neurol. 404, 449-458 (1999).</li><li>Thomas, J. B., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+altering+synaptic+connectivity+between+identified+neurons+in+Drosophila.">Mutations altering synaptic connectivity between identified neurons in Drosophila.</a> J. Neurosci. 4, 530-538 (1984).</li><li>Baird, D. H., Schalet, A. P., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Passover+locus+in+Drosophila+melanogaster:+complex+complementation+and+different+effects+on+the+giant+fiber+neural+pathway.">The Passover locus in Drosophila melanogaster: complex complementation and different effects on the giant fiber neural pathway.</a> Genetics. 126, 1045-1059 (1990).</li><li>Gorczyca, M., Hall, J. C. <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+a+cholinergic+synapse+in+the+giant+fiber+pathway+of+Drosophila+using+conditional+mutations+of+acetylcholine+synthesis.">Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis.</a> J. Neurogenet. 1, 289-313 (1984).</li><li>Allen, M. J., Murphey, R. 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+chemical+component+of+the+mixed+GF-TTMn+synapse+in+Drosophila+melanogaster+uses+acetylcholine+as+its+neurotransmitter.">The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.</a> The European journal of neuroscience. 26, 439-445 (2007).</li><li>Mejia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+in+vivo+screening+of+toxins+using+the+Drosophila+Giant+Fiber+circuit.">A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit.</a> Toxicon. 56, 1398-1407 (2010).</li><li>Stork, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+function+of+the+blood-brain+barrier+in+Drosophila.">Organization and function of the blood-brain barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

والأحيائي nanoinjection / الكهربية المقدمة هنا يسمح لفحص السريع للمركبات في الجهاز العصبي من ذبابة الفاكهة. هذه هي رواية في تقنية الجسم الحي الذي يتطلب كميات صغيرة من مركب للحصول تأثير على مجموعة متنوعة من الأهداف الجزيئية في دائرة العصبية جيدا تتميز. ويمكن استخد...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> أقطاب الزجاج تسجيل: البورسليكات الزجاج الشعيرات الدموية </td><td> الآلات الدقيقة العالم </td><td> 1B100F-4 </td><td> 1.0mm التطوير التنظيمي، 0.58mm معرف </td></tr><tr><td> منشط </td><td> العشب آلات </td><td> نموذج S48 </td><td></td></tr><tr><td> مكبر الصوت </td><td> الحصول على صكوك شركة </td><td> نموذج 5A </td><td></td></tr><tr><td> الحصول على البيانات البرمجيات: Digidata </td><td> الجزيئية الأجهزة </td><td> نموذج 1440A </td><td></td></tr><tr><td> جمع البيانات والبرمجيات: pCLAMP </td><td> الجزيئية الأجهزة </td><td> الإصدار 10 </td><td></td></tr><tr><td> Stereomicroscope ثإيث من الألياف البصرية خاتم مجهر إضاءة </td><td> AmScope </td><td> SM-4T نموذج HL250-AR </td><td></td></tr><tr><td> تشريح مجالا لتصاعد </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> طائرة ورقية دليل Micromanipulator وإمالة قاعدة </td><td> الآلات الدقيقة العالم </td><td> نموذج # M3301 طائرة ورقية: نموذج # KITE-M3-L </td><td></td></tr><tr><td> ذبابة الفاكهة السوداء البطن الوراثي 10E البرية (البرية سلالة نوع) </td><td> بلومنغتون سهم مركز </td><td> الاسهم # 3892 </td><td></td></tr><tr><td> عمودي ماصة مجتذب </td><td> ديفيد Kopf الآلات </td><td> 700c نموذج </td><td></td></tr><tr><td> بال micropipettes حقن الزجاج: الزجاج البورسليكات الشعيرات الدموية </td><td> الآلات الدقيقة العالم </td><td> كتالوج # 4878 </tد&gt; <td> OD 1.14mm، 0.5mm معرف </td></tr><tr><td> السيليكون النفط </td><td> الصياد </td><td> كتالوج # S159-500 </td><td></td></tr><tr><td> Beveler </td><td> سوتر شركة الصك </td><td> KT براون نوع نموذج # BV-10 </td><td></td></tr><tr><td> Nanoliter2000 </td><td> الآلات الدقيقة العالم </td><td> كتالوج # B203XVY </td><td></td></tr><tr><td> أزرق تلوين الطعام </td><td> مكورميك </td><td> N / A </td><td> المكونات: الماء، بروبيلين جلايكول، FD &amp; C الأزرق 1، و 0.1٪ بروبيل بارابين (حافظة). </td></tr><tr><td> Methyllycaconitine سترات (MLA) </td><td> العلوم البيولوجية Tocris </td><td> كتالوج # 1029 </td><td></td></tr><tr><td> الشمع العصي البلاستيكية </td><td> هجنيك شركة (أكرون أوهايو الولايات المتحدة الأمريكية) </td><td></td><td></td></tr></tboدى&gt; </table>

paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the drosophila melanogaster giant fiber system

ويمكن استخدام مركبات الكشف عن النشاط في الجسم الحي، كخطوة أولى لتحديد المرشحين التي يمكن وضعها في 1،2 كلاء الدوائية. وضعنا رواية nanoinjection / الكهربية الفحص الذي يسمح للكشف عن الآثار تغييري من المركبات النشطة بيولوجيا على وظيفة الدائرة العصبية التي تتوسط استجابة الهروب في ذبابة الفاكهة الدروسوفيلا 3،4. لدينا في فحص الجسم الحي، والذي يستخدم الألياف العملاق ذبابة الفاكهة نظام (GFS، الشكل 1) يسمح للفحص من أنواع مختلفة من المركبات، مثل الجزيئات الصغيرة أو الببتيد، ويتطلب سوى كميات ضئيلة للحصول تأثير. بالإضافة إلى ذلك، GFS ذبابة الفاكهة يقدم مجموعة كبيرة ومتنوعة من الأهداف الجزيئية المحتملة على الخلايا العصبية أو العضلات. الألياف العملاق (GFS) المشبك كهربائيا (GAP المفارق)، فضلا عن كيميائيا (الكوليني) في الصعود إلى عصبون Synapsing الطرفية (PSI) وTrochanteral Tergo الخلايا العصبية العضلية (TTMn) 5 </سوب&gt;. هذه المبادرة إلى الاتصال (الخلايا العصبية الظهرية العضلات الطولية) DLMn يعتمد على Dα7 acetylcholine المستقبلات النيكوتينية (nAChRs) 6. وأخيرا، فإن الوصلات العصبية والعضلية (NMJ) من TTMn وDLMn ومع القفزة (الايراد) وعضلات الطيران (DLM) هي glutamatergic 7-12. هنا، نحن لشرح كيفية حقن كميات نانولتر من مركب، في حين الحصول على تسجيلات داخل الخلايا الكهربية من نظام الألياف العملاق 13 وكيفية رصد آثار مجمع على وظيفة من هذه الدائرة. نعرض خصوصية مقايسة مع سترات methyllycaconitine (MLA)، وهو خصم nAChR، والذي يعطل هذه المبادرة إلى DLMn اتصال ولكن ليس إلى GF TTMn اتصال أو وظيفة NMJ في الوثب أو عضلات الطيران. قبل البدء في هذا الفيديو من الأهمية بمكان أن تشاهد بعناية وتصبح مألوفة مع الفيديو [جوف] بعنوان &quot;تسجيلات الكهربية من مسار الألياف العملاق من melan د.والمقصود ogaster &quot;من أوغسطين وآخرون 7، كما قدم شريط الفيديو هنا كما توسع لهذه التقنية القائمة. هنا نستخدم طريقة التسجيلات الكهربية والتركيز في التفاصيل فقط على إضافة nanoinjections تقرن وتقنية الرصد.

Watch this Scientific Journal Video about Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System at JoVE.com

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

سريع<em&gt; في الجسم الحي</em&gt; فحص لاختبار المركبات neuromodulatory باستخدام نظام الألياف العملاق (GFS) من<em&gt; ذبابة الفاكهة السوداء البطن</emيوصف&gt;. يمكن Nanoinjections في رأس الحيوان جنبا إلى جنب مع التسجيلات الكهربية من GFS كشف النشاط الحيوي لمركبات على الخلايا العصبية أو العضلات.

تقرن Nanoinjection والفحص الكهربية للكشف عن النشاط الحيوي للمركبات باستخدام ذبابة الفاكهة السوداء البطن نظام الألياف العملاق

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

paired-nanoinjection-electrophysiology-assay-to-screen-for

Research

JoVE Journal

Neuroscience

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

13.1K Views.  Florida Atlantic University. سريع في الجسم الحي فحص لاختبار المركبات neuromodulatory باستخدام نظام الألياف العملاق (GFS) من ذبابة الفاكهة السوداء البطن. يمكن Nanoinjections في رأس الحيوان جنبا إلى جنب مع التسجيلات الكهربية من GFS كشف النشاط الحيوي لمركبات على الخلايا العصبية أو العضلات....

Video: Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

تسجيلات الكهربية من الألياف المسار عملاق د. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

كله من جبل إعداد الكبار ذبابة الفاكهة الحبل الاعصاب الاماميه للحقن صبغ الألياف العملاق

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

وضع العلامات واحدة من الخلايا في الجهاز العصبي المركزي<em&gt; ذبابة الفاكهة الدروسوفيلا</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

تحديد العفوي الحركي آخر في<em&gt; ذبابة الفاكهة السوداء البطن</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

الفحص مباشرة للالكمي لتجنب الاجتماعي في<em&gt; ذبابة الفاكهة السوداء</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

عالية الدقة الكمي للسلوك رائحة الموجهة في<em&gt; ذبابة الفاكهة السوداء البطن</em&gt; استخدام<em&gt; Flywalk</em&gt; بارادايم

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

النسخ في والسلوكية الفحص لMechanosensation من MARCM المستندة<em&gt; ذبابة الفاكهة السوداء البطن</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

عالية الإنتاجية الفحص لفحص تفضيلات بيضة زرع الفردي<em&gt; ذبابة الفاكهة</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

طريقة بسيطة لقياس التغيرات في السلوك المكافأة التماس طريق<em&gt; ذبابة الفاكهة</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

الشعيرات الدموية المغذية الفحص يقيس كمية الطعام في<em&gt; ذبابة الفاكهة</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...