وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح NS - 38849 - 076160 وDK ، ومؤسسة أبحاث السكري الأحداث مركز لدراسة مضاعفات في مرض السكري ، وبرنامج للبحوث الأعصاب واكتشاف وألفريد أ. توبمان معهد البحوث الطبية في جامعة ميتشيغان. استخدم هذا العمل المورفولوجيا وصورة كور تحليل ميشيغان مركز الأبحاث والتدريب للسكري ، بتمويل من المعاهد الوطنية للصحة منح 5P60 DK - 20572 من المعهد الوطني للسكري وأمراض الجهاز الهضمي والكلى. المؤلفان بالشكر سكوت كلارك T. من المسابر الجزيئية / Invitrogen لنصائحه القيمة في مختلف انقر EDU - IT مجموعات التوسيم والتبرع السخي من الكواشف لدعم التطوير الأولي للتقنية تضخيم ايدو.

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J., Bai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Number+matters:+control+of+mammalian+mitochondrial+DNA+copy+number.">Number matters: control of mammalian mitochondrial DNA copy number.</a> J. Genet. Genomics. 36 (3), 125-131 (2009).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc Natl Acad Sci U.S.A. 105, 2415-2420 (2008).</li><li>Buck, S. B., Bradford, J., Gee, K. R., Agnew, B. J., Clarke, S. T., Salic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+S-phase+cell+cycle+progression+using+5-ethynyl-2'-deoxyuridine+incorporation+with+click+chemistry,+an+alternative+to+using+5-bromo-2'-deoxyuridine+antibodies.">Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.</a> Biotechniques. 44, 927-929 (2008).</li><li>Yu, Y., Arora, A., Min, W., Roifman, C. M., Grunebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EdU+incorporation+is+an+alternative+non-radioactive+assay+to+[(3)H]thymidine+uptake+for+in+vitro+measurement+of+mice+T-cell+proliferations.">EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations.</a> J Immunol Methods. 350 (1-2), 29-35 (2009).</li><li>Heusden, J. V. a. n., Jong, P. d. e., Ramaekers, F., Bruwiere, H., Borgers, M., Smets, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein-labeled+tyramide+strongly+enhances+the+detection+of+low+bromodeoxyuridine+incorporation+levels.">Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels.</a> J. Histochem. Cytochem. 45 (2), 315-319 (1997).</li><li>Cappella, P., Gasparri, F., Pulici, M., Moll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+based+on+click+chemistry,+which+overcomes+limitations+of+cell+cycle+analysis+by+classical+determination+of+BrdU+incorporation,+allowing+multiplex+antibody+staining.">A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining.</a> Cytometry A. 73, 626-636 (2008).</li><li>Kaiser, C. L., Kamien, A. J., Shah, P. A., Chapman, B. J., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Ethynyl-2'-deoxyuridine+labeling+detects+proliferating+cells+in+the+regenerating+avian+cochlea.">5-Ethynyl-2'-deoxyuridine labeling detects proliferating cells in the regenerating avian cochlea.</a> Laryngoscope. 119, 1770-1775 (2009).</li><li>Lentz, S. I., Edwards, J. L., Backus, C., McLean, L. L., Haines, K. M., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+(mtDNA)+Biogenesis:+Visualization+and+Duel+Incorporation+of+BrdU+and+EdU+Into+Newly+Synthesized+mtDNA+In.">Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In.</a> Vitro. J Histochem Cytochem. 58 (2), 207-218 (2010).</li><li>Edwards, J. L., Quattrini, A., Lentz, S. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+mitochondrial+DNA+occurs+throughout+the+mitochondria+of+cultured+human+cells.">Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells.</a> Exp. Cell. Res. 289, 133-142 (2003).</li><li>Amiri, M., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+biogenesis+in+the+axons+of+vertebrate+peripheral+neurons.">Mitochondrial biogenesis in the axons of vertebrate peripheral neurons.</a> Dev. Neurobiol. 68, 1348-1361 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

طورنا مقايسة الحساسة لتسمية و mtDNA توليفها حديثا في خلايا فردية باستخدام التضخيم إشارة tyramide من ايدو. كانت واحدة من أكبر القضايا خلال الاستفادة المثلى من هذا البروتوكول نتائج غير متناسقة بين coverslips. تم إجراء تغييرات على عدة بروتوكولات Invitrogen لتجنب الخلط أحجام صغيرة على coverslips الفردية وجعل الحلول الرئيسية لاستخدامها لكافة coverslips. وميكرولتر 75-80 المستخدمة لكل 12 مم ساترة زجاجية دائرية هو حجم مثالي لتغطية كامل مساحة السطح في الوقت الذي توفر حلا كافيا لاعطاء نتائج متسقة بين العينات. وكانت الأمثل مرات الحضانة وتركيزات كاشف لكنها قد تكون رؤية تحسينات مع تعديلات عليها. ويهدف البروتوكول لتشغيل كل عملية واحدة. ومع ذلك ، فقد تكررت بعض الخطوات مع حلول جديدة لاستعادة العينات التي فشلت بعد المحاولة الأولى. على وجه الخصوص ، وقد تكررت ردود الفعل فوق الخطوات ايدو (3،4-3،6) من دون زيادة كبيرة في مضان الخلفية. المناهضة للحضانة ولاية أوريغون الضد الأخضر HRP (4،1-4،3) والتضخيم tyramide (4،4-4،5) الخطوات التي تكررت أيضا ، ولكن في أكثر الأحيان. النتائج في إشارة إلى نسبة الضوضاء الفقراء بسبب زيادة مضان الخلفية الكواشف الأكثر حساسية هي الاحتياطي انقر EDU - IT المضافة (G مكون) والمضادة للأرنب أوريغون الأخضر HRP الضد (العنصر الأول) من عدة انقر EDU - IT Microplate الفحص. الاحتياطي انقر ايدو - IT المضافة يتحول تدريجيا مع مرور الزمن الأصفر عند تخزينها في 4 درجات مئوية وبين 6-12 شهرا يظلم كثيرا. تخزين اضغط المضافة لتقنية المعلومات في مخزن ايدو -20 درجة مئوية وسوف تخفف من هذه المسألة. ووضع العلامات والخطوات التضخيم tyramide تعمل بشكل أفضل عندما يتم استخدام المضادة للأرنب أوريغون الأخضر HRP الأضداد في غضون 4-6 أشهر من إعادة تشكيل في 2 MilliQ O. درهم وسم خفيف من ايدو في و mtDNA يسمح للمقارنات مباشرة مع علامات إضافية الخلوية 11/09 ويعزز من جدوى هذا الأسلوب أكثر من النظير ثيميدين أخرى ، مثل 5 برومو deoxyuridine - 2 (BrdU) ، الأمر الذي يتطلب معاملة قاسية لاسترداد حاتمة لها داخل الحمض النووي. مختبرنا 11-12 13-15 وغيرها قد استخدمت بنجاح لتسمية BrdU و mtDNA. تقنية وضع العلامات BrdU مشابهة لتلك المستخدمة في ايدو ولكن بعض الاختلافات الهامة (الشكل 5). بعد الخطوة permeabilization المذكورة أعلاه (3،1-3،2) ، يتم استرداد حاتمة BrdU مع خطوة تمسخ (2 N حمض الهيدروكلوريك لمدة 30 دقيقة عند 37 درجة مئوية تليها ثلاث غسلات في برنامج تلفزيوني 1X). هو المسمى ثم BrdU مع الأجسام المضادة ضد BrdU الابتدائية (مخفف ناقل 01:50 مختبرات في محلول 1 ٪ من حجب عدة TSA وحضنت بين عشية وضحاها في 4 درجات مئوية). وهناك حاجة إلى خطوة الضد الثانوية لتضخيم إشارة BrdU (الماعز المضادة للماوس على مترافق مفتش HRP من مجموعات TSA # # 2 و 5 و 1:100 المخفف في محلول 1 ٪ الحجب والمحتضنة لمدة 45 دقيقة في درجة حرارة الغرفة) قبل الخطوات TSA (4،3-4،5 أعلاه). بعد النظير ثيميدين اثنين ، وايدو BrdU ، لو mtDNA التسمية هو مفيد لإجراء التجارب التسمية المزدوجة حيث يمكن تسمية و mtDNA بالتسلسل في النبض وضع العلامات نماذج 11. مختبرنا يستخدم ايدو وBrdU توسيم و mtDNA لدراسة تنظيم الميتوكوندريا نشوء حيوي في سياق الاعتلال العصبي السكري والمضاعفات الشائعة لمرض السكري. وقد استخدمنا بنجاح تضخيم إشارة إلى قياس التغيرات في نشوء حيوي و mtDNA في الخلايا العصبية الفردية 11-12. هذه التقنية سوف تكون مفيدة في تجارب أخرى تهدف إلى استكشاف آليات النسخ و mtDNA ودوران أو لتحديد الأدوية التي تمنع و mtDNA التوليف. وبالإضافة إلى ذلك ، يمكن تطبيق المبادئ الأساسية للإشارات وايدو BrdU تضخيم لدراسات أخرى لقياس تكرار الحمض النووي أو إصلاح.

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<td>12 mm coverslips</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-82</td>
<td>&nbsp;</td>
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<td>245 mm x 245 mm BioAssay Dish</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>431111</td>
<td>&nbsp;</td>
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<td>Parafilm M</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>PM-996</td>
<td>&nbsp;</td>
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<td>paraformaldehye</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6148</td>
<td>&nbsp;</td>
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<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T-8532</td>
<td>&nbsp;</td>
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<td>Phosphate buffered saline 10X solution</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP399</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-711-7M</td>
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<td>Hydrogen peroxide, 30% in water</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP2633</td>
<td>&nbsp;</td>
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<td>Click-iT EdU Microplate Assay Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C10214</td>
<td>&nbsp;</td>
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<td>TSA Kit #12, with HRP&#x2014;goat anti-rabbit IgG and Alexa Fluor 488 tyramide</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T20922</td>
<td>&nbsp;</td>
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<td>TSA Kit #15, with HRP&#x2013;goat anti-rabbit IgG and Alexa Fluor 594 tyramide</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T20925</td>
<td>&nbsp;</td>
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<td>ProLong Gold antifade reagent with DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P-36931</td>
<td>&nbsp;</td>
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<td>Polyclonal rabbit Neurofilament antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>AB1981</td>
<td>&nbsp;</td>
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<td>Mouse monoclonal anti-BrdU antibody</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>VP-B209</td>
<td>&nbsp;</td>
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<td>TSA Kit #2, with HRP&#x2014;goat anti-mouse IgG and Alexa Fluor 488 tyramide</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T20912</td>
<td>&nbsp;</td>
<tr>
<td>TSA Kit #5, with HRP&#x2014;goat anti-mouse IgG and Alexa Fluor 594 tyramide</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T20915</td>
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visualization of mitochondrial dna replication in individual cells by edu signal amplification

الميتوكوندريا هي المنظمين الرئيسيين للطاقة الخلوية ونشوء حيوي الميتوكوندريا هي عنصر أساسي من عناصر تنظيم أعداد الميتوكوندريا في الخلايا السليمة 1-3. نهج واحد لرصد نشوء حيوي الميتوكوندريا هو قياس معدل الحمض النووي (و mtDNA) تكرار 4. طورنا تقنية حساسة للتسمية و mtDNA تصنيعه حديثا في الخلايا الفردية ، من أجل دراسة و mtDNA نشوء حيوي. أسلوب يجمع بين إدماج - 5 - إيثينيل 2&#39; - deoxyuridine (ايدو) 5-7 مع تضخيم إشارة tyramide (TSA) 8 و mtDNA بروتوكول لتصور تكرارها داخل المقصورات التحت خلوية من الخلايا العصبية. EDU متفوقة على النظير ثيميدين أخرى ، مثل 5 برومو deoxyuridine - 2 (BrdU) ، لأن رد الفعل الأولي لايدو فوق التسمية 5-7 لا تتطلب علاجات حمض القاسية أو هضم أنزيم التي مطلوبة من أجل فضح حاتمة BrdU . وأكثر اعتدالا توسيم ايدو يسمح للمقارنة مباشرة لتأسيسها مع علامات الخلوية الأخرى 9-10. القدرة على تصور وتحديد نشوء حيوي و mtDNA يوفر أداة أساسية عن التحقيق في الآليات المستخدمة لتنظيم نشوء حيوي الميتوكوندريا وستوفر نظرة ثاقبة على المرضية المرتبطة سمية الدواء ، والشيخوخة والسرطان وأمراض الاعصاب. أسلوبنا ينطبق على الخلايا العصبية الحسية ، وكذلك أنواع الخلايا الأخرى. استخدام هذه التقنية لقياس و mtDNA نشوء حيوي لها انعكاسات كبيرة في تعزيز فهم الفيزيولوجيا الخلوية العادية وكذلك الحالات المرضية غير جيدة.

Watch this Scientific Journal Video about Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification at JoVE.com

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

طورنا تقنية حساسة لتسمية الحمض النووي توليف حديثا (و mtDNA) في الخلايا الفردية ، من أجل دراسة و mtDNA نشوء حيوي. أسلوب يجمع بين إدماج ايدو مع بروتوكول إشارة tyramide (TSA) التضخيم لتصور و mtDNA تكرارها داخل المقصورات التحت خلوية من الخلايا العصبية.

تصور المتماثل الحمض النووي في خلايا فردية عن طريق تضخيم الإشارات EDU

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

visualization-mitochondrial-dna-replication-individual-cells-edu

Research

JoVE Journal

Neuroscience

22.8K Views.  University of Michigan. طورنا تقنية حساسة لتسمية الحمض النووي توليف حديثا (و mtDNA) في الخلايا الفردية ، من أجل دراسة و mtDNA نشوء حيوي. أسلوب يجمع بين إدماج ايدو مع بروتوكول إشارة tyramide (TSA) التضخيم لتصور و mtDNA تكرارها داخل المقصورات التحت خلوية من الخلايا العصبية.

Video: Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

المؤتمتة عرض الفيديو التفاعلي للدراسات الاتصالات الحيوانية

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

ترنسفكأيشن من خلايا الشبكية بواسطة الماوس العقدة في الجسم الحي Electroporation

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

تعيش خلية التصوير الحسي للخلايا السليمة في الجهاز السلائف ذبابة الفاكهة شرانق

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

تصور في Proprioceptors<em&gt; ذبابة الفاكهة</em&gt; يرقات وشرانق

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

استهدفت وصفها من الخلايا العصبية في المجال الوظيفي محددة من القشرة المخية الحديثة مايكرو من خلال الجمع بين الإشارات الجوهرية والتصوير ثنائي الفوتون

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

وهناك طريقة للتحكم عالي الدقة Optogenetic من الخلايا العصبية الفردية الهرمية<em&gt; في الجسم الحي</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

تقييم وظيفة الطاقة البيولوجية في خلايا دماغي الأوعية الدموية غشائي

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

تصوير راتيوميتريك الكالسيوم من الخلايا العصبية الفردية في التصرف ايليجانس كاينورهابديتيس

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

التصور للوحدات القشرية في الأرض كورتيسيس الثدييات

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.