עבודה זו נתמכה על ידי המכונים הלאומיים לבריאות מענקים NS-38849 ו DK-076160, Juvenile Diabetes Research Foundation המרכז לחקר סיבוכים של סוכרת, את תוכנית לנוירולוגיה מחקר גילוי אלפרד טאובמן א מכון המחקר הרפואי בבית אוניברסיטת מישיגן. עבודה זו השתמשו מורפולוגיה וניתוח תמונה Core המרכז לחקר הסוכרת והכשרה מישיגן, שמומן על ידי המכונים הלאומיים לבריאות גרנט 5P60 DK-20572 מן המכון הלאומי של סוכרת עיכול מחלות כליה. המחברים מודים סקוט קלארק מ ט בדיקות מולקולריות / Invitrogen עצה יקר שלו על השונות לחץ Edu-IT תיוג ערכות התרומה הנדיבה של ריאגנטים כדי לתמוך בפיתוח הראשוני של הטכניקה הגברה edu.

<ol>
	<li>Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fusion+and+fission+in+mammals.">Mitochondrial fusion and fission in mammals.</a> Annu. Rev. Cell Dev. Biol. 22, 79-99 (2006).</li><li>Dimmer, K. S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(De)constructing+mitochondria:+what+for.">(De)constructing mitochondria: what for.</a> Physiology (Bethesda). 21, 233-241 (2006).</li><li>Suen, D. F., Norris, K. L., Youle, 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=Mitochondrial+dynamics+and+apoptosis.">Mitochondrial dynamics and apoptosis.</a> Genes Dev. 22, 1577-1590 (2008).</li><li>Clay Montier, L. L., Deng, J. 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. I., Figueroa-Romero, C., Cerri, F., Backus, C., Hong, Y., 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=Diabetes+regulates+mitochondrial+biogenesis+and+fission+in+mouse+neurons.">Diabetes regulates mitochondrial biogenesis and fission in mouse neurons.</a> Diabetologia. 53 (1), 160-169 (2010).</li><li>Davis, A. F., Clayton, 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=In+situ+localization+of+mitochondrial+DNA+replication+in+intact+mammalian+cells.">In situ localization of mitochondrial DNA replication in intact mammalian cells.</a> J. Cell Biol. 135, 883-893 (1996).</li><li>Magnusson, J., Orth, M., Lestienne, P., Taanman, J. 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>

אין ניגודי אינטרסים הכריז.

פיתחנו assay רגיש תווית mtDNA מסונתז החדש של תאים בודדים באמצעות הגברה של האות tyramide edu. אחת הבעיות הגדולות במהלך אופטימיזציה של פרוטוקול זה היה תוצאות לא עקביות בין coverslips. שינויים נעשו הפרוטוקולים ערכת Invitrogen כדי למנוע ערבוב בנפחים קטנים על coverslips הפרט ולהפוך פתרונות הורים לשימוש coverslips כל. ΜL 75-80 בשימוש עבור כל 12 מ&quot;מ זכוכית עגול coverslip הוא נפח אידיאלי כדי לכסות לחלוטין את פני השטח, תוך מתן פתרון מספיק כדי לתת תוצאות עקביות בין דגימות. פעמים דגירה ריכוזי מגיב היו אופטימיזציה אבל השיפורים ניתן לראות עם התאמות להם. הפרוטוקול נועד לרוץ כל תהליך פעם. עם זאת, כמה צעדים כבר חזר עם פתרונות חדשים כדי להתאושש דגימות לאחר שנכשלו בניסיון הראשון. בפרט, תגובת לחץ צעדים EDU (3.4-3.6) כבר חזר ללא עלייה משמעותית הקרינה רקע. אנטי אורגון Green-HRP הדגירה נוגדן (4.1-4.3) לבין הגברה tyramide (4.4-4.5) יש גם צעדים חוזרים ונשנים, אך לעתים קרובות יותר תוצאות אות עני יחס רעש בגלל הקרינה רקע מוגבר. ריאגנטים הרגישים ביותר הם תוסף לחץ Edu-IT חוצץ (Component G) הארנב נגד אורגון Green-HRP נוגדן (Component I) מן ערכת לחץ זה Assay microplate edu. תוסף זה לחץ Edu הצפת הופך בהדרגה צהוב לאורך זמן כאשר מאוחסנים 4 ° C ו בין 6-12 חודשים מכהה במידה ניכרת. אחסון של תוסף לחץ Edu-IT הצפת ב -20 ° C יהיה להקל על בעיה זו. תיוג צעדים הגברה tyramide העבודה הטובה ביותר כאשר הארנב נגד אורגון Green-HRP הנוגדן משמש בתוך 4-6 חודשים של ומשחזר אותו MilliQ DH 2 O. תיוג קלה של Edu ב mtDNA מאפשר השוואה ישירה עם סמנים נוספים הסלולר 9-11 ומשפר את השירות של טכניקה זו על אנלוגים thymidine אחרים, כגון 5-bromo-2-deoxyuridine (BrdU), אשר דורש טיפול קשה להתאושש שלה epitope בתוך הדנ&quot;א. המעבדה שלנו 11-12 ועוד 13-15 השתמשו בהצלחה BrdU לתייג mtDNA. הטכניקה תיוג BrdU דומה לזו שבוצעה על Edu אבל יש כמה הבדלים חשובים (איור 5). לאחר השלב permeabilization המפורטים לעיל (3.1-3.2), epitope BrdU הוא התאושש בצעד denaturation (2 N HCl למשך 30 דקות ב 37 ° C ואחריו שלושה שוטף ב 1X PBS). BrdU מסומן ואז עם נוגדן ראשוני נגד BrdU (וקטור מעבדות מדוללת 1:50 בתמיסה של 1% חסימה של ערכת ה-TSA ו מודגרות לילה ב 4 ° C). צעד משני הנוגדנים יש צורך להגביר את האות BrdU (אנטי עכבר עז IgG מצומדות כדי HRP של ערכות ה-TSA # 2 ו - # 5, מדולל 1:100 בתמיסה של 1% חסימה מודגרות 45 דקות בטמפרטורת החדר) לפני TSA צעדים (4.3-4.5 לעיל). שיש שני אנלוגים thymidine, Edu ו BrdU, כדי mtDNA תווית יתרון לביצוע ניסויים תווית כפול שבו mtDNA יכול להיות מתויג ברצף ב הדופק תיוג פרדיגמות 11. המעבדה שלנו היא באמצעות Edu ו BrdU תיוג של mtDNA לבחון את ויסות biogenesis המיטוכונדריה בהקשר של נוירופתיה סוכרתית, סיבוך נפוץ של מחלת הסוכרת. השתמשנו בהצלחה הגברה אות כדי למדוד שינויים mtDNA biogenesis נוירונים בודדים ב 11-12. טכניקה זו יהיה שימושי בניסויים אחרים שנועדו לחקור את המנגנונים של שכפול mtDNA מחזור או לזיהוי תרופות המעכבות סינתזה mtDNA. בנוסף לכך, העקרונות הבסיסיים של הגברה אותות Edu ו BrdU ניתן להחיל מחקרים אחרים כי ה-DNA למדוד שכפול או לתקן.

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visualization of mitochondrial dna replication in individual cells by edu signal amplification

המיטוכונדריה הם הרגולטורים העיקריים של אנרגיה תאית biogenesis המיטוכונדריה היא מרכיב חיוני של ויסות מספר המיטוכונדריות בתאים בריאים 1-3. גישה אחת לניטור biogenesis המיטוכונדריה היא למדוד את שיעור ה-DNA המיטוכונדריאלי (mtDNA) שכפול 4. פיתחנו טכניקה רגישה תווית mtDNA מסונתז החדש של תאים בודדים כדי ללמוד biogenesis mtDNA. הטכניקה משלבת שילוב של 5-ethynyl-2&#39;-deoxyuridine (EDU) 5-7 עם הגברה אות tyramide (TSA) 8 פרוטוקול לדמיין שכפול mtDNA בתוך תאים subcellular של נוירונים. Edu עדיפה אנלוגים thymidine אחרים, כגון 5-bromo-2-deoxyuridine (BrdU), כי התגובה הראשונית לחץ Edu התווית 5-7 אינו מחייב את הטיפולים הקשים חומצה או אנזים מעכל הנדרשים חשיפת epitope BrdU . תיוג מתונה יותר של EDU מאפשרת השוואה ישירה ההתאגדות שלה עם סמנים תאיים אחרים 90-10. היכולת לחזות ולכמת biogenesis mtDNA מספקת כלי הכרחי לחקירת המנגנונים המשמשים להסדיר biogenesis המיטוכונדריה וגם יספק תובנות בפתוגנזה הקשורות רעילות התרופה, הזדקנות, סרטן ומחלות ניווניות. הטכניקה שלנו הוא ישים עצב סנסורי כמו גם סוגי תאים אחרים. השימוש בטכניקה זו כדי למדוד biogenesis mtDNA יש השלכות משמעותיות לקדם את ההבנה של הפיזיולוגיה הן הסלולר הרגיל, כמו גם מדינות מחלה לקוי.

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

פיתחנו טכניקה רגישה תווית הדנ&quot;א המיטוכונדרי מסונתז החדש (mtDNA) בתוך תאים בודדים כדי ללמוד biogenesis mtDNA. הטכניקה משלבת שילוב של Edu יחד עם פרוטוקול tyramide הגברה (TSA) אות לדמיין שכפול mtDNA בתוך תאים subcellular של נוירונים.

ויזואליזציה של שכפול ה-DNA מיטוכונדריאלי של תאים בודדים על ידי הגברה אות 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

22.9K Views.  University of Michigan. פיתחנו טכניקה רגישה תווית הדנ"א המיטוכונדרי מסונתז החדש (mtDNA) בתוך תאים בודדים כדי ללמוד biogenesis mtDNA. הטכניקה משלבת שילוב של Edu יחד עם פרוטוקול tyramide הגברה (TSA) אות לדמיין שכפול mtDNA בתוך תאים subcellular של נוירונים.

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.

ניגון אוטומטי של וידאו אינטראקטיבי ללימודי תקשורת של בעלי חיים

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.

Transfection של תאים עכבר רשתית גנגליון על ידי 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 ...

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

Live-תא הדמיה של תאים מבשר חושי האיברים אינטקט תסיסנית גלמים

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.

ויזואליזציה של Proprioceptors ב<em&gt; תסיסנית</em&gt; זחלים וגלמים

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.

תיוג ממוקד של תאי עצב בתחומים ספציפיים מייקר פונקציונליים של הניאוקורטקס ידי שילוב אותות פנימיים והדמית שני פוטונים

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.

שיטה לנאמנות גבוהה בקרת optogenetic של פרט פירמידת נוירונים<em&gt; בvivo</em

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

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

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

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

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

בו זמנית<em&gt; Vivo לשעבר</em&gt; בדיקות פונקציונליות של שתי הרשתיות על ידי<em&gt; In vivo</emמערכת electroretinogram&gt;

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

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.

הערכה של תפקוד Bioenergetic בתאי האנדותל מוחות דם

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 ...

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

Ca מיטוכונדריאלי2 + שמירה קיבולת Assay ו- Ca2 +-עוררה Assay נפיחות מיטוכונדריאלי

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

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;

סידן רציומטרי הדמיה של נוירונים בודדים ב מתנהג Caenorhabditis Elegans

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

ויזואליזציה של שכפול ה-DNA מיטוכונדריאלי של תאים בודדים על ידי הגברה אות Edu

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ניגון אוטומטי של וידאו אינטראקטיבי ללימודי תקשורת של בעלי חיים

Transfection של תאים עכבר רשתית גנגליון על ידי In vivo Electroporation

Live-תא הדמיה של תאים מבשר חושי האיברים אינטקט תסיסנית גלמים

ויזואליזציה של Proprioceptors ב

תיוג ממוקד של תאי עצב בתחומים ספציפיים מייקר פונקציונליים של הניאוקורטקס ידי שילוב אותות פנימיים והדמית שני פוטונים

שיטה לנאמנות גבוהה בקרת optogenetic של פרט פירמידת נוירונים

בו זמנית

הערכה של תפקוד Bioenergetic בתאי האנדותל מוחות דם

Ca מיטוכונדריאלי^{2 +} שמירה קיבולת Assay ו- Ca^{2 +}-עוררה Assay נפיחות מיטוכונדריאלי

סידן רציומטרי הדמיה של נוירונים בודדים ב מתנהג Caenorhabditis Elegans