אנו מודים ברנרד Charroux לדיונים. עבודה זו מומנה על ידי בית הספר ביו KCl.

1. Dissection של הרשתית תסיסנית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מילוי אחד טוב של שקופיות 3-ובכן זכוכית עם פוספט שנאגרו מלוחים (PBS 1X), ולאחר מכן להרדים זבובים על ידי ה-CO 2. שני זכרים ונקבות ניתן להשתמש לצורך הניסוי הזה. </li><li style=";text-align:right;direction:rtl"> לאחר הזבובים מורדמים, להרים זבוב אחד באמצעות מלקחיים ו זרוק אותו לתוך צלחת קטנה של PBS. </li><li style=";text-align:right;direction:rtl"> תחת בהיקף לנתח, לצבוט את חוטם עם מלקחיים, ולאחר מכן לנתק את הראש מהגוף על ידי ניתוק הצוואר באמצעות סט שני של מלקחיים. </li><li style=";text-align:right;direction:rtl"> החזקת הראש לטוס חוטם, בחצי לאורך קו האמצע שמאלה / ימינה ראש לטוס עם microscissors לחשוף את המוח. </li><li style=";text-align:right;direction:rtl"> השתמש קצה פיפטה לחתוך להרים את הרשתית, ולהעביר שנייה גם של 3-Slide ובכן זכוכית מלאים בינוני שניידר. </li><li style=";text-align:right;direction:rtl"> ראשית, לקלף ביותר לציפורן ראש ולאחר מכן להסיר את המוח עם מלקחיים. שמור את הרשתית דחוקה בין lamina לבין הקרנית. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, להעביר את הרשתית על ירידה (50 μl) של המדיום Schnieder טרי ממוקם ישירות על שקופיות הזכוכית. </li></ol> 2. Dissection של האומטידיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בהתאם למטרה הסופית של הניתוח (בין אם באופן מיידי או הדמיה תרבות) ועל אופטיקה של מיקרוסקופ פלואורסצנטי (ישיר או הפוך), שלב זה יכול להתרחש גם על המיקרוסקופ שקופית או בצלחת 24 באר. למטרה הסופית של הניתוח הזה, האומטידיה יהיה גזור ישירות על גבי שקופיות הזכוכית. </li><li style=";text-align:right;direction:rtl"> אחוז או סוף הגב או הגחון ביותר של הרשתית עם מלקחיים. שימוש microscissors, לחתוך את הרשתית במחצית לאורך קו האמצע הגב / הגחון לחשוף את האומטידיה באמצע העין. </li><li style=";text-align:right;direction:rtl"> המשך לתפוס את הרשתית כזה הקרנית פונה למטה lamina הוא פונה כלפי מעלה. באמצעות מחט טונגסטן בסדר, לנתק את האומטידיה מ lamina לבין הקרנית. </li><li style=";text-align:right;direction:rtl"> האומטידיה יחיד וקבוצות האומטידיה תאסוף בתחתית הבאר או שקופיות. הסר את כל פסולת גדול שייתכן שנאספו לפני שתמשיך. </li></ol> 3. , טיפול מכתים הדמיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לנתח autophagy, לדלל 1:1000 Lysotracker בירידת שניידר (הראשון לדלל 1:10 ב שניידר ולאחר מכן להוסיף 0.5 μl עד טיפת בשקופית), לערבב ולהמתין 10 שניות. </li><li style=";text-align:right;direction:rtl"> הסר את נוזל עודף בשקופית, נזהר לצאת מאחורי האומטידיה. זה נעשה בצורה הטובה ביותר עם טיפ ג&#39;ל עדין טוען. </li><li style=";text-align:right;direction:rtl"> הוסף ירידה של Vectashield כדי לכסות את האומטידיה, להחיל coverslip וסוגרים עם nailpolish. </li></ol> 4. נציג תוצאות: ביצוע טוב של הפרוטוקול צריך להשאיר מספר האומטידיה יחיד של קבוצות קטנות של האומטידיה שמרו יחד על ידי שברי lamina אבל מופרדים יפה בצד הקרנית. אלה יכולים להיות צילמו כמו בדוגמה זו כדי להמחיש Atg8: GFP ו Lysotracker, מראה autophagosomes, lysosomes ו autophagolysosomes (איור 3). השיטה יכולה לשמש הדמיה לחיות, אולם במקרה זה יש לציין כי autofluorescence של המדיום של שניידר יקשה להבחין בין אותות בעוצמה נמוכה ניאון. סוגיה נוספת היא כי גרגרי פיגמנט שנותרו המחוברים photoreceptors הם פלורסנט מאוד, במיוחד במסלול האדום. תמונה brightfield עשויים להידרש להבדילם האברונים אמיתי. <img src="/files/ftp_upload/2882/2882fig1.jpg" alt="איור 1"/> באיור 1. תרשים זרימה של הליך לנתיחה כדי להשיג האומטידיה בודד או קבוצות קטנות של האומטידיה מן הרשתית לעוף. <img src="/files/ftp_upload/2882/2882fig2.jpg" alt="איור 2"/> איור 2. וריאציות אפשריות של פרוטוקול פשוט לתאר כאן שכוללות גנטיקה לטוס והזדקנות במעלה הזרם של דיסקציה ו מכתים או culturing במשך עד 24 שעות והתרופות במורד הזרם של הניתוח. <img src="/files/ftp_upload/2882/2882fig3.jpg" alt="איור 3"/> איור 3 לסרוק Confocal של autophagosomes ו lysosomes ב אומטידיום יחיד גזור מן aw;-GMR Gal4, כטב&quot;מ-Atro75QN; כטב&quot;מ-GFP:.: Atg8 / + לעוף, להביע polyglutamine Atrophin מוטנטי בגילאי 12 ימים ב 29 ° C. לוח brightfield (מימין למעלה) מציג את מבנה כמעט בשלמותה של אומטידיום ואת המסגרת מצביע על האזור שנסרק. האדום מסמן ליזוזומים, ירוק autophagosomes, Auto-lysosomes, וכתוצאה מכך על ידי איחוי של שני אברונים, מופיעים צהוב. ראשי חץ מציינים שני אירועים היתוך מתמשך בין autophagosomes ו lysosomes.

<ol>
	<li>Marsh, J. L., Thompson, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+in+the+study+of+neurodegenerative+disease.">Drosophila in the study of neurodegenerative disease.</a> Neuron. 52, 169-178 (2006).</li><li>Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>Nisoli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+by+polyglutamine+Atrophin+is+not+rescued+by+induction+of+autophagy.">Neurodegeneration by polyglutamine Atrophin is not rescued by induction of autophagy.</a> Cell Death Differ. 17, 1577-1587 (2010).</li><li>Charroux, B., Fanto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+line+between+waste+disposal+and+recycling:+DRPLA+fly+models+illustrate+the+importance+of+completing+the+autophagy+cycle+for+rescuing+neurodegeneration.">The fine line between waste disposal and recycling: DRPLA fly models illustrate the importance of completing the autophagy cycle for rescuing neurodegeneration.</a> Autophagy. 6, 667-669 (2010).</li><li>Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+potassium+channels+in+Drosophila+photoreceptors.">Voltage-sensitive potassium channels in Drosophila photoreceptors.</a> J Neurosci. 11, 3079-3095 (1991).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends in neurosciences. 24, 251-254 (2001).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1237 (1993).</li><li>Gambis, A., Dourlen, P., Steller, H., Mollereau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+in+vivo+imaging+of+photoreceptor+apoptosis+and+development+in+Drosophila.">Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila.</a> Dev Biol. 351, 128-1234 (2011).</li><li>Napoletano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyglutamine+Atrophin+provokes+neurodegeneration+in+Drosophila+by+repressing+fat.">Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat.</a> The EMBO journal. 30, 945-958 (2011).</li><li>Ravikumar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+mTOR+induces+autophagy+and+reduces+toxicity+of+polyglutamine+expansions+in+fly+and+mouse+models+of+Huntington+disease.">Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease.</a> Nature. 36, 585-595 (2004).</li><li>Zou, S., Meadows, S., Sharp, L., 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=Genome-wide+study+of+aging+and+oxidative+stress+response+in+Drosophila+melanogaster.">Genome-wide study of aging and oxidative stress response in Drosophila melanogaster.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 13726-13731 (2000).</li></ol>

אין ניגודי אינטרסים הכריז.

דיסקציה אומטידיום יחיד המוצגת כאן מאפשרת איסוף מידע התא עמוק יותר על תהליכים ביולוגיים כמו ניוון מוחיים, כאשר הדגם בעין תסיסנית. הנוירונים הם photoreceptor נגיש יותר בקלות מאשר נוירונים אחרים והם הציטופלסמה הוא גדול במיוחד, מתאים ובכך ללמוד הדינמיקה שלפוחית, בתאי בדי...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th><th> הערות (אופציונלי) </th></tr><tr><td> Dispenser CO2 ניידת </td><td> Flystuff </td><td> 59-150 </td><td> מילוי גם זמין </td></tr><tr><td> דומונט פינצטה לא 5 </td><td> מדעי אגר </td><td> T5034 </td><td></td></tr><tr><td> 3-ובכן זכוכית נושאת </td><td> EMS </td><td> 71561-01 </td><td></td></tr><tr><td> מיקרו מספריים </td><td> VWR </td><td> HAMMHSB516-09 </td><td></td></tr><tr><td> שניידר בינוני </td><td> VWR </td><td> 733-1663 </td><td></td></tr><tr><td> LysoTracker האדום DND-99 </td><td> Invitrogen </td><td> L7528 </td><td></td></tr><tr><td> Superfrost שקופיות </td><td> VWR </td><td> 631-0108 </td><td></td></tr><tr><td> Vectashield עם Dapi </td><td> וקטור Labs </td><td> H-1200 </td><td></td></tr><tr><td> Coverslips </td><td> VWR </td><td> 631-1336 </td><td></td></tr></tbody></table>

single drosophila ommatidium dissection and imaging

זבוב הפירות דרוזופילה melanogaster תרם תרומות יקר המוח במחקר כבר בשימוש נרחב כמודל מחלות ניווניות בגלל הגנטיקה שלה עוצמה 1. העין לטוס בפרט כבר האיבר של בחירה עבור מחקר ניוון מוחיים, להיות חלק נגיש ביותר החיים dispensable של מערכת העצבים תסיסנית. עם זאת, אזהרה גדולה של העיניים שלם הוא הקושי, בגלל autofluorescence האינטנסיבי של הפיגמנט, באירועים הדמיה תאיים, כגון autophagy הדינמיקה 2, אשר עליונה הבנה של ניוון מוחיים. השתמשנו לאחרונה לנתיחה והתרבות של האומטידיה אחד 3 כי כבר חיוני להבנת תפקוד autophagic במודל זבוב של ניוון Dentatorubro-Pallidoluysian (DRPLA) 3, 4. כעת אנו מדווחים תיאור מקיף של טכניקה זו (איור 1), המותאמים ממחקרים אלקטרו 5, אשר צפוי להרחיב באופן משמעותי את האפשרות של מודלים לטוס ניוון מוחיים. שיטה זו יכולה להתאים לדימוי חי אירועים subcellular כדי לפקח והתרופות אפקטיבי על התאים קולטי האור (איור 2). אם בשילוב עם טכניקות פסיפס 6-8, התגובות של תאים שונים גנטית יכול להיות assayed במקביל (איור 2).

Watch this Scientific Journal Video about Single Drosophila Ommatidium Dissection and Imaging at JoVE.com

Single Drosophila Ommatidium Dissection and Imaging

הגורם המגביל בשימוש של המבוגר<em&gt; תסיסנית</em&gt; עין ללמוד ניוון מוחיים, ביולוגיה של התא הוא קשה הדמיה של תהליכים תאיים. אנו מתארים את דיסקציה של האומטידיה בודד כדי ליצור בתום-בתום תא ראשוני תרבות העצבית, אשר יכול להיות נושא לטיפול תרופתי והדמיה מתקדמים.

אחד תסיסנית אומטידיום Dissection והדמיה

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

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Neuroscience

Single Drosophila Ommatidium Dissection and Imaging

14.0K Views.  King's College London. הגורם המגביל בשימוש של המבוגר תסיסנית עין ללמוד ניוון מוחיים, ביולוגיה של התא הוא קשה הדמיה של תהליכים תאיים. אנו מתארים את דיסקציה של האומטידיה בודד כדי ליצור בתום-בתום תא ראשוני תרבות העצבית, אשר יכול להיות נושא לטיפול תרופתי והדמיה מתקדמים.

Video: Single Drosophila Ommatidium Dissection and Imaging

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Sensillum הקלטות סינגל חרקים תסיסנית melanogaster ו אנופלס gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

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

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

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

Dissection והדמיה של אזורי פעיל תסיסנית Neuromuscular צומת

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

נתיחה ואימונוהיסטוכימיה של זחל, גלמים ומבוגרים<em&gt; דרוזופילה</em&gt; רשתיות

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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.

תיוג של תאים בודדים במערכת העצבים המרכזית של<em&gt; דרוזופילה melanogaster</em

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

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

יחיד לטוס Assay עבור התנהגות שיחור מזון ב<em&gt; דרוזופילה</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Live Imaging of Drosophila Larval Neuroblasts

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

Live Imaging of Drosophila Larval Neuroblasts

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

הדמיה חיה של<em&gt; דרוזופילה</em&gt; הזחל Neuroblasts

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

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

צעד-אחד פשוט פרוטוקול לנתיחה עבור כל הר הכנה למבוגרים<em&gt; תסיסנית</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

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

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

קרינה פלואורסצנטית תא בודד רזולוציה לחיות הדמיה של שעונים היממה דרוזופילה בתרבות המוח זחל

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

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

ניתוח של סיבי שריר שלד יחיד עבור ניתוחים אימונופלואורסצנטיים ומורפומטריים של צמתים נוירו-שריריים בהר שלם

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

ניטור אירועים אפילפטיים בדרוזופילה באמצעות הדמיית סידן

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

זרקור על המחבר: תובנות לגבי הטכניקות והממצאים של ההתקדמות האחרונה בחקר האפילפסיה 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

אקס ויוו הדמיית סידן למודל דרוזופילה לאפילפסיה

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...