Name: use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule
Uploaded: 2015-11-25T15:00:00.000+00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

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The authors have nothing to disclose.

השימוש של assay ראמזי, יחד עם אלקטרודות יון ספציפי, מאפשר המדידה של שיעורי הפרשת נוזל והנתיבים יון בMalpighian tubules חרקים מבודדים (כליות). ניתן assayed עשרים או יותר tubules בזמן, המאפשר תפוקה גבוהה יותר בהשוואה לassay של פרט בtubules המבחנה microperfused. בנוסף, אלקטרודות יון ספציפי תאפשר ק...

<html> תחבורת יון אפיתל כליות בבסיס iono- האורגניזם וויסות האוסמוטי. אבובית דרוזופילה melanogaster Malpighian (כליות) מציעה הזדמנות שאין כמותו כדי לחקור את המנגנונים המולקולריים של תחבורת יון אפיתל. זאת בשל השילוב של הגנטיקה החזקה של דרוזופילה, יחד עם הנגישות של tubules הכליות שלה למחקר פיסיולוגי. Assay ראמזי, על שמו של החוקר שהיה חלוץ טכניקת 1, מודד שיעורי הפרשת נוזל מצינוריות Malpighian מבודדות, והוקם בשנת 1994 על ידי תסיסנית דאו ועמיתים 2. זה סלל את הדרך למחקרים נוספים באמצעות תסיסנית כלים גנטיים, כגון מערכת GAL4-כטב&quot;מ 3,4, להגדיר מסלולי איתות תא ספציפי המסדירים את הפרשת נוזלים. דוגמא כוללת איתות סידן בתגובה להורמון פפטיד 5, בקרב רבים אחרים 6,7. ve_content &quot;&gt; שילוב של טכניקות גנטיות ומחקר פיסיולוגי קלאסי הוכיח כי דור שתן בזבוב מתרחש באמצעות ההפרשה של נוזל כלוריד-עשיר אשלגן מהמגזר העיקרי של אבובית. זו מתרחשת באמצעות הפרשת transepithelial המקבילה של קטיונים, בעיקר K + אלא גם Na +, דרך התא העיקרי, וCl -. ההפרשה דרך תא stellate 8-12 היכולת למדוד בנפרד והנתיבים transepithelial K + וNa + מאפשרת אפיון מפורט יותר של מנגנוני תחבורה מאשר המדידה של הפרשת נוזל יש לבד. לדוגמא, בtubules תסיסנית unstimulated, + Na / K + -ATPase מעכב ouabain אין כל השפעה על הפרשת נוזל 2, גם כאשר ספיגתו לתוך תאים עיקריים היא מעוכבת על ידי taurocholate מעכב טרנספורטר אניון האורגני 13. עם זאת, לינטון ו אודונל הראה כי ouabain depolarizesפוטנציאל הממברנה basolateral, ומגביר Na + שטף 9. כפי שניתן לראות בנציגי התוצאות, אנו משוכפלים ממצאים אלה, והראו כי K + שטף הוא במקביל ירד 14; שטף Na + המוגבר וירידת K + שטף יש השפעות מנוגדות על הפרשת נוזל, וכתוצאה מכך אין שינוי נטו בהפרשה. לפיכך, יש שתי החלטות ל&quot; פרדוקס ouabain, &quot;כלומר, ההתבוננות הראשונית שouabain אין כל השפעה על הפרשת נוזלים באבובית תסיסנית:. ראשונה, בtubules המגורה, ההשפעה של ouabain על הפרשת נוזל אינה נראית לעין עקב הספיגה שלה על ידי טרנספורטר אניון האורגני 13; ושנייה, בtubules unstimulated, ouabain יש השפעות מנוגדות על transepithelial Na + K + ושטף, וכתוצאה מכך אין שינוי נטו בהפרשת נוזל (ראה תוצאות ונ&quot;צ נציג. 9). לכן, התפקיד העיקרי של + Na / K + -ATPASE בtubules unstimulated הוא להוריד תאיים ריכוז Na + ליצור שיפוע ריכוז נוח לNa + -coupled תהליכי הובלה על פני קרום basolateral. ואכן, על ידי מדידה בנפרד Na + K + ונתיבים, שהוכחנו כי tubules חסר cotransporter נתרן, אשלגן-2-כלוריד זבוב (NKCC) ירד K + שטף transepithelial, ללא ירידה נוספת לאחר תוספת ouabain, ולא חלו שינוי בtransepithelial Na + שטף 14. ממצאים אלה נתמכים מסקנתנו כי Na + כניסה לתא דרך NKCC ממוחזרת דרך Na + / K + -ATPase. בדוגמא אחרת, Ianowski נצפה et al. כי הורדת K אמבטיה + ריכוז מ -10 מ&quot;מ ועד 6 מ&quot;מ ירד K transepithelial + שטף וtransepithelial המוגבר שטף Na + בצינוריות מprolixus Rhodnius, ללא שינוי נטו בהפרשת נוזל <sup&gt; 15. אפקטי ההפרש בשטף Na + K + ושטף על פני tubules זחל גם נצפו בtubules דרוזופילה בתגובה לדיאטות מלח שונה 16 ובשני מיני יתושים בתגובה לגידול מליחות 17. האתגר הגדול ביותר במדידת שטף יון transepithelial בהכנת assay רמזי הוא קביעת ריכוזי יון בתוך הנוזל המופרש. אתגר זה כבר נפגש עם פתרונות שונים, כולל photometery להבה 18, שימוש ביונים רדיואקטיביים 19, וגל אלקטרון הבדיקה ספקטרוסקופיה נפיצה 20. טכניקות אלה דורשות העברה של ירידת הנוזל המופרשת למכשיר למדידת ריכוזי יון. מאז את נפח הנוזל המופרש על ידי אבובית תסיסנית unstimulated הוא קטן, בדרך כלל ~ 0.5 NL / דקה, זה מציב אתגר טכני וגם מציגה שגיאה אם חלק מהנוזל המופרש הואאיבד על העברה. לעומת זאת, השימוש באלקטרודות יון ספציפי מאפשרת המדידה של פעילות יון (שממנו ניתן לחשב ריכוז יון) באתרו. הפרוטוקול הנוכחי הותאם מזה בשימוש על ידי Maddrell ועמיתים למדידת K transepithelial + שטף על פני אבובית Rhodnius באמצעות valinomycin כK + ionophore 21, ומתאר גם את השימוש ב-butylcalix 4 טרט [4] חומצת arene-tetraacetic Na מבוסס אסתר tetraethyl + אלקטרודה יון ספציפי -specific המאופיינת Messerli et. אל. 22. אלקטרודות יון ספציפיות יש גם שימשו למדידת ריכוזי יון בנוזל המופרש על ידי צינוריות Malpighian בassay רמזי ב9,23 מבוגרים ו -16 זחל תסיסנית melanogaster, ניו זילנד Alpine Weta (Hemideina מאורי) 24 וביתושים 17. כאן, אנו מתארים בפירוט את השימוש ברמזים כאומר למדוד שיעורי הפרשת נוזל בtubules Malpighian מדרוזופילה melanogaster, כמו גם שימוש באלקטרודות יון ספציפי כדי לקבוע את הריכוזים של K + וNa + בתוך הנוזל המופרש ולכן החישוב של נתיבי יון transepithelial. סקירה של assay מסופקת באיור 1. <img alt="איור 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> איור 1. סכמטי של קשיות Malpighian וAssay רמזי עם שימוש ביון ספציפי אלקטרודות למדידת ריכוזי יון. נתון זה ממחיש את ההתקנה לassay רמזי. (א) לכל זבוב ארבעה tubules, זוג הצינוריות קדמית וזוג הצינוריות אחורית, לצוף שבחלל הבטן המוקף hemolymph. בכל זוג, שתי הצינוריות להצטרף בשופכן, אשר לאחר מכן מרוקן את השתן בצומת של midgut וhindguלא. Tubules הם הסתיים עיוור. שתן נוצר על ידי המגזר העיקרי מפרישי הנוזל (באדום), וזורם לכיוון השופכן והחוצה אל הבטן. לאחר נתיחה, זוג אבובית הוא ניתק מהבטן על ידי חוצה את השופכן. (ב) זוג הצינוריות מועבר לאחר מכן לטיפה של ים מלוח בתוך היטב של צלחת assay. אחד משתי הצינוריות, המכונה כאן &quot;אבובית העוגן,&quot; עטוף סביב סיכת מתכת והוא אינרטי. אבובית האחרות היא אבובית הסתרתו. הקטע הראשוני (שאינו מפריש נוזל) והקטע העיקרי של אבובית מפרישות להישאר בתוך הטיפה של ים מלוח. יונים ומהלך מים מתמיסת מלח הים ולתוך לום אבובית של המגזר העיקרי, ולאחר מכן לנוע לכיוון השופכן, כפי שהיו קורים בvivo. הקטע התחתון (הכחולה) הוא מחוץ למלוח רחצה ולכן אדיש. מאז שופכן הוא לחתוך, הנוזל המופרש מתגלה כטיפה מסוף החתך של השופכן. Tהוא מופרש טיפת נוזל מגדילה לאורך הזמן כהפרשה ממשיכה, וקוטרה נמדד באמצעות מיקרומטר עיני. שכבה של שמן מינרלים מונעת אידוי של הנוזל המופרש. אלקטרודות ספציפיות ההתייחסות והיון למדוד את ריכוז היון של הנוזל המופרש. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

אפנון של תחבורת יון אפיתל כליות מאפשר אורגניזמים לשמור על הומאוסטזיס יוני והאוסמוטי בפרצוף של משתנה תנאים חיצוניים. אבובית דרוזופילה melanogaster Malpighian (כליות) מציעה הזדמנות שאין כמותו כדי לחקור את המנגנונים המולקולריים של תחבורת יון אפיתל, בשל הגנטיקה החזקה של האורגניזם הזה ואת הנגישות של tubules הכליות שלה למחקר פיסיולוגי. כאן, אנו מתארים את השימוש של assay רמזי למדידת שיעורי הפרשת נוזל מtubules הכליות לטוס מבודד, עם השימוש באלקטרודות יון ספציפי למדידת ריכוזי נתרן ואשלגן בנוזל המופרש. assay זה מאפשר לימוד והנתיבים נוזל transepithelial ויון של ~ 20 tubules בזמן, ללא הצורך להעביר את הנוזל המופרש למנגנון נפרד למדידת ריכוזי יון. מבחינה גנטית ניתן לנתח tubules השונה כדי להעריך את התפקיד של גנים ספציפיים בתהליכי הובלה. בנוסף, במלוח athing יכול להיות שונה כדי לבחון את ההשפעות של מאפייניו הכימיים, או תרופות או הורמונים הוסיפו. לסיכום, טכניקה זו מאפשרת האפיון המולקולרי של מנגנונים בסיסיים של תחבורת אפיתל יון באבובית דרוזופילה, כמו גם הסדרת מנגנוני תחבורה אלה.

איורים 7 ו -8 להפגין כי שימוש בassay רמזי עם אלקטרודות יון ספציפי למדידת ריכוזי K + וNa + יכול להבחין K גנטי ופרמקולוגית נפרד + והנתיבים Na +, מידע שאינו נתפס על ידי מדידת שיעורי הפרשת נוזל לבד. איור 7 מראה כי ירידה בהפרשת נוזלים בצינורי...

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

פרוטוקול זה מתאר את השימוש של assay רמזי למדידת שיעורי הפרשת נוזל מצינוריות Malpighian (כליות) מבודדות מmelanogaster תסיסנית. בנוסף, השימוש באלקטרודות יון ספציפי למדידת ריכוזי נתרן ואשלגן בנוזל המופרש, המאפשר חישוב של שטף יון transepithelial, מתואר.

שימוש של assay רמזי כדי למדוד את הפרשת נוזלים ויון שטף מחירים ב<em&gt; תסיסנית melanogaster</em&gt; אבובית Malpighian

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

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Research

JoVE Journal

Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.8K Views.  University of Texas Southwestern Medical Center.  פרוטוקול זה מתאר את השימוש של assay רמזי למדידת שיעורי הפרשת נוזל מצינוריות Malpighian (כליות) מבודדות מmelanogaster תסיסנית. בנוסף, השימוש באלקטרודות יון ספציפי למדידת ריכוזי נתרן ואשלגן בנוזל המופרש, המאפשר חישוב של שטף יון transepithelial, מתואר. 

Video: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

שיטות Assay תסיסנית התנהגות

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Neuroscience

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

מדידת תגובות פיזיולוגיות של<em&gt; תסיסנית</em&gt; עצב סנסורי כדי ליפידים פרומונים שימוש סידן הדמיה חיה

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Immunology and Infection

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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

את assay נימי מזין מודד צריכת המזון ב<em&gt; תסיסנית</em

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

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

מדידה של הזחל פעילות ב<em&gt; דרוזופילה</em&gt; צג פעילות

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

PH כימות של תאיים אופטי בדרוזופילה melanogaster צנורית Malpighian Epithelia עם pH מקודד פלורסנט גנטית מחוון

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

חקר הפעילות של נוירופפטידים ורגולטורים אחרים של מערכת ההפרשה יתוש למבוגרים

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

ניטור החמצת מעיים במעי דרוזופילה למבוגרים

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

דרוזופילה ניתוח משקעי צואה לגילוי תרופות

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

מחבר זרקור: קידום מחקר נוגד שלשולים עם מודל דרוזופילה 

Here, a method is described to feed Drosophila melanogaster with drugs and plant extracts and assess their effect on the gastrointestinal tract by analyzing the fruit flies fecal deposits. The drug-treated flies can be used as a model for further research.

הערכת ההשפעה של תרופות נוגדות שלשול ותמציות צמחים על Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...

שימוש של assay רמזי כדי למדוד את הפרשת נוזלים ויון שטף מחירים ב

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שיטות Assay תסיסנית התנהגות

מדידת תגובות פיזיולוגיות של

את assay נימי מזין מודד צריכת המזון ב

מדידה של הזחל פעילות ב

PH כימות של תאיים אופטי בדרוזופילה melanogaster צנורית Malpighian Epithelia עם pH מקודד פלורסנט גנטית מחוון

חקר הפעילות של נוירופפטידים ורגולטורים אחרים של מערכת ההפרשה יתוש למבוגרים

ניטור החמצת מעיים במעי דרוזופילה למבוגרים

הערכת ההשפעה של תרופות נוגדות שלשול ותמציות צמחים על Drosophila melanogaster

הערכת ההשפעה של תרופות נוגדות שלשול ותמציות צמחים על Drosophila melanogaster

הערכת ההשפעה של תרופות נוגדות שלשול ותמציות צמחים על Drosophila melanogaster