עבודה זו נתמכה על ידי המכונים הלאומיים לבריאות מענקים RO1GM-33205 ו MH-LC עד 067,284 גריפית על ידי מלגה לתואר ראשון באוניברסיטת ברנדייס בקיץ מחקר NJ הורנשטיין. ניסויים ראשוניים של טכניקה זו בוצעו בשלב המעבדה לביולוגיה ימית במסגרת מערכות עצביות 2008 ובקיץ התנהגות כמובן (NIMH מענק: R25 MH059472) ב וודס הול, מסצ&#39;וסטס.

חלק 1: טיפול בבעלי חיים וחוצה גנטי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שמור כטב&quot;מ-ChR2 ו-OK371 GAL4 קווי לטוס בבקבוקים נפרד המכיל התקשורת לטוס תקן 8. </li><li style=";text-align:right;direction:rtl"> איסוף בתולות OK371-GAL4 קווי לעוף הזכרים של כטב&quot;מים-ChR2 הקווים. </li><li style=";text-align:right;direction:rtl"> שים הן זכרים ונקבות בבקבוקון המכיל התקשורת לטוס רגיל מעורבב עם 1 מ&quot;מ כל טרנס הרשתית (ATR). מזון ATR צריכה להיעשות על ידי הראשון קבוע ההיתוך התקשורת לטוס במיקרוגל למשך דקה 1 ~. לאחר מותך, להתקרר למשך כ 30 שניות עד דקה אחת ולאחר מכן להוסיף 100 μl של 100 mM ATR ב ETOH 100 מ&quot;ל 10% עבור כל התקשורת לעוף. צלוחיות מקום על הקרח, אז חנות באזור כהה 4 ° C. </li><li style=";text-align:right;direction:rtl"> בואו להזדווג הזבובים להטיל את הביצים באזור חשוך בשעה 22-25 ° C. </li><li style=";text-align:right;direction:rtl"> חכה 3-4 ימים עד 3 instar הזחלים גלויים, בנקודה זו, תשליך זבובים בוגרים. </li></ol> חלק 2: הגדרת Rig <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צרף כל עין 10x לנתח היקף היצירה (במקרה שלנו, של Carl Zeiss Inc, www.zeiss.com, Thornwood, ניו יורק), אל מקור אור LED כחולות עם גוף הקירור (Thor מעבדות, www.thorlabs.com, ניוטון, NJ ). צרף בסיס מגנטי עם פירסום מהדק. כיסוי גוף הקירור עם מגן הארקה חשמלית כדי להפחית את הרעש החשמלי שנוצר על ידי מקור האור. </li><li style=";text-align:right;direction:rtl"> מניחים בסיס מגנטי עם מקור אור על שולחן אוויר של electrophysiology מעטה. </li><li style=";text-align:right;direction:rtl"> חיבור מקור האור לשלוט מעגל ומתחברים מעגל בקרה למקור מתח חיצוני (Powerlab 4 / 30, ADInstruments, www.adinstruments.com, קולורדו ספרינגס, CO). </li><li style=";text-align:right;direction:rtl"> תן 1-5 פולסים V לשלוט מעגל להפעיל אור כחול. </li><li style=";text-align:right;direction:rtl"> התאם בסיס מגנטי מקור אור עד אלומת אור כחול הוא התמקד באזור להיות כבוש על ידי לנתיחה הזחל. </li></ol> חלק 3: Dissection <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Six פלייס 0.1 מ&quot;מ סיכות חרקים לתוך הרצפה של צלחת sylgard מרופד. </li><li style=";text-align:right;direction:rtl"> הסרת 3 rd הזחלים instar מהתקשורת מזון ומקום לתוך כל צלחת פלסטיק-פטרי. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את הזחלים עם מלח כדי להסיר את המזון. </li><li style=";text-align:right;direction:rtl"> המקום זחלים ב לנתח צלחת ליד סיכות משך ולהוסיף מלח לרמה ½. </li><li style=";text-align:right;direction:rtl"> אוריינט הזחלים, כך שאתה יכול לראות שני צינורות כסופים (קנה הנשימה) פועל על פני השטח הגבי של החיה. </li><li style=";text-align:right;direction:rtl"> הכנס פין גדול ישירות לתוך הזנב בין צינורות לקנה הנשימה. החזק את הזחלים למטה במקום סיכה גדולה נוספת לתוך הראש שלה, להיות בטוח כדי למתוח את הגוף החוצה לאורכו. </li><li style=";text-align:right;direction:rtl"> לעשות חתך קטן ליד הזנב, ולהמשיך אותו אורך הגוף. ודא טיפים של המספריים גדלים כדי לא בטעות לחתוך עצבים הגחון ו / או קיר שרירי הגוף. </li><li style=";text-align:right;direction:rtl"> מקום ארבע סיכות על ארבע פינות של החיה עכשיו הבטן פתוחה. הגדר אותם לתוך התבשיל לנתח כדי פילה החי. הכנה פין לימד החוצה. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים ומספריים, להסיר את האומץ של החיה, קנה הנשימה וגורמים שומן. </li><li style=";text-align:right;direction:rtl"> שוטפים את הכנה עם תמיסת מלח. </li><li style=";text-align:right;direction:rtl"> אתר את האונות הקדמיות (כפי שניתן לראות באיור) ו הגנגליון הגחון של הכנה. </li><li style=";text-align:right;direction:rtl"> באמצעות מיקרו מספריים, לחתוך בזהירות הגנגליון הגחון ממש מאחורי האונות הקדמיות. </li></ol> חלק 4: הקלטה שרירים וגירוי אור כחול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> משוך אלקטרודה 10-20 μω באמצעות אלקטרודה חולץ אלקטרוניים (סאטר מכשירים). </li><li style=";text-align:right;direction:rtl"> מלאו את האלקטרודה משך עם 3M KCl. </li><li style=";text-align:right;direction:rtl"> המקום מלא בעל אלקטרודה אלקטרודה אלקטרודה קצה לתמרן עם micromanipulator ליד הכנה הזחל תחת מיקרוסקופ לנתח על electrophysiology מעטה. </li><li style=";text-align:right;direction:rtl"> זיהוי שרירים 6 (M6) כל גוף במגזר הקיר (איור). </li><li style=";text-align:right;direction:rtl"> בזהירות להכניס אלקטרודה לתוך M6 ולצפות עבור hyperpolarization מהירה. מנוחה שרירים הפוטנציאלים הממברנה צריך להיות בכל מקום מ -30 mV ל -70 mV. </li><li style=";text-align:right;direction:rtl"> התאם את האור הכחול, כך הכנה גזור מרוכז קרן אור. </li><li style=";text-align:right;direction:rtl"> תן 20-100 פעימות ms מתח לשלוט מעגל. בהדרגה להגדיל את מתח המסופק לשלוט מעגל. Watch עבור פוטנציאלים מעוררים צומת בתא השריר (איור 1 ב). </li></ol> נציג תוצאות: איור 1 א מראה סכימטי של הגדרת הקלטה הכנה נשלף. 1B איור מראה אופייני EJP עורר על ידי להבזקי האור קצר. משרעת EJP המוצג הוא סיכם משרעת משני הנוירונים המנוע ידוע כאחד innervate M6. לעוצמות אור תחתון בלבד מופעל יחידה מוטורית (מידע לא מוצג). <img src="/files/ftp_upload/1133/figure1.png" alt="איור 1" width="600" height="373" /> איור 1: סכמטית) כללי של המתקן הקלטה תאיים עם LED כחול. המוח (בר) מוסר לעכב פעילות קצבית ב הגנגליון הגחון (VG). ChR2 מתבטא הנוירונים המנוע באמצעות מערכת GAL4-כטב&quot;מ. ב) הקלטה תאיים מ שריר M6. 40 ms כחול להבזקי האור (ב 127 μW / מ&quot;מ 2) אמין לעורר פוטנציאלים סינפטיים גדול (כוכביות) ב M6.

<ol>
	<li>Keshishian, H., Broadie, K., Chiba, A., Bate, 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+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35 (2007).</li><li>Lagow, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+a+hydrophobic+layer+by+a+point+mutation+in+syntaxin+1A+regulates+the+rate+of+synaptic+vesicle+fusion.">Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.</a> PLoS Biol. 5 (4), e72 (2007).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279 (2005).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940 (2003).</li><li>Schroll, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16 (17), 1741 (2006).</li><li>Lin, D. M., Auld, V. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+neuronal+cell+ablation+in+the+Drosophila+embryo:+pathfinding+by+follower+growth+cones+in+the+absence+of+pioneers.">Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.</a> Neuron. 14 (4), 707 (1995).</li><li>Ralph Greenspan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics, 2nd ed. , (2004).</li></ol>

אין לנו שום ניגודי עניינים לחשוף.

צעדים קריטיים לערב גם את לנתיחה הראשוני של הכניסה לתאי שריר. אם עצבים נחתכים או שריר פגום במהלך הניתוח הראשוני קשה כדי להמשיך את שאר הניסוי. במהלך הניתוח, יש להיזהר מאוד זווית לנתח מספריים כלפי מעלה ככל האפשר במהלך חתך הגבי. במהלך השלב המכריע השני, נכנסים לתא השריר, יש ...

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comment</td>
</tr>	
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Ellsworth adhesives </td>
<td>4019862</td>
<td><a href="http://www.ellsworth.com" target="_blank">www.ellsworth.com</a></td>
</tr>
<tr>
<td>Minutens Pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td><a href="http://www.finescience.com" target="_blank">www.finescience.com</a></td>
</tr>
<tr>
<td>Dissecting Dish</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td><a href="http://www.fishersci.com" target="_blank">www.fishersci.com</a></td>
</tr>
<tr>
<td>Neuroprobe Intracellular Amplifier and Head Stage</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>680100</td>
<td><a href="http://www.a-msystems.com" target="_blank">www.a-msystems.com</a></td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">Powerlab 4/30 data acquisition system</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com" target="_blank">AD instruments</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com" target="_blank">www.adinstruments.com</a></td>
</tr>
<tr>
<td>Grass stimulator</td>
<td>&nbsp;</td>
<td>Grass instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.grasstechnologies.com" target="_blank">www.grasstechnologies.com</a></td>
</tr>
<tr>
<td>Desktop Computer</td>
<td>&nbsp;</td>
<td>Dell</td>
<td>&nbsp;</td>
<td><a href="http://www.dell.com" target="_blank">www.dell.com</a></td>
</tr>
<tr>
<td>Dissecting Scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td><a href="http://www.leica-microsystems.com" target="_blank">www.leica-microsystems.com</a></td>
</tr>
<tr>
<td>Light Source</td>
<td>&nbsp;</td>
<td>Dolan-Jenner</td>
<td>41446-062</td>
<td><a href="http://www.dolan-jenner.com" target="_blank">www.dolan-jenner.com</a></td>
</tr>
<tr>
<td>Fly Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>All-Trans-Retinal</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>116-31-4</td>
<td><a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a></td>
</tr>
<tr>
<td>OK-371 Gal4 Flies</td>
<td>&nbsp;</td>
<td>Aberle lab, Griffith lab, Bloomington stock center </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>UAS-ChR2 Flies</td>
<td>&nbsp;</td>
<td>Fiala lab, Griffith lab </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>LED controller circuit</td>
<td>&nbsp;</td>
<td>Built in Griffith lab</td>
<td>&nbsp;</td>
<td><a href="http://www.ledsupply.com" target="_blank">http://www.ledsupply.com</a> 
<a href="http://www.futureelectronics.com" target="_blank">http://www.futureelectronics.com</a> 
Composed of: 
1. 200 mA Buck Puck 
2. Blue LED 
3. Insulated wire 
4. Circuit bread board </td>
</tr>
<tr>
<td>LED Heat Sink</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>&nbsp;</td>
<td><a href="http://www.thorlabs.com/" target="_blank">http://www.thorlabs.com/</a></td>
</tr>
<tr>
<td>Air Table</td>
<td>&nbsp;</td>
<td>TMC</td>
<td>&nbsp;</td>
<td><a href="http://www.techmfg.com/products/accessories/intro3.html" target="_blank">http://www.techmfg.com/products/accessories/intro3.html</a></td>
</tr>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>Built in Griffith lab</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Leica Leitz M Micro-Manipulator</td>
<td>&nbsp;</td>
<td>Leica Leitz</td>
<td>ACS01</td>
<td><a href="http://www.leica-microsystems.com" target="_blank">www.leica-microsystems.com</a></td>
</tr>
<tr>
<td>Electrode Holder</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.axon.com" target="_blank">www.axon.com</a></td>
</tr>
<tr>
<td>Borosilicate Glass</td>
<td>&nbsp;</td>
<td>FHC</td>
<td>&nbsp;</td>
<td><a href="http://www.fh-co.com/p14-15.pdf" target="_blank">www.fh-co.com/p14-15.pdf</a></td>
</tr>
<tr>
<td>Electrode Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.sutter.com" target="_blank">www.sutter.com</a></td>
</tr>
<tr>
<td>HL 3.1 Saline with 0.8mM Ca2+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Contents (mM): 
NaCl:70 KCl:5 CaCl2: 0.8 MgCl2:4Sucrose:115 
NaHCO3: 10 Trehalose: 5 HEPES
</td>
</tr>
<tr>
<td>Micro-Dissection Tools</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td><a href="http://www.finescience.com" target="_blank">www.finescience.com</a></td>
</tr>		
</tbody>
</table>

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

<em&gt; תסיסנית</em&gt; הכנה הזחל neuromuscular הוכיחה להיות כלי שימושי לחקר הפיזיולוגיה הסינפטי<sup&gt; 1,2,3</sup&gt;. נכון לעכשיו, רק את האמצעים לעורר junctional פוטנציאלים מעוררים (EJPs) בהכנה זו כרוכה בשימוש אלקטרודות יניקה. הן במחקר והן מעבדות ההוראה, התלמידים מתקשים לעיתים קרובות תמרונים וטיפול זה סוג של אלקטרודה מגרה. בעבודה הנוכחית, אנו מראים כיצד מרחוק לעורר פוטנציאלים סינפטיים ב NMJ הזחל ללא שימוש אלקטרודות יניקה. על ידי הבעת channelrhodopsin2 (ChR2)<sup&gt; 4,5,6</sup&gt; ב<em&gt; תסיסנית</em&gt; מנוע הנוירונים באמצעות<em&gt; GAL4-כטב&quot;מ</em&gt; מערכת<sup&gt; 7</sup&gt;, וביצוע שינויים קלים לבוש electrophysiology בסיסיים, הצלחנו לעורר אמין EJPs עם פולסים של אור כחול. טכניקה זו יכולה להיות שימוש מסוים במעבדות הוראה הנוירופיזיולוגיה שם תלבושת תלמיד לתרגל זמן ומשאבים מוגבלים.

Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

פרוצדורה זו משתמשת ערוץ תכלת המופעל אצות תא ספציפי כלי הביטוי הגנטי לעורר פוטנציאלים סינפטיים עם להבזקי האור בצומת neuromuscular (NMJ) ב<em&gt; תסיסנית</em&gt; הזחלים. טכניקה זו היא זולה וקלה לשימוש חלופה גירוי יניקה אלקטרודה ללימודי פיזיולוגיה הסינפטי מחקר ומעבדות הוראה.

Channelrhodopsin2 גירוי מתווכת של פוטנציאלים Synaptic בבית תסיסנית Neuromuscular כלאיים

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

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Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

13.6K Views.  Brandeis. פרוצדורה זו משתמשת ערוץ תכלת המופעל אצות תא ספציפי כלי הביטוי הגנטי לעורר פוטנציאלים סינפטיים עם להבזקי האור בצומת neuromuscular (NMJ) ב תסיסנית הזחלים. טכניקה זו היא זולה וקלה לשימוש חלופה גירוי יניקה אלקטרודה ללימודי פיזיולוגיה הסינפטי מחקר ומעבדות הוראה.

Video: Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

טוען תסיסנית מסופי עצב עם אינדיקטורים סידן

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Operant Learning of Drosophila at the Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

למידה אופרנטית של תסיסנית במד מומנט

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

מערכת מגנטית לקשור לחקור חזותית חוש הריח בקרת טיסה מתווך תסיסנית

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

שיטות אלקטרו עבור הקלטה פוטנציאלים Synaptic מן NMJ של הזחלים תסיסנית

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

ראייה ריח מעקב מתווכת במהלך טיסה ב תסיסנית

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

לדמיין את חיים תסיסנית גליה-neuromuscular צומת עם צבעי ניאון

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

בתחום ההדמיה vivo של הזחלים תסיסנית אינטקט ברזולוציה משנה הסלולר

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

השוואה כמותית של Cis-רגולטוריות (Cre) אלמנט פעילות טראנסגנטי תסיסנית melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering הומולוגי רקומבינציה בבונה<em&gt; דרוזופילה</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

שימוש Coculture לזהות אינטראקציות interspecies המתווכת כימית

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...