This work was supported by NIH P20GM103643 to I. Meng.

1. إعداد يرقات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتحليل يرقة المطلوب للتحرك أو موقف تفضيل، وتنمو اليرقات في ظل ظروف قياسية لعمر المطلوبة لفحص 10 باستخدام ذبابة الغذاء القياسية 11. </li><li style=";text-align:right;direction:rtl"> إجراء التصفية شبكة التي تمتد الشاشة الحريرية الصف النايلون شبكة فوق قمع. تأمين شبكة في الرقبة قمع مع الشريط المطاطي. وضع قمع في كوب. </li><li style=";text-align:right;direction:rtl"> لجمع اليرقات لتحليلها، مغرفة ملعقة-كاملة من الأغذية التي تحتوي على يرقات تستخدم علفا من زجاجة الثقافة ويغسل بالماء RT الصنبور على تصفية شبكة، وجمع اليرقات الفردية مباشرة من شبكة مع الرسام 10. </li></ol> 2. إعداد الفحص أنابيب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الاستعداد لتوصيل أنابيب الاختبار، وتغلي بعناية 4٪ هلام أجار وتصب في طبق بتري على عمق 1.5 سم. هذا وسوف توفر المكونات لجانبي الأنبوب عندما يتم وضع الحيوان. الحل 4٪ كثيفة بما فيه الكفاية لمنع اليرقات من صenetrating المقابس. </li><li style=";text-align:right;direction:rtl"> التأكد من أن أنابيب نظيفة واضحة قبل إدخال اليرقة. إذا لم تكن كذلك، وهذا قد منع الحركات من يجري تسجيلها. </li><li style=";text-align:right;direction:rtl"> لضمان اليرقات لديها ما يكفي من الرطوبة للحركة، وإعداد زجاجة ضغط تحتوي على كمية صغيرة من الماء الساخن. قلب الزجاجة، توجيه بعناية منفذ نحو بالوعة والضغط برفق لطرد الماء من أنبوب صغيرة. </li><li style=";text-align:right;direction:rtl"> إدراج منفذ زجاجة في أنبوب الاختبار. ضغط على زجاجة لتقديم بخار الماء في أنبوب الاختبار حتى تظهر طبقة رقيقة من التكثيف على جدار الأنبوب. الفيديو 1 يظهر يرقة تتحرك في أنبوب مع كمية مناسبة من الرطوبة. </li><li style=";text-align:right;direction:rtl"> لاغلاق اليرقة في الأنبوب، وإزالة هلام أجار 1.5 سم سميكة من طبق بتري ومكان على سطح شبكة لتسمح بتدفق الهواء تحت آغار بحيث يمكن إدراج قابس أجار في أنبوب. اضغط احدة من نهاية أنبوب الاختبار في هلام مرتين لإدراج اثنين من المقابس من هلام إلى نهاية واحدة. </li><li style=";text-align:right;direction:rtl"> مع paintbالاندفاع، ضع يرقة واحدة في أنبوب ما يقرب من 1.5 بوصة العميق وختم الأنبوب عن طريق الضغط على نهاية أقرب اليرقة إلى هلام أجار. فإن الضغط الناتج طرد المكونات الثاني من هلام أجار من الطرف المقابل وختم الحيوان داخل الأنبوب. </li><li style=";text-align:right;direction:rtl"> أنابيب مكان في الجهاز MB5 متعدد شعاع ذبابة الفاكهة مراقبة النشاط (DAM)، وتعديل المواقف الأنابيب &#39;حتى أن الحيوانات لا يمكن أن تتحرك خارج نطاق من أجهزة الاستشعار. </li><li style=";text-align:right;direction:rtl"> للتأكد من أن فحص الأنابيب لا تنزلق من تحت الحمراء إطار القراءة للجهاز أثناء النقل، وعقد أنبوب في مكان بواسطة الربط حلقة من المعجون حول كل أنبوب حيث إنها تجري اتصالات مع جهاز تسجيل. </li></ol> 3. قياس آخر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> سجل النشاط في حاضنة لمنع قراءات دقيقة التي قد تحدث بسبب الظلال، ومصابيح الفلورسنت أو لتغيرات درجة الحرارة في المختبر. انظر الشكل 5 لترتيب المقترح للنظام. </li><li style=";text-align:right;direction:rtl"> مجموعة حاضنة إلى 20 درجة مئوية (انظر الشكل 1). لتجنب تسجيلات كاذبة بسبب تدخل من مصادر الضوء ساطع أثناء التسجيل، إطفاء الأنوار حاضنة الفلورسنت واستخدام مصدر مستقل ضوء LED أثناء التسجيل في الحاضنة. إجراء المحاكمة دون أي الحيوانات لضمان ظروف الإضاءة لا تثير aberrantly أجهزة الاستشعار. يجب أن تكون هناك أية بيانات حركة سجلت بعد هذا الاختبار. </li><li style=";text-align:right;direction:rtl"> السماح اليرقات حتى يتأقلم إلى 20 ° C إعدادات الحاضنة لمدة 5 دقائق قبل بدء الفحص. </li><li style=";text-align:right;direction:rtl"> لضبط النظام DAM لفترات التسجيل المطلوب (على سبيل المثال، 1 دقيقة)، تأكد من تحميل DAM برنامج تسجيل نظام لاستضافة الكمبيوتر 12 وفتح ملف النظام DAM قبل ربط غرفة تسجيل واجهة PSIU. </li><li style=";text-align:right;direction:rtl"> لضبط التردد التسجيل المطلوب الذي سيتم حفظ البيانات، حدد تفضيلات ثم انقر فوق أو تحت خيار الفاصل الزمني القراءة لتحديد الأطر الزمنية المختلفة التي البياناتسيتم تخزين. على سبيل المثال، حدد القراءة الأوقات الفاصلة من 1 ثانية إلى 1 ساعة. </li><li style=";text-align:right;direction:rtl"> لتحديد المعلمة متفاوتة لتسجيل البيانات، اختر تفضيلات ثم حدد المربعات المقابلة تحت نوع بيانات الناتج (التهم، والتحركات والمواقف ويسكن، انظر الجدول 1). توفر كل معلمة تحليلا فريدا للنشاط اليرقات داخل الأنبوب. </li><li style=";text-align:right;direction:rtl"> لبدء التسجيل، توصيل جهاز العرض إلى وحدة واجهة التيار الكهربائي (PSIU) باستخدام كابل الهاتف CAB6. ربط PSIU إلى التيار الكهربائي. وهناك ضوء أخضر تشير الاتصال المناسب. </li><li style=";text-align:right;direction:rtl"> ربط PSIU من خلال الناقل التسلسلي العالمي (USB) برقية إلى ماكنتوش أو جهاز كمبيوتر ويندوز لتسجيل البيانات. فتح برنامج DAM SystemMB1v6x على الكمبيوتر لبدء تلقائيا تسجيل البيانات. </li><li style=";text-align:right;direction:rtl"> بعد وقت جمع المطلوبة كاملة، حدد إنهاء، ثم قم بإنهاء الآن على الشاشة النشاط DAM. وبعد ذلك يتم حفظ البيانات تلقائيا تحت DAM بيانات النظام. خذ هذا الملف البيانات (على سبيل المثال، مراقبة 1) واسحب<html> في مجلد منفصل. وسوف تخزن البيانات الأولية من برنامج DAM بحيث يمكن بعد ذلك تتم معالجتها. </li></ol> 4. إعداد بيانات للتجهيز (DAM FileScan) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لمعالجة البيانات الخام إلى صيغة مفهومة فتح التطبيق DAM FileScan واختر تحديد مجلد بيانات الإدخال. ثم اختر مجلد البيانات والملف المطلوب، وحدد الخيار المسح الضوئي. وأخيرا تحديد طول بن لفترة القراءة المطلوبة. وهذا تنظيم البيانات الخام إلى المعايير المحددة من قبل المشغل وهذا البرنامج سوف يقدم تقريرا البيانات التي تم جمعها في هذا النطاق معين. </li><li style=";text-align:right;direction:rtl"> حدد نوع بيانات الناتج لتحليل وضع الحركة المطلوبة (مراقبة التهم، والتحركات، يسكن). ضمن القائمة قراءات إضافية، حدد المبلغ إلى بن (هذا إذا كان طول بن أكبر من نظام الفاصل الزمني للقراءة مختارة). </li><li style=";text-align:right;direction:rtl"> اسم الملف بشكل مناسب وحفظها. سيتم تخزين الملف في نفس الموقع مثل مجلد من الخطوة 3.9. </li></ol>_title &quot;&gt; 5. الوصول إلى البيانات لتحليل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> من أجل جمع متوسط ​​التحركات، عملية البيانات الخام الناتجة عن الخطوة 4. لإنشاء جدول في برنامج جداول البيانات، فتح ملف تم حفظه سابقا (الخطوة 4.3)، وعندما تظهر نافذة معالج استيراد النص، حدد النهاية لفتح ملفات البيانات في شكل جداول. ملاحظة: اعتمادا على نوع البيانات التي تم تحليلها، وسيتم قراءة جدول البيانات بشكل مختلف. عادة لقياس تحركات الشاشة أو التهم الموجهة إليه، وسوف يتم تسجيل الفترة الزمنية للدراسة من الناحية العددية، في حين أن كل أنبوب القراءة الفردية سيحصل بريد إلكتروني معين. عند عرض البيانات في جدول البيانات، والأعمدة KZ تمثل فتحات الأنابيب فحص 16/01 على التوالي. الصفوف تمثل نقاط البيانات التي تم جمعها. </li><li style=";text-align:right;direction:rtl"> على سبيل المثال، إذا تم جمع التحركات لمدة 20 دقيقة في 1 دقيقة فترات، ومتوسط ​​20 الصفوف لحساب متوسط ​​عدد التحركات / دقيقة وقياسات أخرى. </li></ol>

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Science Education Database Available from: <a target="_blank" href="https://www.jove.com/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans">https://www.jove.com/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans</a> (2015)</li><li>Woods, J. K., Kowalski, S., Rogina, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+spontaneous+locomotor+activity+in+Drosophila+melanogaster.">Determination of the spontaneous locomotor activity in Drosophila melanogaster.</a> J. Vis. Exp. (86), e51449 (2014).</li><li>Troncoso, B., Godoy-Herrera, R., Waldo, 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+development+of+larval+movement+patterns+in+Drosophila).">The development of larval movement patterns in Drosophila).</a> Heredity. 58 (1), 321-329 (1987).</li><li>Helfrich, C., Engelmann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+the+locomotor+activity+in+Drosophila.+melanogaster.and+its+mutants+&#8216;sine+oculis&#8217;+and+&#8216;small+optic+lobes.">Circadian rhythm of the locomotor activity in Drosophila. melanogaster.and its mutants &#8216;sine oculis&#8217; and &#8216;small optic lobes.</a> Physiological Entomology. 8 (3), 257-272 (1983).</li><li>Johnson, A. W., Carder, W. 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The authors declare that they have no competing financial interests.

ويتأثر النشاط من يرقات ذبابة الفاكهة من قبل مجموعة متنوعة من العوامل بما في ذلك النمط الجيني 8، سن 13 ودرجة الحرارة المحيطة 2. على الرغم من أن وسائل videographic قوية قادرة على تحليل مفصل للغاية تم تطويرها من قبل أولئك الذين يدرسون الحركة 5، هذا ال...

The use of Drosophila as a genetic tool has transformed scientific knowledge of biological systems. Drosophila larvae have been used in a variety of studies including nociception1, development2 and as a model for the study of human disease genes3. Drosophila activity encompasses a range of behaviors that vary under different conditions including temperatures2, exposure to drugs4 and amongst different genotypes. Yet, despite the significant use of the larva as a model organism, a simple, standardized method to analyze larval activity has not been available. Presently, many larval locomotion studies employ sophisticated video analysis software5. While powerful, the complexity of such automated tools may discourage labs that are not already equipped to study locomotion from including analysis of informative activity parameters in their studies. In other current non-automated methods, such as the grid crawling analysis, motion is scored by a human observer, which introduces the possibility of subjectivity and limits throughput to one animal at a time6-7. A similar study used a 5-lane crawling assay, which measured the time it took larvae to travel a certain linear distance8. In such non-automated assays, displacement is measured but this does not account for non-linear travel between the start and end points. As discussed below, the method described here accounts for more of the actual larval movement, is objective, easy to operate, and offers robust throughput.
To easily study larval activity behavior without the compromise of accuracy, efficiency, or cost, this method employs the TriKinetics Drosophila Activity Monitor (DAM), a device often used to study adult activity. Using one device to study both adult and larval activity is cost-effective, and allows direct comparison of motion by animals at these two life stages. The system, featuring the manufacturer&#8217;s highest level of resolution, makes use of 17 infrared detection beams per assay tube, which record larval activity when sensor beams are broken within the 16 individual tubes. The system then automatically saves recorded information to a computer, making it available for manipulation with basic graphing software. The data obtained represents the beams that were broken by individual larvae (which can be converted into a rate), movement when a larva stays within a detector beam and the position of the animals within the assay chambers during a recording period (allowing one to calculate position preference). The system is efficient and relatively simple to operate, and brings highly reproducible basic activity analysis within the reach of any laboratory studying Drosophila larvae.
To demonstrate the power of this assay, data are presented that show its use to verify differences in activity resulting from varying ambient temperatures, as well as through the comparison of a mutant previously described as hypoactive (iav1)9 with a widely analyzed control (w1118).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila Activity Monitor, Multibeam, 16 tubes, including wires</td>
			<td>TriKinetics Inc.&#160;</td>
			<td>MB5</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply Interface for Activity Monitor&#160;</td>
			<td>TriKinetics Inc.&#160;</td>
			<td>PSIU24</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass 80 x 5 mm tubes for Activity Monitor (100)</td>
			<td>TriKinetics Inc.&#160;</td>
			<td>PGT 5x80</td>
			<td></td>
		</tr>
		<tr>
			<td>DAMsystemMB1v6x Data Acquisitions Software for Macintonsh OSX (Intel)</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>DAMFileScan 108x software for Macintosh</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>USB software (PSIUdrivers.zip).</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>DAMSystem Notes 308</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>Zeiss Stemi 2000C- Stereo Microscope</td>
			<td>Spectra Services</td>
			<td>SP-STEMI2000C-BS</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide</td>
			<td>Maine Oxy</td>
			<td></td>
			<td>anaesthesia</td>
		</tr>
		<tr>
			<td>Fly Pad</td>
			<td>Genesee</td>
			<td>59-114</td>
			<td>surface for sorting anaesthetized flies</td>
		</tr>
		<tr>
			<td>Small paint brush&#160;</td>
			<td>Winsor &#38; Newton</td>
			<td>#2 ROUND</td>
			<td>or similar, used for sorting anaesthetized flies</td>
		</tr>
		<tr>
			<td>Silk Screen Printing Mesh (160)</td>
			<td>msj-gallery.com</td>
			<td>SM160W63-3YD</td>
			<td>pore sized used in this protocol was ~ 0.1 mm</td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Genesee</td>
			<td>20-258</td>
			<td>preservative</td>
		</tr>
		<tr>
			<td>Ethanol (190proof)</td>
			<td>Pharmco</td>
			<td>111000190</td>
			<td>used to dissolve Tegosept</td>
		</tr>
		<tr>
			<td>6 oz Square Bottom Bottle (PP)</td>
			<td>Genesee</td>
			<td>32-130</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Flugs&#34; for Plastic Fly bottles</td>
			<td>Genesee</td>
			<td>49-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Vials, Wide (PS)</td>
			<td>Genesee</td>
			<td>32-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Flugs for wide plastic vials</td>
			<td>Genesee</td>
			<td>49-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Degerminated Corn Meal</td>
			<td>Gold Medal</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila agar</td>
			<td>LabScientific</td>
			<td>FLY 8020</td>
			<td></td>
		</tr>
		<tr>
			<td>Baker&#39;s Yeast - Red Star</td>
			<td>King Arthur Flour</td>
			<td>1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated Sugar - Extra Fine</td>
			<td>Domino</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

ويبين الشكل 1 نتائج من دراسة استجابة الحرارة من سيطرة الطور الثالث اليرقات، ث 1118، وذلك باستخدام جهاز مراقبة للكشف عن الاختلافات في الحركة اليرقات في سبع درجات حرارة مختلفة. تم غسلها اليرقات ووضعها في الجهاز النشاط DAM كما هو موضح أعلاه، ووضعها في حاضنة ?...

Watch this Scientific Journal Video about Measurement of Larval Activity in the Drosophila Activity Monitor at JoVE.com

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.

قياس اليرقات آخر في<em&gt; ذبابة الفاكهة</em&gt; مراقبة النشاط

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

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Measurement of Larval Activity in the Drosophila Activity Monitor

9.3K Views.  University of New England. 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.

Video: Measurement of Larval Activity in the Drosophila Activity Monitor

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

تشريح الجهاز العصبي المركزي اليرقات في Melanogaster ذبابة الفاكهة

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ذبابة الفاكهة تشريح اليرقات NMJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

ذبابة الفاكهة اليرقات NMJ المناعية

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

رصد وظيفة القلب في اليرقات ذبابة الفاكهة السوداء البطن للدراسات فسيولوجية

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

تشريح وتلون ذبابة الفاكهة اليرقات المبايض

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

يعاير النشاط كيناز من LRRK2 في المختبر</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

قياس عمر في<em&gt; ذبابة الفاكهة الدروسوفيلا</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

A طريقة Permeabilization من<em&gt; ذبابة الفاكهة</em&gt; الأجنة للفحوصات الصغيرة جزيء آخر

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

إعادة إعماره الوظيفية وآخر قناة قياسات تنقية Wildtype والمسخ CFTR البروتين

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

في فيفو جهاز الاستشعار البيولوجي المسارات غير أفكارك كاسباس آخر في ذبابة الفاكهة

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...