Name: the capillary feeder assay measures food intake in drosophila melanogaster
Uploaded: 2017-03-17T13:00:00
Description: Watch this Scientific Journal Video about The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster at JoVE.com

We thank the past and present members of the Scholz lab for discussion and Helga D&#246;ring for excellent technical support. We especially thank the members of the Biocenter workshop of the University of Cologne for their support and creativity. The work is supported by SFB 1340, SysMedAlc, and DAAD-Siemens.

1. الفحص CAFE ملاحظة: يتكون الاختبار من ثلاثة عناصر هي: قارورة التجريبية، غطاء معين والشعيرات الدموية الدقيقة. ويستخدم مربع من البلاستيك مع غطاء لنقل قنينات مستعدة وللسيطرة على الرطوبة بشكل أكثر كفاءة. <ol style=";text-align:right;direction:rtl"><li styl...

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ويصف التقرير فحص CAFE بطريقة خطوة بخطوة، مع التركيز على الإعداد التقني والأداء الناجح في المختبر. نظرا لبساطته، ويمكن أيضا أن هذا الاختبار أن تستخدم تربويا كتجربة المدرسة. وتبين الأمثلة التي مقايسة يسمح التحقيق في الاستشعار عن الطعام، وتفضيل والاستهلاك في ذبابة ال?...

الأكل ضروري. ومع ذلك، رفع القيود عن تناول الطعام مما يؤدي إلى اضطرابات الأكل مثل الشره المرضي، وفقدان الشهية أو الاتجاه العام إلى وجبة دسمة يفرض تكاليف على الأفراد والمجتمع 1، 2، 3. الهدف من هذا البحث هو الكشف عن الآليات التنظيمية لتناول الطعام، وتوفير استراتيجية للتحايل على تشكيل اضطراب. وقد وفرت العديد من الدراسات باستخدام الكائنات نموذج الثدييات رؤى جديدة من الدوائر ودور نظم الإنذار في اضطرابات الأكل 4، 5، 6. ومع ذلك، لدينا معرفة الأسس العصبية والجزيئية الكامنة وراء هذه الاضطرابات لا تزال بعيدة عن الاكتمال. في السنوات الأخيرة، وذبابة الفاكهة ذبابة الفاكهة أصبح نظام نموذجا قيما للكشف رؤى الآلية الأساسية في تنظيم metabolisم 7، 8، 9. تأسست الشعرية المغذية (CAFE) فحص لذبابة الفاكهة في مختبر سيمور بنزر في عام 2007 مستوحاة من العمل في وقت سابق من قبل DETHIER في الذبابة 10 و 11. جعل فحص CAFE من الممكن قياس مباشرة تناول الطعام في ذبابة الفاكهة. في هذا النظام الاختبار السلوكي، الذباب تتغذى على المواد الغذائية السائلة الواردة في الشعيرات الدموية الزجاج متدرجة وضعها داخل القارورة. تراجع الغضروف المفصلي الشعرية يشير فقدان حل الغذائية عن طريق التبخر والاستهلاك الغذائي. تحديد سعر التبخر التي كتبها قارورة دون الذباب يسمح للتقدير دقيق لتناول الطعام. فحص CAFE هو واحد من عدة نماذج سلوكية تستخدم لقياس التغذية في ذبابة الفاكهة ويجب على الباحثين اختيار الأنسب لالمحددة لهاسؤال. قرار استخدام فحص معين يجب النظر في النقاط التالية: طبيعة الطعام المقدم؛ في حالة التغذية. قياس كمية أو امتصاص العناصر الغذائية والتحقيق استهلاك الغذاء أو الاستجابة للطعام. فحص CAFE كما هو موضح في هذا التقرير هو المثالي لتناول الطعام التالية من مصدر المواد الغذائية السائلة في ظل حالة التغذية في وضع مستقيم. بدلا من تناول الطعام يمكن قياسها لمجموعة ذبابة على مصدر الغذاء الملون في قارورة أو على طبق من ذهب. والذباب قتل عادة أو تخدير بعد الرضاعة ويتم تحديد كمية من الصبغة تناولها من قبل الطيف أو الفحص البصري من البطن الملون. الذباب تبدأ في إفراز المواد الغذائية تناولها سوى 30 دقيقة بعد تناول وبالتالي هذا النهج من الصعب استخدام لتحليل التغذية أطول مستمرة السلوكيات 12، 13. في المقابل يتم الاحتفاظ الذباب على حالها عند صبغ امتصاصيتم استخدام الصورة مع استشفاف المشعة وسجل استهلاكها من النظائر المشعة في عداد التلألؤ 14 و 15. امتصاص radiolabel من قبل الجهاز الهضمي ذبابة يجعل طويلة الأجل قياس امتصاص الغذاء ممكن، ولكن قد يؤدي إلى التقليل من استهلاك بسبب جزيئات غير استيعابها وتفرز التتبع. وثمة نهج آخر لقياس استجابة في الغذاء في ذبابة الفاكهة هو الرد خرطوم تمديد (PER)، والذي يحدث عادة لتناول الطعام (16). يقيس هذا الأسلوب الأنيق الاستجابة الأولية لحافز الطعام ولكن لا يسجل كمية السعرات. يتم ضبط الاستهلاك الغذائي حيوي أثناء الرضاعة باستخدام عدة إشارات ردود الفعل بعد الهضمية التي تعتبر بالغة الأهمية لتنظيم التغذية 17 و 18. وقد بذلت عدة محاولات في السنوات الأخيرة لجمع البيانات شبه أتمتة في مقايسة PER <sحتى الطبقة = &quot;XREF&quot;&gt; 19، 20. يتم الكشف عن PER عن طريق لوحة كهربائية أو مجموعة من الأقطاب الكهربائية وتحسب عن طريق الكمبيوتر. الجمع بين فحص PER مع امتصاص النظائر المشعة كشف أن هذا الاختبار محدودة بسبب انخفاض الحساسية للكشف عن الاختلافات التغذية كمية 18. الفحص اليدوي التغذية (MAFE) 21، والتي يتم تغذيتها ذبابة يدويا مع الزجاج الشعرية، وقد وضعت مؤخرا لقياس امتصاص المواد الغذائية في ذبابة يجمد واحد. فحص MAFE يلغي التدخلات من البحث عن الطعام والتغذية بدء ولديه قرار وقت ثانية، وبدء PER ويمكن رصد استهلاك المواد الغذائية بشكل مستقل في الفحص. ومع ذلك، فإن الطريقة التي تجميد الطاير يؤثر على جوانب معينة من السلوك التغذية (مثل الحركة، والتحفيز) لا تزال بحاجة إلى التحقيق. للاستعراضات المقارنة ممتازة من فحوصات مختلفة لقياس استهلاك الغذاء في ذبابة الفاكهة ليlanogaster ولمساعدة الباحثين في العثور على واحدة الأنسب، انظر التقارير التي ديشباندي وماركس 13، 22. فحص CAFE يتجنب بعض من مساوئ فحوصات أخرى المذكورة أعلاه، ويجمع بين سهولة الاستخدام مع قياس موثوق من تناول الطعام. هنا، يتم تقديم وصفا مفصلا لفحص CAFE ونظهر تعديل الإعداد بسيطة للحد من تبخر. وأظهرت نتائج ممثلة بما في ذلك اختيار لفحص اثنين من المواد الغذائية (على المدى القصير والطويل) وامتصاص السكروز من الذباب. في مناقشة قارنا طريقة موضح لدينا مع طرق بديلة لأداء الفحص CAFE، وتسليط الضوء على أوجه القصور المحتملة.

<table border="0" cellpadding="0" cellspacing="0" width="582">
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Vials (breeding)</td>
			<td style="width:117px;">Greiner Bio-One</td>
			<td style="width:123px;">960177</td>
			<td style="width:183px;">www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (CAFE assay)</td>
			<td>Greiner Bio-One</td>
			<td>217101</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Lid-CAFE assay</td>
			<td>Workshop</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Plastic box, low wall</td>
			<td>Plastime</td>
			<td>353</td>
			<td>www.plastime.it</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cover for the plastic box</td>
			<td>Workshop</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Capillaries</td>
			<td>BLAUBRAND&#160;</td>
			<td>REF 7087 07</td>
			<td>www.brand.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Pipette tips</td>
			<td>Greiner Bio-One</td>
			<td>771290</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Filter paper circles</td>
			<td>Whatman</td>
			<td>10 311 804</td>
			<td>www.sigmaaldrich.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">D(+)-Sucrose</td>
			<td>AppliChem</td>
			<td>57-50-1</td>
			<td>www.applichem.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Ethanol absolute</td>
			<td>VWR Chemicals</td>
			<td>20,821,330</td>
			<td>www.vwr.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (red, E124)</td>
			<td>Backfun</td>
			<td>10027</td>
			<td>www.backfun.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (blue, E133)</td>
			<td>Backfun</td>
			<td>10030</td>
			<td>www.backfun.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Soap solution (CVK 8)</td>
			<td>CVH</td>
			<td>103220</td>
			<td>www.cvh.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Digital caliper</td>
			<td>GARANT</td>
			<td>412,616</td>
			<td>www.hoffmann-group.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (breeding)</td>
			<td></td>
			<td></td>
			<td>Height 9.8 cm, diameter 4.8 cm&#160;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (CAFE assay)</td>
			<td></td>
			<td></td>
			<td>Height 8 cm, diameter 3.3 cm</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Lid-CAFE assay</td>
			<td></td>
			<td></td>
			<td>Produced in university workshop, technical drawing supplied</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Plastic box, low wall</td>
			<td></td>
			<td></td>
			<td>A plastic grid inlay was custom-made for 8 x 10 vial positions&#160;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cover for the plastic box</td>
			<td></td>
			<td></td>
			<td>Dimensions (37 x29 x18 cm)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Capillaries</td>
			<td></td>
			<td></td>
			<td>DIN ISO 7550 norm,&#160; IVD-guideline 98/79 EG, ends polished</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Pipette tips</td>
			<td></td>
			<td></td>
			<td>Pipettes for the outer circle are cut according to the lid</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Filter paper circles</td>
			<td></td>
			<td></td>
			<td>45 mm diameter works nicely if folded for the vials used</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">D(+)-Sucrose</td>
			<td></td>
			<td></td>
			<td>Not harmful</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Ethanol absolute</td>
			<td></td>
			<td></td>
			<td>Highly flammable liquid and vapor</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (red, E124)</td>
			<td></td>
			<td></td>
			<td>Not stated</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (blue, E133)</td>
			<td></td>
			<td></td>
			<td>Not stated</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Soap solution (CVK 8)</td>
			<td></td>
			<td></td>
			<td>Odor neutral soap</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Digital caliper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Standard fly food</td>
			<td></td>
			<td></td>
			<td>(for 20 L)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Agar</td>
			<td></td>
			<td></td>
			<td>160 g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Brewer`s Yeast</td>
			<td></td>
			<td></td>
			<td>299.33 g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cornmeal</td>
			<td></td>
			<td></td>
			<td>1200g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Molasses</td>
			<td></td>
			<td></td>
			<td>1.6 L</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Propionic acid</td>
			<td></td>
			<td></td>
			<td>57.3 mL</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Nipagin 30%</td>
			<td></td>
			<td></td>
			<td>160 mL</td>
		</tr>
	</tbody>
</table>

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.

وتستخدم الذباب من النمط الجيني ث 1118 لشرح كيفية يتم تنفيذ الفحص. وتستخدم المسوخ ث 1118 عادة لتوليد خطوط المعدلة وراثيا وللسيطرة على الخلفية الوراثية الجينات المحورة ملحوظ مع الجين الأبيض. عادة، لإجراء التجارب السلوكية، وbackcr...

Watch this Scientific Journal Video about The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster at JoVE.com

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.

الشعيرات الدموية المغذية الفحص يقيس كمية الطعام في<em&gt; ذبابة الفاكهة</em

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

The overall goal of the CApillary FEeder assay is to observe and measure liquid food intake of adult flies. This method can help to answer key questions in metabolic research such as how actual feeding behavior is initiated and how food consumption is terminated. The main advantages of this technique are that it's easy to use and highly reliable.Visual demonstration of the method is critical as several preparatory steps need to be carefully executed for consistent results. To begin the experiment, obtain a drosophila melanogaster culture plastic vial and seal it with a manufactured Plexiglass lid containing an O-ring. Use five microliter microcapillaries with one microliter marks to present the liquid food.Cut off the top of a two to 20 microliter pipette tip and insert the tip into the whole of the lid to position the capillaries in the conical openings. Insert an uncut two to 20 microliter pipette tip into the same opening to prevent flies from escaping. Load eight or 20 flies by trapping or with a blow pipe through the lid's central opening and close the hole with a sponge bung.Next, place the multiple prepared vials into a plastic box with a gridded inlay to handle them safely. To control breeding conditions, introduce 35 virgin females and 15 males for each experimental group into a plastic culture vial containing 50 milliliter fly food. Allow the flies to lay eggs for the first three days, then transfer adult flies to fresh food vials and let them lay eggs for two more days.After two days, repeat the transfer. On day seven, discard the adult flies. Next, anesthetize two to three day old adult flies using a CO2 pad and collect 100 flies sorted by sex in a 1.5 milliliter plastic tube.Measure their weight with a standard laboratory scale. Determine the wet weight of at least four independent fly groups sorted by sex and use the weight to calculate the microliter food consumption per milligram fly. For a three hour assay, use 20 flies in two filled capillaries.For a long term experiment, use a group of eight flies with a supply of four filled capillaries. Fill up to 10 capillaries simultaneously with previously prepared colored food solution. Insert the capillaries into the food solution and then tilt them to a 45 degree angle to accelerate filling.Stop when the liquid reaches the top five microliter mark of the capillary. Remove excess solution on the outside and inside of the capillary with tissue paper. Carefully remove the pipette tip that is closing one of the outer openings and insert a filled glass capillary, bottom end first.Secure the capillary by placing the pipette tip back next to the capillary. Repeat this step for all food solutions tested. Next, place the capillary ends inside all of the vials at the same level three to four centimeters from the lid.Leave some distance between the filter paper to prevent the capillary from leaking. Use a marker pen to label the upper end of the colored liquid. Label them individually using a stripe code to ensure the different capillaries can be identified.Place prepared CAFE assays into a plastic box with a gridded inlay. Use a sponge bung to apply 100 microliters of fresh water every 24 hours to keep the humidity constant inside the assay. Use four separate vials filled with 30 milliliters of double distilled H2O as humidity devices and place them next to the CAFE assays in the plastic box.Cover the plastic box to create a humidity controlled environment during the experiment. Transfer the box to a secure position in a temperature, light, and humidity controlled climate chamber for the experimental period. Before each 24 hour interval, make note of the dead flies and use the number of live flies to calculate the consumption per fly for the following period.At the end of the assay and before replacing the capillary, keep the CAFE assay in the upright position. Do not remove the lid and mark the lower meniscus of the capillary. Discard the data if the mark end is not below the initial mark.Replace the used capillaries with freshly filled capillaries for long term experiments every 24 hours. Measure the decline of the meniscus, then discard the old capillaries. Then carefully remove the capillaries from the assay and store them for data collection.Check if the liquid inside the capillary reached the lower end. If the liquid inside the capillary did not reach the lower end, discard the data. After the experiment, dismantle the setup and wash the vials, lids, and sponge bungs in a soap bath.Then dry them overnight at room temperature for further use. Repeat experiments with the same genotypes on at least three different days. Using a caliper, measure the distance between mark beginning and mark end on the capillary.Finally, discard the capillaries after the measurements. Two food choices were offered, a 1 molar sucrose solution and a 1 molar sucrose solution with 15%ethanol. Visual examination of the abdomen indicates the flies feed on both solutions.However food consumption per fly is nearly two folds greater for the solution containing ethanol. The ethanol preference was also observed in long term experiments where a group of eight flies had access to similar food sources for four days. The CApillary FEeder assay also reveals that male and female flies prefer higher sucrose concentrations over lower sucrose concentrations.And male flies consume significantly more of the high sucrose concentrations than their female counterparts. While attempting this procedure, it's important to remember to perform the assay in a controlled fashion and to repeat the experiment with the same genotypes on at least three different days. In addition, when testing mutant flies, it's important to consider whether the flies can actually perform the task required to obtain the food, such as sending the food source or climbing up and down the capillary.

Research

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Neuroscience

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

تسجيلات Sensillum واحدة في الحشرات ذبابة الفاكهة السوداء البطن و الأنوفيلة الغامبية</em

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Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

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تقرن Nanoinjection والفحص الكهربية للكشف عن النشاط الحيوي للمركبات باستخدام ذبابة الفاكهة السوداء البطن نظام الألياف العملاق

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Optogenetic تحفيز السلوك في الهروب<em&gt; ذبابة الفاكهة الدروسوفيلا</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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تحديد العفوي الحركي آخر في<em&gt; ذبابة الفاكهة السوداء البطن</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

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الفحص مباشرة للالكمي لتجنب الاجتماعي في<em&gt; ذبابة الفاكهة السوداء</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

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Conditions Affecting Social Space in Drosophila melanogaster

الظروف التي تؤثر في الفضاء الاجتماعي<em&gt; ذبابة الفاكهة السوداء البطن</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

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Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

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Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

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We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...