ونحن ممتنون لT. Stürner للتصحيحات مفيدة، المناقشات والتعليقات على المخطوطة. SL Zipursky لتوفير مخزون ذبابة. M Schölling لأداء معالجة الصور. تم تنفيذ جزء من تحليل الصور في مختبر A. Kakita ل. وأيد هذا العمل من قبل الكسندر فون همبولت مؤسسة وJSPS المنح الدراسية للبحوث في الخارج (AS)، JSPS الزملاء (SH-S)، المنح التحتية المعونة من أجل البدء (24800024)، على مناطق المبتكرة (25110713)، موتشيدا، تاكيدا، اينامورى، دايتشي سانكيو-، أسس توراي (TS)، DZNE التمويل الأساسي (GT) ومرفق DZNE ضوء المجهر (CM).

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The authors have nothing to disclose.

في هذه الدراسة، وأظهرت لنا كيفية إعداد ظروف الإضاءة لفضح الذباب لشدة الضوء متساوية. نحن كميا ليس فقط على عدد من نقاط وعلامة متشابك ولكن يمكن أيضا حل مكانيا كثافة نقاط الاشتباك العصبي على طول المحاور وقياس مستوى تمركز البروتين علامة في المناطق هيولية. هذه التقييمات ث?...

<html> التشكيل وظيفة متشابك يساهم في قدرة ملحوظة في الجهاز العصبي للرد على وجه التحديد أو التكيف مع تغير المحفزات البيئية. ضبط قبل المشبكي إطلاق سراح الحويصلة احتمال هي طريقة واحدة للسيطرة على قوة متشابك 1. متشابك الافراج عن حويصلة يحدث في المنطقة النشطة (AZ)، وهي منطقة متخصصة للغشاء قبل المشبكي 2. يتميز من الألف إلى الياء من قبل كاسيت بروتينات معينة 3، 4. معظم البروتينات التي تسهم في التجمع من الالف إلى الياء والحفظ جدا في الديدان الخيطية والحشرات والثدييات 5. وتشير الدراسات الحديثة إلى أن مستوى نشاط الخلايا العصبية وينظم التركيب الجزيئي من الألف إلى الياء، وهذا بدوره يساهم في تنظيم الانتاج وظيفية على حد سواء في المختبر والمجراة 6، 7،&gt; 8. لقد وجدنا سابقا أن AZS مبصرة تخضع إعادة الجزيئي في ذبابة الفاكهة بعد التعرض لفترات طويلة للضوء المحيط الطبيعية 9. في هذه الحالة، لاحظنا أن عدد Bruchpilot (بدر) -positive AZS تم تخفيض في المحاور مبصرة. البروتينات بدر / CAST / عائلة الايائل هي اللبنات الأساسية للAZS في الفقاريات واللافقاريات نقاط الاشتباك العصبي 10. في المسوخ BRP ذبابة الفاكهة، أثار إطلاق سراح الحويصلة يتم منعها 11 و 12. بقايا الأحماض الأمينية C-محطة 17 من بدر ضرورية لتجميع حويصلة متشابك في ذبابة الفاكهة الوصل العصبي العضلي (NMJ) 13، 14. وأظهرت هذه الدراسات على الدور المحوري لهذا الجزيء في المؤسسة من الألف إلى الياء وظيفة. مع أداة الوراثية التي تم تطويرها مؤخرا، متشابك توصيف مع إعادة التركيب (STAR)، بدريمكن ملاحظتها في الجسم الحي في أنواع معينة من الخلايا، في مستويات التعبير الذاتية وعلى قرار المشبك واحد 15. هذه الأداة يجعل من الممكن لتقييم ديناميات الذاتية من نقاط الاشتباك العصبي كميا في الجهاز العصبي المركزي معقدة. كانت هناك العديد من الدراسات بما في ذلك التحديد الكمي المشبك استنادا إلى بيانات تم الحصول عليها من الفحص المجهري متحد البؤر. تم تقييم التعديلات متشابك من خلال قياس الطول، المنطقة، وحجم، وكثافة وتحصي عدد استنادا إلى تطبيقات البرمجيات المتطورة. على سبيل المثال، يوفر يماغيج مجانية طريقة القياس الكمي لمجموع مساحة متشابك والتدابير كثافة متشابك في ذبابة الفاكهة NMJ 16. وقد تم تقدير عدد المواقع colocalization من علامات قبل وبعد المشبكي باستخدام البرنامج المساعد &quot;نقاط ومحلل&quot; متاح على منصة برنامج ImageJ 17. بدلا من ذلك، متعدد قدم المساواةبرنامج يستند إلى adigm العددية بيئة الحوسبة، المشبك الكاشف (SYND)، ويمكن تتبع تلقائيا التشعبات من الخلايا العصبية المسماة مع علامة فلوري، ثم يقيس مستويات بروتين متشابك بوصفها وظيفة من المسافة من جسم الخلية 18. وقد تم تصميم البرنامج متشابك تحليل نقاط و(SynPAnal)، لتحليل السريع للصور 2D من الخلايا العصبية المكتسبة من متحد البؤر المجهري أو الفلورسنت. وتتمثل المهمة الرئيسية لهذا البرنامج هي الكمي التلقائي والسريع في كثافة وشدة من البروتين نقاط و19. مؤخرا، تم إنشاء القائم على التعلم خوارزمية الكشف المشبك التلقائي لتقدير عدد متشابك في 3D 20، والاستفادة من البرنامج المساعد التصور-3D تحليل (Vaa3D) 21. صورة التجارية برامج التحليل أيضا أدوات قوية لالتحديد الكمي متشابك. على سبيل المثال، fluorescentlyتم كميا مستقبلات الناقل العصبي المسمى أو مكون من الالف إلى الياء قبل المشبكي في ثلاثة أبعاد مع الدقة المشبك واحد في C. ايليجانس 22 أو نظام ذبابة الفاكهة حاسة الشم 23، 24، مما سمح لمئات من نقاط الاشتباك العصبي إلى أن تتسم بسرعة في عينة واحدة. هنا، فإننا نقدم وسيلة من برنامج تحليل صورة مخصصة المكونات في تنفيذها في عدة نموذج بيئة الحوسبة العددية التي تسمح لتحليل جوانب شبه تلقائيا متعددة من AZS، بما في ذلك عددهم والتوزيع ومستوى التخصيب من المكونات الجزيئية لل الألف إلى الياء. وهكذا، هذا التحليل المعقد سمح لنا لتقييم ديناميات مكونات متشابك في محطات محوار تحت ظروف بيئية مختلفة. نحن التحقيق في تأثير التعرض للضوء على نقاط الاشتباك العصبي الناتج من خلايا مستقبلة للضوء ذبابة الكبار. يتم تنفيذ الإجراء في ثلاث خطوات: 1)استعدادا لالتعرض للضوء، 2) تشريح، المناعية والتصوير متحد البؤر، و3) تحليل الصور.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vial</td>
			<td>Hightech, Japan</td>
			<td>MKC-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Plug</td>
			<td>Thermo Fisher Sciehtific, USA</td>
			<td>AS-275</td>
			<td></td>
		</tr>
		<tr>
			<td>Customized transparent rack made of acrylic resin&#160;</td>
			<td>Shin-Shin Corporation, Japan</td>
			<td></td>
			<td>a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step</td>
		</tr>
		<tr>
			<td>Cool incubator</td>
			<td>MITSUBISHI ELECTRIC, Japan</td>
			<td>CN-40A</td>
			<td></td>
		</tr>
		<tr>
			<td>LED panel</td>
			<td>MISUMI, Japan</td>
			<td>LEDXC170-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital light meter</td>
			<td>CEM</td>
			<td>DT-1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly pad</td>
			<td>Tokken, Japan</td>
			<td>TK-HA03-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (35 x 10 mm)</td>
			<td>Greiner Bio-One International, Germany</td>
			<td>627102</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablet</td>
			<td>Takara, Japan</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Wako, Japan</td>
			<td>160-24751</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Sarstedt, Germany</td>
			<td>A. 152X</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 16%</td>
			<td>NEM, Japan</td>
			<td>3152</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-200</td>
			<td>Gilson</td>
			<td>F123601&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-2</td>
			<td>Gilson</td>
			<td>F144801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-chaoptin antibody</td>
			<td>DSHB</td>
			<td>24B10</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa568-conjugated anti-mouse antibody</td>
			<td>Life Technologies</td>
			<td>A-11031</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide (76 x 26 mm)</td>
			<td>Thermo Fisher Scientific Gerhard Menzel B.V. &#38; Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip (18 x 18 mm, 0.17 mm)</td>
			<td>Zeiss, Germany</td>
			<td>474030-9000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Industrial Microscopes</td>
			<td>Olympus, Japan</td>
			<td>SZ61-C-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope Lighting</td>
			<td>Olympus, Japan</td>
			<td>KL 1600 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscopy</td>
			<td>Zeiss, Germany</td>
			<td>LSM780</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane, Switzerland</td>
			<td></td>
			<td>Version 7.6.4 or above</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>The MathWorks, Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel for Mac&#160;</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing synaptic modulation of drosophila melanogaster photoreceptors after exposure to prolonged light

الجهاز العصبي لديه قدرة ملحوظة على التكيف والاستجابة للمؤثرات المختلفة. ويتحقق هذا التعديل العصبي إلى حد كبير من خلال اللدونة على مستوى متشابك. المنطقة النشطة (أ-ي) هي المنطقة في الغشاء قبل المشبكي التي تتوسط الافراج عن العصبي ويتكون من مجموعة كثيفة من البروتينات سقالة. AZS من ذبابة الفاكهة (ذبابة الفاكهة) المستقبلات الضوئية تخضع لإعادة الجزيئي بعد التعرض لفترات طويلة للضوء المحيط الطبيعي. وبالتالي فإن مستوى نشاط الخلايا العصبية يمكن إعادة ترتيب التركيب الجزيئي للمن الالف إلى الياء والمساهمة في تنظيم الانتاج وظيفية. بدءا من التعرض للضوء إعداد انشاء لالمناعية، تفاصيل هذا البروتوكول كيفية تحديد عدد، والتوزيع المكاني، ومستوى تمركز جزيئات متشابك في AZS في المستقبلات الضوئية ذبابة الفاكهة. عن طريق تحليل الصور البرمجtware، تم تحديد مجموعات من المكون AZ-تنصهر GFP Bruchpilot لكل R8 مبصرة (R8) محطة محور عصبي. تم تعيين الكشف عن البقع Bruchpilot تلقائيا إلى محاور R8 الفردية. لحساب توزيع تردد بقعة على طول المحور، نفذنا البرنامج المساعد البرمجيات المخصصة. وقد تم تحديد بداية نقطة لكل محور عصبي ونقطة النهاية يدويا، وكان من المتوقع على الخط الذي يربط بين بداية ونقطة النهاية موقف كل بقعة Bruchpilot. إلى جانب عدد من مجموعات Bruchpilot، ونحن أيضا كميا مستوى تمركز Bruchpilot-GFP ضمن مجموعات. وتعكس هذه القياسات بالتفصيل ديناميات متشابك حل مكانيا في الخلايا العصبية واحدة تحت ظروف بيئية مختلفة للمؤثرات.

العين مجمع ذبابة الفاكهة تضم ~ 780 ommatidia، تحتوي كل منها على ثمانية أنواع من المستقبلات الضوئية (R1-8). R7 وR8 مشروع محاور لالعقدة البصرية الثانية، النخاع، حيث أنها تشكل نقاط الاشتباك العصبي في طبقات M6 و M3، على التوالي 26. لدراسة تأثير التعر...

Watch this Scientific Journal Video about Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light at JoVE.com

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

نحن هنا تظهر كيفية تحديد عدد والتوزيع المكاني للمناطق نشطة متشابك في المستقبلات الضوئية البطن ذبابة الفاكهة، سلط الضوء مع الواسمات الجزيئية المشفرة وراثيا، وتعديل على بعد التعرض لفترات طويلة للضوء.

تحليل متشابك التحوير من<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء بعد التعرض للضوء لفترات طويلة

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

analyzing-synaptic-modulation-drosophila-melanogaster-photoreceptors

Research

JoVE Journal

Neuroscience

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

6.5K Views.  Niigata University.  نحن هنا تظهر كيفية تحديد عدد والتوزيع المكاني للمناطق نشطة متشابك في المستقبلات الضوئية البطن ذبابة الفاكهة، سلط الضوء مع الواسمات الجزيئية المشفرة وراثيا، وتعديل على بعد التعرض لفترات طويلة للضوء. 

Video: Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

في التصور المجراة من الحويصلات متشابك ضمن محاور عصبية ذبابة الفاكهة القطاعي اليرقات

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

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 ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

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 ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

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 ...

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

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

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

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

عالية الإنتاجية الفحص لفحص تفضيلات بيضة زرع الفردي<em&gt; ذبابة الفاكهة</em

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

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

تستخدم علفا بروتوكول مسار طول ل<em&gt; ذبابة الفاكهة</em&gt; يرقات

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

الطريقة الكهربية لالردود تسجيل بين الخلايا الجهد ل<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء وInterneurons إلى النور المحفزات<em&gt; في فيفو</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

طريقة الكهربية لالكامل خلية الجهد المشبك تسجيلات من<em&gt; ذبابة الفاكهة</em&gt; المستقبلات الضوئية

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

أزاله الحساسية والتعافي من المستقبلات الضوئية جراد الصيد عند تسليم التحفيز الخفيفة

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.