تدعمه المنحة R01 MH 099557 المعاهد الوطنية للصحة

1-"تصنيع أقطاب تسجيل" بروتوكول <ol><li>سحب أقطاب الزجاج <ol><li>استخدام ما يلي ساحبة ميكرويليكترودي (انظر الجدول للمواد): <ol><li>1 خط: الحرارة سحب 510-السرعة 30 مرة 250؛ السطر 2: الحرارة سحب 490-السرعة 30 مرة 250- 
			ملاحظة: وحدات الوقت تناظر 0.5 مللي ثانية كل وحدة؛ وحدات أخرى النسبية. وينبغي تعديل قيمة الحرارة لكل خيوط بعد إجراء اختبار الانحدار.</li>
		</ol></li> <li>استخدام مجهر (35 x التكبير) للتأكد من أن القطر الداخلي لمسرى سحبت في حدود 7-10 ميكرون ( الشكل 1A). تخزين الشعيرات الدموية في حاويات مغلقة بأحكام لمنع تراكم الغبار.</li>
	</ol></li> <li>النار لتلميع <ol><li>النار البولندية الشعيرات الدموية (80-90% قيمة الحد الأقصى من الحرارة s 1-2) استخدام الصغرى-فورج (انظر الجدول للمواد). تأكد من القطر الداخلي النهائي القطب مصقول 5 ميكرومتر ( الشكل 1A) 27-</li>
	</ol></li> <li>الانحناء ملاحظة: الانحناءات اثنين مصنوعة من أجل موقف مسرى أعلى عضلة تحت هدف تكبير عالية.
	<ol><li>استخدام الجهاز المبين في الشكل 1 باء. الإصلاح الكهربائي في المناور وضع الطرف الخيط، لمس عدم إجرائه.</li>
		<li>مجموعة الحرارة القيمة إلى 60-70 في المائة من الحد الأقصى، واضغط على دواسة الصغرى-فورج ل s 1-2 (انظر الجدول للمواد) لتسخين الشعيرة. استخدام إبرة على شكل L لهدم بلطف غيض من قطب كهربائي ( الشكل 1B الموسع). جعل منعطف في حوالي 90 س ( الشكل 1).</li>
		<li>عقد مسرى على لهب الشعلة باليد باستخدام الملقط وجعل منحنى الثانية على مسافة 7-10 ملم من أول منعطف وزاوية حوالي 120 o ( الشكل 1).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg"/> الرقم 1. الخطوات النهائية لتصنيع ميكروبيبيتي. نصائح القطب (A) بعد سحب والتلميع النار. (باء-1) الإعداد لنصيحة الانحناء. ويرد (باء-2) منطقة محاصر الموسع على الحق. مسرى والشعيرة ثابتة حيث يكون تلميح القطب وضعه أعلى قليلاً من الأسلاك وعدم لمس. (جيم 1) مسرى التسجيل. ويرد (جيم 2) منطقة محاصر الموسع على الحق. أول منعطف بزاوية 90 درجة تقريبا، والمسافة بين أول منعطف وتلميح القطب حوالي 1 مم-مقياس بار = 3 مم-<a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a>
 2-"خطوات تحضيرية إضافية" <ol><li>تحضير مثل هياموليمف (HL3) الحل (مم): كلوريد الصوديوم 70, 5 بوكل و 20 مجكل 2، 10 NaHCO 3، 5 تريهالوسي، السكروز 115، هيبيس 5 و 1 مم كاكل 2؛ ضبط درجة الحموضة إلى 7.3- 7.4. يبقى الحل في الثلاجة وجعلها جديدة كل أسبوع.</li>
	جعل <li>بيبيتس التحفيز نفس الطريقة كتسجيل الزجاج بيبيتس، إلا أن هناك لا ضرورة ينحني لهم. 
	ملاحظة: إعداد أقطاب التحفيز يرد بالتفصيل في 28 و 27. قطر نهائي بعد التلميع النار ينبغي أن تكون في حدود 5-7 ميكرون.</li>
	<li>إدراج متصلاً ماصة تحفيز في حامل ميكروليكترودي حقنه.</li>
	<li>تصنيع لوحات التشريح من أطباق بيتري صغيرة (35 × 10 مم) مغلفة بالمطاط سيليكون الإيبوكسي (انظر الجدول للمواد) كما هو موضح في 28- 
	ملاحظة: يجب أن يكون تصلب تماما قبل استخدام المطاط سيليكون.</li>
	<li>بيك يرقات الطور الثالث تجول وتشريح كما هو موضح في 27 ، ، من 28 29 ، 30- 
	ملاحظة: لتصور بوتونس، استخدام سلالة يطير CD8-التجارة والنقل (انظر الجدول للمواد) <ol><li>استخدام الملقط (انظر الجدول للمواد)، اختيار يرقات الطور rd 3 من قنينة.</li>
		<li>دبوس اليرقات، وضع دبوس الأول الخلفي وآخر دبوس الأمامي (قريبة من خطاطيف الفم)-</li>
		<li>الحل إضافة HL3-</li>
		<li>جعل خفض استخدام مقص الربيع (انظر الجدول للمواد) على طول الطريق من دبوس أعلى إلى أسفل أحد في الجهة الظهرية من اليرقات.</li>
		<li>دبوس فيليه اليرقات، وضع دبابيس إضافية 2 على الجانبين الأيمن والأيسر من اليرقات.</li>
		<li>إزالة الشجاعة واستخدام الملقط تراتشياس.</li>
		<li>قص الأعصاب خارج الحبل البطني العصب باستخدام مقص الربيع-</li>
	</ol></li></ol> 3. التسجيلات الكهربائية من إيكس <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg"/> الرقم 2. إعداد تسجيل. نموذج NMJ المثبتة السيليكون وضع طبق بتري المغلفة (السهم) عبر المرحلة منقولة من المجهر مجمع تستقيم مزودة بقدرات ابيفلوريسسينسي وهدفا تضخم عالية، وهما ميكرومانيبولاتورس. وتتمركز المجهر على طاولة المضادة اهتزاز. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a>
<ol><li>تسجيل <ol><li>مكان طبق بيتري مع إعداد على المسرح المجهر ( الشكل 2). إدراج مرجع مسرى في الحمام-</li>
		<li>ملء مسرى التسجيل مع الحل HL3. تحت الهدف 10 x (انظر الجدول للمواد)، تزج قطب كهربائي في الحمام ووضعه على عضلات 6 و 7 من شرائح البطن 2 أو 3 أو 4 باستخدام ميكرومانيبولاتور ((انظر الجدول للمواد) الشكل 3 ألف-1 وألف-2)-</li>
		<li>التبديل الهدف إلى 60 x (انظر الجدول للمواد). التركيز على مجال الاهتمام باستخدام برنامج التحصين الموسع-الأسفار أو البصريات DIC. ضع طرف القطب على رأس بوتون متشابك ( الشكل 3 باء-1-3)-</li>
		اضغط <li>الكهربائي بلطف جداً على العضلات. قد تلحق الضرر NMJ الضغط المفرط أو الحث على زيادة في نشاط متشابك عفوية. تأكد من غيض مسرى ليس انسداد--إذا كان، يستعاض عن ذلك</li>
		<li>التبديل على مكبر للصوت والمجلس A/D، والكمبيوتر-</li>
		<li>اختيار وضع المشبك الجهد على مكبر للصوت.</li>
		<li>بدء تشغيل البرنامج اقتناء واختيار &#39; خالية من الفجوة &#39; الوضع.</li>
		<li>مراقبة مظهر ميكس على شاشة الكمبيوتر.</li>
		<li>التأكد من أن السعة ميكس في حدود 0.2-0.7 na. 
		ملاحظة: تشير إلى أصغر إيكس أن مسرى تسجيل العيوب أو أن عدم وضعها بشكل صحيح.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg"/> الرقم 3. تصور boutons متشابك. صورة برايتفيلد (A) A قطعة هيمي تحت 10 x التكبير (أ-1) ومنطقة محاصر الموسع عرض عضلات 6 و 7 (ألف-2، يمثل السهم مسرى التسجيل). شريط المقياس = 50 ميكرومتر. (ب) boutons متشابك هي تصور في خط المورفولوجية مع علامة العصبية مشفرة جينياً (CD8-التجارة والنقل) باستخدام برنامج التحصين الموسع-الأسفار التصوير (باء-1) أو DIC البصريات (باء-2). يتم أخذ صور مع 60 × الهدف والمكعب تصفية للتصوير بالتجارة والنقل (انظر الجدول للمواد). سينابتييتم وضع علامة بتونس ج مع الأسهم، ويظهر تراكب الفلورسنت والصور DIC في باء-3. شريط المقياس = 10 ميكرون. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a>
<ol start="2"> <li>التحفيز <ol><li>باستخدام ميكرومانيبولاتور، تحت عنصر تحكم visual، ضع التحفيز الكهربائي قرب إكسون إينيرفاتينج شرائح البطن 2-4-</li>
		<li>تطبيق ضغط سلبي عن طريق سحب المكبس للمحاقن متصلاً بصاحب القطب، بحيث أن يتم سحبها في إكسون داخل قطب كهربائي ( الشكل 2).</li>
		<li>بدوره على مشجعا. أدر في وحدة العزل (انظر الجدول للمواد) لتعيين صفر الحالي، وثم بلطف زيادة، حتى تظهر إيكس (أو حتى يتم التوصل إلى العتبة).</li>
		<li>إجراء التحفيز في نظام سوبراثريشولد، مع تحفيز الحالي ازدادت حوالي مرتين، مقارنة بالحد الأدنى لمراقبة اجكس. 
		ملاحظة: في تجربتنا، مثل كثافة التحفيز الأمثل لتجنب فشل إمكانات العمل وإمكانات عمل إطلاق النار. على سبيل المثال، إذا اجكس تظهر في التحفيز الحالية 0.2 mA، استخدام تيار 0.4 mA طوال التجربة.</li>
	</ol></li> <li>ختم المقاومة <ol><li>قياس المقاومة ختم القطب ماكروباتش التسجيل قبل تشغيل التبديل المقاومة الكهربائي من مكبر للصوت إلى &#34; ختم اختبار &#34; الموقف. ملاحظة أنه سيتم عرض قيمة ختم المقاومة في GΩ في &#34; الحالية &#34; النافذة.</li>
		تأكد من ختم المقاومة في حدود 0.5-2 م <li>&#8486;. قيم خارج هذا النطاق تشير إلى أن مسرى مصقول غير صحيح أو غير صحيح المتمركزة.</li>
		<li>رصد المقاومة الختم خلال التسجيل، والتأكد من أنها تظل ثابتة طوال التجربة.</li>
	</ol></li></ol> 4. تحليل <ol><li>تحليل تسجيلات توظيف تخصيص برامج لتحليل (انظر الجدول للمواد)- 
	ملاحظة: لدينا مختبر يستخدم برامج داخلية كوانتان 31، التي يتم تخصيصها للكشف عن إيكس وسجلت فوكالي ميكس ( الشكل 4). هذا البرنامج يتضمن عامل تصفية رقمية ضبابي ويتيح الكشف عن قمم كانتال في تداخل الأحداث المتعددة كانتال ( الشكل 4 أ). نهوج أخرى موصوفة في 17-</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg"/> الرقم 4. تحليل كانتال. الكشف عن ميكس (A) وايكس (ب) بالبرمجيات كوانتان. منطقة الحدث يتسم باللون الأخضر، وتميزت قمم رؤوس الأسهم الحمراء. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions.">Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.</a> J Comp Physiol A. 175 (2), 179-191 (1994).</li><li>Pawlu, C., DiAntonio, A., Heckmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postfusional+control+of+quantal+current+shape.">Postfusional control of quantal current shape.</a> Neuron. 42 (4), 607-618 (2004).</li><li>Akbergenova, Y., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+maintains+the+reserve+vesicle+pool+and+spatial+segregation+of+the+recycling+pool+in+Drosophila+presynaptic+boutons.">Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons.</a> Brain Res. 1178, 52-64 (2007).</li><li>Akbergenova, Y., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+endosomal+endocytic+pathway+increases+quantal+size.">Enhancement of the endosomal endocytic pathway increases quantal size.</a> Mol Cell Neurosci. 40 (2), 199-206 (2009).</li><li>Vasin, A., Volfson, D., Littleton, J. T., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+Complexin+Accessory+Helix+with+Synaptobrevin+Regulates+Spontaneous+Fusion.">Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.</a> Biophys J. 111 (9), 1954-1964 (2016).</li><li>Wong, K., Karunanithi, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+unit+populations+at+the+Drosophila+larval+neuromuscular+junction.">Quantal unit populations at the Drosophila larval neuromuscular junction.</a> J Neurophysiol. 82 (3), 1497-1511 (1999).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction.">The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.</a> Pflugers Arch. 391 (1), 35-40 (1981).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Ca2++inflow+to+quantal,+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle.">Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.</a> Pflugers Arch. 422 (2), 129-142 (1992).</li><li>Marrero, H. G., Lemos, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Loose-Patch-Clamp method. , (2007).</li><li>Wu, W. H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle.">Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle.</a> J Vis Exp. (45), (2010).</li><li>Verstreken, P., Ohyama, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Imlach, W., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

الكتاب ليس لها علاقة بالكشف عن.

المورفولوجية يمثل كائن نموذج مفيد لدراسة انتقال متشابك. وقد استخدمت العديد من تكوينات التسجيل في NMJ اليرقات، بما في ذلك تسجيلات داخل الخلايا من إمكانات متشابك، تسجيلات تيارات متشابك مع قطب هما الجهد المشبك33،34، والتنسيق ماكروباتش تسجيلات متشابك التيا...

المورفولوجية نظام نموذجا ممتازا دراسة الآليات الجزيئية السيطرة على انتقال متشابك. النظام العصبي العضلي في المورفولوجية جلوتاماتيرجيك، وذلك يمكن استخدام الوصلات العصبية العضلية المورفولوجية (NMJ) لدراسة ملامح مصانة من الإصدار جلوتاماتيرجيك. منذ يان وجان الدراسة1، استخدمت يرقات الطور الثالث على نطاق واسع لدراسة انتقال متشابك مقولة والعفوية عن طريق رصد إمكانات مفرق ضادات (ايبس) أو التيارات (إيكس). عادة يتم تسجيل ايبس إينتراسيلولارلي مع مايكرو زجاج حادة-قطب كهربائي، وأنها تعكس نشاط NMJ كاملة، بما في ذلك جميع boutons جعل نقاط الاشتباك العصبي في الألياف العضلية معين.
على النقيض من ذلك، يمكن تسجيل النشاط على عدد محدود من المواقع لإطلاق سراح فوكالي عن طريق وضع تلميح ميكروبيبيتي قرب المحطات العصبية أو varicosities متشابك. واستخدمت هذا الأسلوب أصلاً كاتز وميليدي2، والتسجيلات خارج الخلية المحورية قد استخدمت بنجاح في الأعمال التحضيرية NMJ عدة، بما في ذلك الضفدع3،،من45، الماوس6 , 7 , 8و القشريات9،10،11،،من1213،،من1415،16 المورفولوجية17،،من1819،20،21،،من2223. كذلك وضع هذا النهج دوديل، الذين الأمثل إعادة ترميز أقطاب24،25ماكروباتش. في التنفيذ دوديل، تتطابق هذه التقنية طريقة فضفاضة-التصحيح-المشبك26.
NMJ اليرقات المورفولوجية حددت بوضوح boutons متشابك، وخطوط المحورة وراثيا مع العلامات الفلورية العصبية المشفرة جينياً (انظر الجدول للمواد) متوفرة بسهولة. هذه المزايا مكنتنا من تسجيل اجكس وميجكس من21،20،بوتون متشابك محدد22. هنا، يمكننا وصف هذا الأسلوب بالتفصيل.

<table><tbody><tr><td>Sutter P-97</td><td>Sutter instrument</td><td>P-97</td><td>Microelectrode puller</td></tr><tr><td>Narishige MF-830</td><td>Narishige</td><td>MF-830</td><td>Microforge</td></tr><tr><td>WPI MF200</td><td>WPI</td><td>MF200</td><td>Microforge</td></tr><tr><td>Glass capilaries</td><td>WPI</td><td>B150-86-10</td><td>Glass capilaries</td></tr><tr><td>Microtorch 1WG61</td><td>Grainer</td><td>1WG61</td><td>Microtorch</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD 184</td><td>Silicone for dissection plates preparation</td></tr><tr><td>Dissection pins</td><td>Amazon</td><td>B00J5PMPJA</td><td>Pins for larvae positioning</td></tr><tr><td>Tweezers</td><td>WPIINC</td><td>500342</td><td>Tweezers for placing pins, removing the guts and tracheas.</td></tr><tr><td>Scissors</td><td>WPIINC</td><td>501778</td><td>Scissors for cutting the cuticula of the larvae and nerves.</td></tr><tr><td>Olympus BX61WI</td><td>Olympus</td><td>BX61WI</td><td>Upright microscope</td></tr><tr><td>Olympus Lumplan FL N 60x</td><td>Olympus</td><td>UPLFLN 60X</td><td>Microscope objective 60X</td></tr><tr><td>Olympus UPlan FL N 10x</td><td>Olympus</td><td>Uplanfl N 10X</td><td>Microscope objective 10X</td></tr><tr><td>Narishige Micromanipulator</td><td>Narishige</td><td>MHW-3</td><td>Three-axis Water Hydraulic Micromanipulator</td></tr><tr><td>npi Electronic GmbH ELC-03XS</td><td>npi Electronic GmbH</td><td>ELC-03XS</td><td>Electrophysiological amplifier</td></tr><tr><td>A.M.P.I Master 8</td><td>A.M.P.I.</td><td>Master 8</td><td>Electrical stimulator</td></tr><tr><td>A.M.P.I Iso-Flex</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolator</td></tr><tr><td>TMC antivibration table</td><td>TMC</td><td>63-9090</td><td>Antivibration table</td></tr><tr><td>TMC Faraday cage</td><td>TMC</td><td>81-333-90</td><td>Faraday cage</td></tr><tr><td>Digidata 1322A</td><td>Axon Instruments</td><td>Digidata 1322A</td><td>Digidata</td></tr><tr><td>Computer</td><td>Dell</td><td>Dell Dimension 5150</td><td>Computer with Win XP OS</td></tr><tr><td>Electrode holder</td><td>WPI</td><td>MEH3SW</td><td>Electrode holder</td></tr><tr><td>Optical filter</td><td>Omega optical</td><td>XF 115-2</td><td>Filter cube for Green Fluorescent Protein (GFP) detection</td></tr><tr><td>pCLAMP 8</td><td>Axon Instruments</td><td>8.0.0.81</td><td>Software for signal recording</td></tr><tr><td>Quantan</td><td>In-house software</td><td>-</td><td>Software for signal processing</td></tr><tr><td>Canton-S (Wildtype)</td><td>Bloomington Stock Center</td><td>64349</td><td>Control fly line</td></tr><tr><td>cpx SH1</td><td>Generous Gift of J.T. Littleton</td><td>-</td><td>Complexin knock-out fly line with increased spontaneous exocytosis</td></tr><tr><td>CD8-GFP</td><td>Bloomington Stock Center</td><td>5137</td><td>Fly line with neuronal fluorescent (GFP) Tag</td></tr></tbody></table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

الوصلات العصبية العضلية المورفولوجية (NMJ) نظام نموذجا ممتازا لدراسة انتقال متشابك جلوتاماتيرجيك. يصف لنا هذا أسلوب تسجيلات ماكروباتش المحورية للتيارات متشابك من تصور بتونس في NMJ اليرقات المورفولوجية . يتطلب هذا الأسلوب تلفيق مخصصة لتسجيل ميكروبيبيتيس، فضلا عن مجهر المجمع مجهزة بتكبير عالية والهدف غمر المياه البعيدة المدى والبصريات التباين (DIC) التدخل التفاضلية ونيون مرفق. يتم وضع مسرى التسجيل على رأس بوتون متشابك محدد تصور مع DIC البصريات، وبرنامج التحصين الموسع-الأسفار، أو كليهما. وميزة هذا الأسلوب أنه يتيح رصد النشاط متشابك لعدد محدود من المواقع للإفراج. مسرى التسجيل التي يبلغ قطرها عدة ميكرون، والإفراج عن المواقع المتمركزة خارج حافة القطب لا تؤثر كثيرا على التيارات المسجلة. التيارات متشابك مسجل حركية سريعة ويمكن حلها بسهولة. هذه المزايا ذات أهمية خاصة لدراسات المسخ خطوط الطيران مع تعزيز النشاط متشابك عفوية أو غير متزامن.

تسجيلات ماكروباتش المحورية تمكين رصد النشاط متشابك من boutons متشابك مختارة (الشكل 5). عندما يتم وضع مسرى على الجزء العلوي بوتون متشابك (الشكل 5A، موقع 1)، ميكس مسجل (الشكل 5، موقع 1) الاتساع إلى حد كبير يتجاوز مستوى الضوضاء وحادة ?...

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

تسجيلات ماكروباتش المحورية للتيارات متشابك من الوصلات العصبية العضلية اليرقات المورفولوجية

يمكن تسجيل التيارات متشابك فوكالي من تصور boutons متشابك في الوصلات العصبية العضلية يرقات الطور المورفولوجية الثالثة. هذا الأسلوب يتيح رصد نشاط بوتون واحد متشابك.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Universidad de Cartagena UDEC

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JoVE Journal

Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K Views.  Wayne State University. يمكن تسجيل التيارات متشابك فوكالي من تصور boutons متشابك في الوصلات العصبية العضلية يرقات الطور المورفولوجية الثالثة. هذا الأسلوب يتيح رصد نشاط بوتون واحد متشابك.

Video: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

المقترنة تسجيلات المشبك من تصحيح للسيارات ، والخلايا العصبية في العضلات الهيكلية الهدف اسماك الزرد

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

تشريح والتصوير من المناطق النشطة في ذبابة الفاكهة الوصل العصبي العضلي

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

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

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

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

الكهربية طرق تسجيل الإمكانيات متشابك من يرقات ذبابة الفاكهة من NMJ

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

Biology

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 تحفيز ساطة من الإمكانيات في متشابك ذبابة الفاكهة العضلية المفارق

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

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

تشريح والتسجيل من مفرق العضلية جيم ايليجانس

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

إعداد<em&gt; ذبابة الفاكهة</em&gt; الخلايا العصبية المركزي لل<em&gt; في الموقع</em&gt; تصحيح لقط

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

الكهربية في تسجيل ذبابة الفاكهة الأجنة

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

تسجيلات المشبك التصحيح من الخلايا الجنينية الزرد ماوتنر

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

تحسين عملية تحضير العينة للفحص المجهري الإلكتروني النافذ للتقاطعات العصبية العضلية في يرقات ذبابة الفاكهة

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

ذبابة الفاكهة القياس الكمي للوصلة العصبية العضلية (NMJ): طريقة لتقييم مورفولوجيا الوصلة المشبكية ووظيفتها

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...