Önce başlangıç ​​hazırlar: <ul><li> Wandering yhird instar Drosophila larvaları </li><li> HL3.1 (hemolimf-benzeri Değiştirilmiş) çözümü </li><li> Brent ve McCabe (2008) 3 tarafından açıklanan yöntemleri kullanarak küçük (35 x 10 mm) plastik petri kaplarına Sylgard (şeffaf silikon kauçuk) diseksiyonu plakaları hazırlanmıştır. </li><li> Diseksiyonu pimleri kısa kesilmiş </li><li> Elektrot pipetler teşvik </li><li> Keskin kayıt pipetler </li></ul> HL3 Çözüm: <ol><li> 70 NaCl, 5 KCl, 4 MgCl 2, 10 NaHCO 3, 5 trehaloz, 115 sakaroz, 5 Hepes, pH 7,2: diseksiyonlar ve elektrofizyolojik deneyler sırasında larva HL3.1 çözüm 2 (mM) içeren daldırılır. </li><li> CaCl 2 konsantrasyonları ekstraselüler Ca 2 + gerekli konsantrasyonu sağlamak için HL3.1 çözüm eklenir. <ul><li> Larvaların diseksiyonları HL3.1 kullanarak + konsantrasyonları (kas kasılması önlemek için) + 0.25 mM CaCl 2, buz üzerinde tutulmalıdır düşük Ca 2 yapılmaktadır. </li><li> Elektrofizyolojik deneyler genellikle HL3.1 kaydedilir + 1 mM CaCl 2, ama bu 0.4 ile ayarlanabilir - 1 mM Ca 2 + gerektiği gibi. </li></ul></li><li> HL3.1 çözüm filtresi kullanımdan önce sterilize ve 4 saklanan ˚ C </li></ol> Pipetler uyarıcı ve kayıt hazırlanması: <ol><li> Isı = 390, Filament = 4, Hız = 35 = 200 Gecikme ve = 0 çekin: Kayıt ve uyarıcı pipetler aşağıdaki ayarları kullanarak bir Sutter P-2000 Lazer tabanlı mikropipet çektirmesi ile çekilir. </li><li> Uyarıcı elektrot kopmuş motor aksonlar genişliği biraz daha geniş biter ince duvarlı borosilikat cam kapilerleri hazırlanmıştır. Şeklinde uçları sinir hasarı en aza indirmek için düzgün bir yüzey (bir Narashiga pipet parlatıcı kullanarak) vermek firepolished. Uyarıcı elektrot banyo solüsyonu (HL3.1 + 1 mM Ca 2 +) ile doldurulur. </li><li> Kayıt elektrotlar keskin bir pipet (M 30-60) oluşturmak için çekti ve 3M KCl ile dolu 1.2mm borosilikat cam, hazırlanmıştır. </li></ol> Bölüm 1: Drosophila Larva Diseksiyon <ol><li> Üçüncü instar dolaşan larvalar daha önce 4 açıklandığı gibi vücut duvarı kasları ortaya çıkarmak için disseke. </li><li> Diseksiyonlarının HL3.1 yapılmaktadır + 0.25 mM Ca 2 + küçük silikon plakalar (35 x 10 mm). Larva uyuşturan için diseksiyonlarının buz HL3.1 çözüm olmalıdır ve diseksiyonlarının öncesinde ve sırasında buz üzerinde tutulmalıdır. </li><li> Larva HL3.1 diseksiyonu çözüm + 0.25 mM Ca 2 ekleyerek ve daha az olan kısa diseksiyonu pimleri (~ 2 mm uzunluğunda) kullanılarak elektrofizyoloji deneyler için değiştirilmiş Brent ve McCabe (2008), tarafından açıklanan yöntemleri kullanarak disseke bir deney sırasında mikroskop objektif ve elektrotlar vurmak olasıdır. </li><li> Diseksiyonu takiben, larvaların motor aksonlar kopmuş. Bu MSS nazikçe tutarak ve ventral ganglion arka kasları innerve periferik sinirler kasları (Şekil 1) zarar vermeden kesim böylece biraz yükselterek tarafından yapılır. Daha sonra beyin kaldırılır. </li><li> Disseke larval hazırlık HL3.1 ile iki kez yıkanır + 1 mM Ca 2 +. </li></ol><img src="/files/ftp_upload/1109/fig1.png" alt="Şekil 1" /> Şekil 1 disseke larvaların motor aksonlar kesme yer alan adımları gösteren şematik. MSS forseps ile kaldırdı ve motor aksonlar, beyin tabanında kesilir. Bölüm 2: larva kas hücrelerinde intrasellüler kayıtları. <ol><li> Elektrofizyoloji mikroskop ve batığın HL3.1 ile hazırlık diseksiyonu plaka + 1.5 mM Ca 2 + koyun ve çözüm ile temas halinde olduğu, bu nedenle banyo elektrot düzeltmek . </li><li> Bütün deneyler, üçüncü karın segment (A3) içinde kas 6 yapılmaktadır. Periferik sinirler kasları innerve emme elektrot 5 kullanarak uyarılır. </li><li> Intrasellüler elektrot yerinde titreşimleri önlemek için çanak ilk uyarıcı elektrot yerleştirin. 6 kas innerve motor nöron yakınında uyarıcı elektrot yerleştirin ve kesme sinir cam pipet içine kadar nazik vakum uygulamak. Emme uygulandığında sinirleri germek için dikkatli olun. Biraz bu yüzden, bu yüzden kesim sinir lifleri (Şekil 2) üzerine çekerek değil kas ve pozisyon dokunmadan değildir pipet kaldırın. <img src="/files/ftp_upload/1109/fig2.png" alt="Şekil 2" width="600" height="296" /> Şekil 2 Drosophila larva bir karın segment kas ve motor nöronlar innerve şematik. Kas 6 uyarıcı pozisyonda pipet ve sinirler kopmuş ve kayıt elektrot pozisyonu gösterilmiştir. </li><li> İntra3 M KCl ile dolu keskin Mikroelektronlar hücresel kayıtları yapılır. Üçüncü karın segment (A3) kas 6 merkezinin üstünde kayıt elektrodu yerleştirin ve banyo çözümü için sıfırı ofset girişi ayarlayabilirsiniz. Elektrot yavaş yavaş alt ve kas kas yüzeyi dokunana kadar yüksek optik büyütme altında yaklaşım. Kas nüfuz doğrulamak için osiloskop izleyin. Dinlenme zar potansiyeli -60 mV en az olmalı, ya da hayvan kullanılmamalıdır. Sporadik minyatür son plağın potansiyelleri şimdi görünür olmalıdır. Kayıt yapmaya başlamadan önce bir dakikalık stabilize olması için hücre bırakın. </li><li> Membran potansiyeli değişiklikler bir Axon HS-2A kafa sahne ve Axoclamp 2B amplifikatör ile tespit ve Clampex v 8.2.0.235 (Axon Instruments) ile kaydedilir. Axoclamp 2B pCLAMP yazılım ve Digidata 1322A arayüzü ile birlikte çalışır çalışan bir bilgisayar arayüzü. </li><li> Hücreler mEJP tepkileri ölçmek için herhangi bir uyarana olmadan 3 dakika kaydedildi. İzler Mini analiz yazılımı (Synaptosoft, v 6.0.3) ve belirlenen mEJP genlik ve frekans kullanılarak analiz edildi. </li></ol> Sinir Uyarım: <ol><li> Motor nöron kopmuş sonunda tüm kas tutarlı tepkiler uyandırmak, hem de motor aksonlar için yeterli bir yoğunluk kare voltaj darbeleri (0.3 ms süresi) bir dizi ile uyarılır. </li><li> Uyaran süresi ve aralığı kez göre sürekli darbeler sunmak için programlanmış Master-8 darbe jeneratör, ile üretilir. </li><li> NMJ sinaptik kurtarma uyaranlar arasında beş saniye bekleyin. </li><li> Uyaran gücünü belirlerken, en düşük seviyesi ile başlar ve birkaç denemeden içinde yavaş yavaş yoğunluğunu arttırmak. Uyarılmış bir tepki olarak sonuçları en az uyaran yoğunluğu deneylerde kullanılması gerekir. </li><li> Uygun bir uyaran şiddeti ulaşıldığında, bir bileşik EJP ve bir kas kasılması uyaran tarafından uyarılmış olması gerekir. </li><li> Her kas ve ortalama genlikleri en az 10 uyarılmış potansiyeller kaydedin. </li></ol> Bölüm 3: Temsilci Sonuçlar <img src="/files/ftp_upload/1109/fig3.png" alt="Şekil 3" width="600" height="358" /> Şekil 3 uyarılmış EJP gösteren kas 6 Temsilcisi intraselüler kayıt segmental sinirin elektriksel stimülasyon ve sporadik minyatür son plağın potansiyelleri, ya da mEJP yanıt. Kanton G gibi sağlıklı yabani tip larvaları, 6 kas EJP genlikleri, genellikle yaklaşık 40 mV ve 1-3 mV arasında mEJP amplitüdleri.

<ol>
	<li>Atwood, H. L., Govind, C. K., 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=Differential+ultrastructure+of+synaptic+terminals+on+ventral+longitudinal+abdominal+muscles+in+Drosophila+larvae.">Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae.</a> J. Neurobiol. 24 (8), 1008-1024 (1993).</li><li>Feng, Y., Ueda, A., 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=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., Ramaswami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+of+dynamin+within+individual+Drosophila+synaptic+boutons+relative+to+compartment-specific+markers.">Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.</a> J Neurosci. 16, 5443-5456 (1996).</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=Protocol+for+dissection+of+Drosophila+larvae.">Protocol for dissection of Drosophila larvae.</a> J Vis Exp. , (2008).</li><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol (Lond). 262, 189-214 (1976).</li><li>Katz, L. C., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+construction+of+cortical+circuits.">Synaptic activity and the construction of cortical circuits.</a> Science. 274, 1133-1138 (1996).</li></ol>

Burada açıklanan yöntemler NMJ sinaptik fonksiyon değişiklikleri algılamak için nispeten hızlı ve geniş bir yol sağlar. In vivo elektrofizyolojik kayıtlar bozulmamış hayvanların kullanarak gerçekleştirmek ve genetik ya da farmakolojik manipülasyonlar yapmak için yetenek, Drosophila nörotransmisyon fizyolojik ve genetik açıdan araştırmak için ideal bir hayvan modeli olun. Kas hücreleri çok büyük olduğundan, bazı iki elektrot voltajı kelepçe (TEVC) kayıt için...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Small Petri dishes (35 x 10 mm)</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>1008</td>
<td>&nbsp;</td>
<tr>
<td>SYLGARD 182 Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>3097366-1004</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>475002-9902</td>
<td>&nbsp;</td>
<tr>
<td>Light for microscope</td>
<td>&nbsp;</td>
<td>Schott</td>
<td>KLI500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>pClamp 9 software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>PCLAMP 9 STANDARD</td>
<td>&nbsp;</td>
<tr>
<td>Dissection scissors: 3mm Vannas Spring Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-0</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillaries, with filament (1.2 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B120F-4</td>
<td>&nbsp;</td>
<tr>
<td>Sutter P-2000 Laser Based Micropipette Puller </td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Model P-2000</td>
<td>&nbsp;</td>
<tr>
<td>Pipette polisher</td>
<td>&nbsp;</td>
<td>Narishiga</td>
<td>MF-83</td>
<td>&nbsp;</td>
<tr>
<td>Axon HS-2A head stage</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>Model HS-2A</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Axoclamp 2B amplifier</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>AXOCLAMP 2B</td>
<td>&nbsp;</td>
<tr>
<td>Clampex Software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>v 8.2.0.235</td>
<td>&nbsp;</td>
<tr>
<td>Mini analysis software. v 6.0.3</td>
<td>&nbsp;</td>
<td>Synaptosoft</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Brownlee Precision Amplifier</td>
<td>&nbsp;</td>
<td>Brownlee</td>
<td>Model 410</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Baker</td>
<td>4058-01</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>p-9333 </td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>s6297-1kg</td>
<td>&nbsp;</td>
<tr>
<td>Trelahose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>TO167</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>bp220-212 </td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>h-3375</td>
<td>&nbsp;</td>
<tr>
<td>MgCl-6H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>m2670-1kg </td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>c79-500</td>
<td>&nbsp;</td>
<tr>
<td>Master-8 Pulse Generator</td>
<td>&nbsp;</td>
<td>A.M.P.I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibration table for electrophysiology set up</td>
<td>&nbsp;</td>
<td>Technical manufacturing corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrophysiological methods for recording synaptic potentials from the nmj of drosophila larvae

Bu video, sinaptik iletim, nöromüsküler Drosophila larva bileşke (NMJ) kayıt için elektrofizyolojik yöntemler açıklanmaktadır. Larva nöromüsküler sistemi için bir model Sinaps, sinaptik fizyolojisi ve nörotransmisyon çalışma ve genetik tanımlanmış ve deneysel manipülasyon erişilebilir değerli bir araştırma aracı. Larva maruz vücut duvarı kas, merkezi sinir sistemi ve periferik sinirlerin disseke edilebilir. Drosophila ve innervasyon desen kasları iyi karakterize edilir ve kasların hücre içi kayıt için erişimi kolay. A7 - Bireysel kasları 8 karın segmentleri segmentlerinde A2 tekrarlanan bir model olarak düzenlenmiş her 30 kas içinde kendi konumunu ve yönünü tespit edilebilir. Disseke Drosophila larvaları, ince ve bireysel kasları ve motor nöron akson demetleri transillüminasyon tarafından görüntülenmiştir<sup&gt; 1</sup&gt;. Transgenik yapıları, görsel kimlik veya özel dokularda gen ürünleri işlemek için hedef hücreler etiket için kullanılabilir. Larvaları, eksitatör birleşme potansiyelleri (EJPs) motor nöronlar sinaps veziküler serbest glutamat yanıt olarak üretilir. Disseke larvaları, EJP kas hücre içi bir elektrot ile kaydedilmelidir. Aksiyon potansiyelleri, ventral ganglion posterior kesilmiş, hafif bir emme bir cam pipet içine çekilir ve bir elektrot ile uyarılmış olan motor nöronların yapay uyarılmış olabilir. Bu motor nöronlar uyarılır farklı ateş eşikleri vardır ve aynı anda ateş, kas bir yanıt oluşturmak. NMJ sinaps boyunca sinyaller motor nöronlar innerve kaslar kaydedilebilir. EJPs ve minyatür eksitatör birleşme potansiyelleri (mEJPs), membran potansiyeli değişiklikleri olarak görülmektedir. Elektrofizyolojik yanıtları modifiye minimal hemolimf gibi çözüm oda sıcaklığında kaydedilir.<sup&gt; 2</sup&gt; (HL3) 5 mM Mg içeren<sup&gt; 2 +</sup&gt; Ve 1.5 mM Ca<sup&gt; 2 +</sup&gt;. Uyarılmış EJPs genliği değişiklikler sinaptik işlev ve yapı farklılıkları gösterebilir. Sayısallaştırılmış kayıtları EJP genlik, mEJP frekans ve genlik ve quantal içeriği analiz edilir.

Watch this Scientific Journal Video about Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae at JoVE.com

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

Burada Drosophila larva nöromüsküler kavşakta sinaptik iletim ölçmek için elektrofizyolojik yöntemler açıklanmaktadır. Uyarılmış serbest motor nöron aksonları uyararak yapay olarak başlatılan ve iletim yoluyla NMJ kas uyarılmış postsinaptik tepki ölçülebilir.

Drosophila Larva NMJ Synaptic Potansiyeller kayıt için Elektrofizyolojik Yöntemler

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

electrophysiological-methods-for-recording-synaptic-potentials-from

Research

JoVE Journal

Biology

20.8K Görüntüleme.  Columbia University College of Physicians and Surgeons. Burada Drosophila larva nöromüsküler kavşakta sinaptik iletim ölçmek için elektrofizyolojik yöntemler açıklanmaktadır. Uyarılmış serbest motor nöron aksonları uyararak yapay olarak başlatılan ve iletim yoluyla NMJ kas uyarılmış postsinaptik tepki ölçülebilir.

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3. Instar Drosophila Larva hayali Disklerin Diseksiyon

Biyoloji

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Kaliks Patch Kelepçe Kapasitans Kayıtlar Yöntemleri

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Yüksek çözünürlüklü Ölçüm Drosophila Larva Koku-Driven Davranış

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Drosophila Larval NMJ Dissection

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

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

Drosophila Larvaların NMJ Diseksiyon

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

Drosophila Larval NMJ Immunohistochemistry

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

Drosophila Larval NMJ Immunohistochemistry

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

Drosophila Larvaların NMJ İmmünohistokimya

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.

Synaptic Potansiyellerinin Channelrhodopsin2 aracılığı Uyarım Drosophila Nöromüsküler Kavşaklar

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

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

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. 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.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Hasat ve hazırlanması Drosophila</emElektrofizyolojik Kayıt ve Diğer İşlemler> Embriyolar

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

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.

Elektrofizyolojik Kayıt Drosophila Embriyo

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Kerevit Açıcı Kas NMJ Tarihsel ve Fizyolojisi gösteri

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

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

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

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

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

Alt hücresel Çözünürlük bozulmamış Drosophila Larva in vivo Görüntüleme

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