NIH grant R01 MH 099557 tarafından desteklenen

1. imalat kayıt elektrotlar <ol><li><ol><li>kullanımı aşağıdaki cam elektrotlar çekerek protokolü için elektrot çektirme (Tablo malzemeleri görmek): <ol><li>Line 1: ısı 510 çek - hız 30 zaman 250; Line 2: 490 çekme - hız 30 ısı zaman 250. 
			Not: Zaman birimi birim başına 0.5 ms karşılık gelir; diğer birimleri görecelidir. Rampa test gerçekleştirildikten sonra ısı değerini her filaman için ayarlanmalıdır.</li>
		</ol></li> <li>Çekti elektrot iç çapı 7-10 µm ( şekil 1A) aralığında olması için mikroskop (35 x büyütme) kullanın. Kılcal damarlar toz birikimi önlemek için sıkıca kapalı kaplarda saklayın.</li>
	</ol></li> <li>Yangın parlatma <ol><li>yangın Lehçe kılcal damarlar (1-2 s için maksimum ısı değeri % 80-90) mikro-demirhane kullanarak (Tablo malzemeleri görmek). Cilalı elektrot son iç çapı 5 µm ( şekil 1A) 27 olduğundan emin olun.</li>
	</ol></li> <li>Bükme Not: iki virajlı elektrot yüksek büyütme objektif altında kas üstüne konumlandırmak için yapılır.
	<ol><li>Kullanımı şekil 1B adımında gösterilen aparatı. Elektrot manipülatör içinde düzeltmek ve ucu onu değil müessir filament üzerinde konumlandırmak</li>
		<li>Set ısı değeri en fazla 60-70 oranında ve mikro-forge pedal için 1-2 s tuşuna basın (filaman ısı Malzemeleri tablo bkz:). L şeklindeki bir iğne yavaşça (büyütülmüş şekil 1B) elektrot uç çekmek için kullanın. Viraj yaklaşık 90 o ( şekil 1 c) olun.</li>
		<li>Elektrot meşale ateşte forseps kullanarak el ile sık ve ikinci bend bir mesafe üzerinden ilk bend 7-10 mm ve bir açıyla yaklaşık 120 o ( şekil 1 c).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> şekil 1. Micropipette imalat son adımlar. (A) elektrot ipuçları çekerek ve yangın parlatma sonra. (B.1) ipucu bükme Kur. (B.2) kutulu alanının sağ tarafta genişlemiş gösterilir. Böylece elektrot ucunu biraz tel üzerinde konumlandırılmış ve değil dokunaklı elektrot ve filament sabittir. (C.1) kayıt elektrot. (C.2) kutulu alanının sağ tarafta genişlemiş gösterilir. İlk bend yaklaşık 90 ° vardır ve ilk bend elektrot ucu arasındaki mesafe yaklaşık 1 mm. ölçek kadar çubuk = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
 2. ek hazırlık adımları <ol><li>hazırla heamolymph gibi (HL3) çözüm (mM): 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 trehalose, 115 Sükroz, 5 HEPES ve 1 mM CaCl 2 7.3 için-pH ayarlama; 7.4. çözüm buzdolabında saklayın ve her hafta taze olun.</li>
	<li>Dışında onları bükmeye gerek yok pipets, aynı şekilde kayıt olarak cam stimülasyon pipets olun. 
	Not: Stimülasyon elektrotlar hazırlanması ayrıntılı 28 ve 27 olarak tanımlanır. Yangın parlatma 5-7 µm aralığında olmalıdır sonra son çapı.</li>
	<li>Bir elektrot tutucu bir stimülasyon pipet bağlı bir şırıngaya ekle.</li>
	<li>Üretim diseksiyon plakalar üzerinden küçük petri yemekler (35 x 10 mm) silikon kauçuk epoksi ile kaplı ( 28 içinde açıklandığı gibi Malzemeleri tablo görmek). 
	Not: Silikon kauçuk kullanmadan önce tamamen sertleşmiş olmalıdır.</li>
	<li>Gezgin bir üçüncü-biçim larva almak ve 27 , 28 , 29 , 30 ' açıklandığı gibi incelemek. 
	Not: boutons görselleştirmek için sinek zorlanma CD8-GFP kullanın (bkz. Tablo reçetesi) <ol><li>kullanma forseps, (bkz: Malzemeler tablo) almak 3 rd INSTAR larvaları bir şişe.</li>
		<li>İlk iğneyi posterior yerleştirerek larvalar, pin ve başka bir pin ağız kanca için anterior (Kapat).</li>
		<li>Ekleyin HL3 çözüm.</li>
		<li>(Bkz. Tablo reçetesi) bahar makas ta üst iğnesinden yukarıdan aşağıya doğru bir larva dorsal tarafta kullanarak bir kesim yapmak.</li>
		<li>Üstünde belgili tanımlık sol ve sağ taraflarındaki, larvalar 2 ek pimleri yerleştirerek larva fileto pin.</li>
		<li>Çıkarmak cesaret ve forseps kullanarak tracheas.</li>
		<li>Sadece bahar makas kullanarak ventral sinir kordonu dışında sinirler kesti.</li>
	</ol></li></ol> 3. Elektrik kayıtları EJC'ler <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> Şekil 2. Kayıt kurulum. Örnek NMJ silikon kaplı petri kabına (ok) dik bileşik mikroskop epifluorescence yetenekleri, yüksek büyütme objektif ve iki micromanipulators ile donatılmış hareketli sahne üzerinde konumlandırılmış sabitlenmiş. Mikroskop bir anti-titreşim tablo üzerinde konuşlu olduğunu. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol><li>Kayıt <ol><li>yer petri kabına hazırlık mikroskop sahnede ( Şekil 2). Referans elektrot banyoda takın.</li>
		<li>Kayıt elektrot HL3 çözüm ile doldurun. 10 x amaç altında (bkz. Tablo reçetesi) elektrot banyo içine sokmak ve 6 ve 7 2, 3 veya 4 micromanipulator kullanarak karın segmentlerinin kaslar yerleştirin (bkz. Tablo reçetesi) () şekil 3 A.1 ve A.2).</li>
		<li>Geçiş yapma amacı için 60 x (Tablo malzemeleri görmek). Epi-floresan veya DIC optik kullanarak ilgi alan üzerinde odaklanın. Sinaptik bouton ( şekil 3 B.1-3) üstüne elektrot ucu yerleştirin.</li>
		<li>Elektrot kas üzerine hafifçe basın. Aşırı basınç NMJ zarar veya spontan sinirsel aktivite artışı teşvik. Elektrot ucu tıkanmış - Eğer o, bunu yerine dikkat edin</li>
		<li>Amplifikatör, A/D yönetim kurulu ve bilgisayar geçiş.</li>
		<li>Amplifikatör gerilim kelepçe konumunda seçin.</li>
		<li>Başlangıç Alım yazılım ve &#39; boşluk içermeyen &#39; modu.</li>
		<li>MEJCs bilgisayar ekranında görünümünü gözlemlemek.</li>
		<li>MEJCs genliği 0,2 - aralıkta olduğunu emin olun 0,7 na 
		Not: Daha küçük EJC'ler kayıt elektrot kusurları vardır veya bu doğru yerleştirilmiyor gösteriyor.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> şekil 3. Görsel olarak sinaptik boutons. (A) A aydınlık alan görüntü, bir hemi-kesim 10 x büyütme (A.1) ve kas 6 ve 7 gösterilen genişlemiş kutulu alanının altında (A.2, ok işaretleri kayıt elektrot). Ölçek çubuğu = 50 µm. (B) sinaptik boutons genetik olarak kodlanmış nöronal marker (CD8-GFP) ile bir Drosophila satırda görüntülenir epi-floresan görüntüleme (B.1) veya DIC optik (B.2) kullanarak. Görüntüleri ile hedef ve filtre küp GFP görüntüleme için x 60 alınır (Tablo malzemeleri görmek). Synaptic boutons oklarla işaretlenmiş ve floresan ve DIC görüntülerin bir bindirme B.3 içinde gösterilir. Ölçek çubuğu 10 µm. = <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol start="2"> <li>stimülasyon <ol><li>görsel bir denetim altında bir micromanipulator kullanarak yer stimülasyon elektrot karın kesimleri 2-4 innerve axon yakınındaki.</li>
		<li>Şırınga piston çekerek negatif basınç uygula bağlı elektrot sahibine böylece akson elektrot ( Şekil 2) içinde çekti.</li>
		<li>Uyarıcı açın. Vana yalıtım Üniteyi açar (bir sıfır akım ayarlamak ve sonra yavaşça, EJC'ler görününceye kadar (veya eşik değerine ulaşılana kadar) artırmak için Malzemeler tablo bkz:).</li>
		<li>Bir suprathreshold rejim stimülasyon geçerli stimülasyon ile artmış yaklaşık iki kez, EJC'ler gözlenmesi için eşik göre gerçekleştir. 
		Not: deneyim, böyle stimülasyon yoğunluğu aksiyon potansiyeli başarısızlıklar ve aksiyon potansiyeli ateş önlemek için en iyi yöntemdir. Örneğin, EJC'ler 0.2 stimülasyon mevcut görünür anne, 0,4 mevcut kullanmak deneme boyunca anne.</li>
	</ol></li> <li>Direnç mühür <ol><li>amplifikatör için elektrot direnç anahtarı çevirerek kayıt macropatch elektrot mühür dayanıklılığını ölçmek &#34; test mühür &#34; pozisyon. GΩ mühür direnç değeri içinde görüntülenir gözlemlemek &#34; geçerli &#34; penceresi.</li>
		<li>0.5 - 2 M aralığında dirençtir mühür emin olun &#8486;. Bu sınırların dışında değerler gösterir elektrot düzgün cilalı veya uygun şekilde konumlandırılmış.</li>
		<li>Mühür direnç kaydı boyunca izlemek, deney boyunca sabit kalır emin olmak istedim.</li>
	</ol></li></ol> 4. Analiz <ol><li>analiz kayıtları istihdam özelleştirilmiş yazılım analiz için (bkz. Tablo reçetesi). 
	Not: Bizim laboratuvar içi yazılım Quantan 31 EJC'ler ve focally kayıt mEJCs ( şekil 4) tespiti için özelleştirilmiş, kullanır. Bu yazılım bir Gauss dijital filtre içerir ve çok quantal olaylar ( şekil 4A) üst üste içinde quantal doruklarına algılama sağlar. Diğer yaklaşımlar 17 ' açıklanan.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> şekil 4. Quantal analizi. , MEJCs (a) ve EJC'ler (B) Quantan yazılım tarafından. Olay alan yeşil işaretlenir ve tepeler tarafından kırmızı ok uçları işaretlenir. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

<ol>
	<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. 262 (1), 189-214 (1976).</li><li>Katz, B., Miledi, R. <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+temperature+on+the+synaptic+delay+at+the+neuromuscular+junction.">The effect of temperature on the synaptic delay at the neuromuscular junction.</a> J Physiol. 181 (3), 656-670 (1965).</li><li>Macleod, G. T., Gan, J., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-associated+proteins+and+quantal+release+at+single+active+zones+of+amphibian+(Bufo+marinus)+motor-nerve+terminals.">Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.</a> J Neurophysiol. 82 (3), 1133-1146 (1999).</li><li>Macleod, G. T., Farnell, L., Gibson, W. G., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+secretion+and+nerve-terminal+cable+properties+at+neuromuscular+junctions+in+an+amphibian+(Bufo+marinus).">Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).</a> J Neurophysiol. 81 (3), 1135-1146 (1999).</li><li>Zefirov, A., Benish, T., Fatkullin, N., Cheranov, S., Khazipov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+active+zones.">Localization of active zones.</a> Nature. 376 (6539), 393-394 (1995).</li><li>Macleod, G. T., Lavidis, N. A., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dependence+of+quantal+secretion+from+visualized+sympathetic+nerve+varicosities+on+the+mouse+vas+deferens.">Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.</a> J Physiol. 480 (Pt 1), 61-70 (1994).</li><li>Samigullin, D., Bill, C. A., Coleman, W. L., 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=Regulation+of+transmitter+release+by+synapsin+II+in+mouse+motor+terminals.">Regulation of transmitter release by synapsin II in mouse motor terminals.</a> J Physiol. 561 (Pt 1), 149-158 (2004).</li><li>Coleman, W. L., 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=Rab3a-mediated+vesicle+recruitment+regulates+short-term+plasticity+at+the+mouse+diaphragm+synapse.">Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.</a> Mol Cell Neurosci. 41 (2), 286-296 (2009).</li><li>Atwood, H. L., Parnas, H., Parnas, I., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+currents+evoked+by+graded+intracellular+depolarization+of+crayfish+motor+axon+terminals.">Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.</a> J Physiol. 383, 587-599 (1987).</li><li>Parnas, H., Dudel, J., Parnas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+release+and+its+facilitation+in+crayfish.+I.+Saturation+kinetics+of+release,+and+of+entry+and+removal+of+calcium.">Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium.</a> Pflugers Arch. 393 (1), 1-14 (1982).</li><li>Wojtowicz, J. M., Marin, L., 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=Activity-induced+changes+in+synaptic+release+sites+at+the+crayfish+neuromuscular+junction.">Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction.</a> J Neurosci. 14 (6), 3688-3703 (1994).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crayfish+neuromuscular+facilitation+activated+by+constant+presynaptic+action+potentials+and+depolarizing+pulses.">Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses.</a> J Physiol. 241 (1), 69-89 (1974).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+statistics+of+transmitter+release+during+facilitation.">Changes in the statistics of transmitter release during facilitation.</a> J Physiol. 229 (3), 787-810 (1973).</li><li>Worden, M. K., Bykhovskaia, M., Hackett, 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=Facilitation+at+the+lobster+neuromuscular+junction:+a+stimulus-dependent+mobilization+model.">Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model.</a> J Neurophysiol. 78 (1), 417-428 (1997).</li><li>Bykhovskaia, M., Hackett, J. T., Worden, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchrony+of+quantal+events+in+evoked+multiquantal+responses+indicates+presynaptic+quantal+interaction.">Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction.</a> J Neurophysiol. 81 (5), 2234-2242 (1999).</li><li>Bykhovskaia, M., Polagaeva, E., Hackett, 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=Mechnisms+underlying+different+facilitation+forms+at+the+lobster+neuromuscular+synapse.">Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse.</a> Brain Res. 1019 (1-2), 10-21 (2004).</li><li>Cooper, R. L., Stewart, B. A., Wojtowicz, J. M., Wang, 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+measurement+and+analysis+methods+compared+for+crayfish+and+Drosophila+neuromuscular+junctions,+and+rat+hippocampus.">Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus.</a> J Neurosci Methods. 61 (1-2), 67-78 (1995).</li><li>Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., 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=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>

Yazarlar ifşa gerek yok.

Drosophila sinaptik iletimi çalışmaya bir avantajlı model organizma temsil eder. Sinaptik potansiyelleri, kayıtları iki elektrot gerilimi kelepçe33,34ve Fokal macropatch ile sinaptik akıntılarının hücre içi kayıtları da dahil olmak üzere larva NMJ, birkaç kayıt yapılandırma kullanılmıştır Burada açıklanan sinaptik akıntılarının kayıtları. İkinci tekniği görüntülenmeyecektir boutons kesin miktar sinaptik iletimi sağla...

Drosophila sinaptik iletimi denetleme moleküler mekanizmaları incelemek için mükemmel model bir sistemdir. Drosophila nöromüsküler sistemde glutamatergic ve bu nedenle Drosophila nöromüsküler kavşak (NMJ) glutamatergic yayın korunmuş özelliklerini incelemek için kullanılabilir. Jan Jan'ın çalışma ve1beri üçüncü INSTAR larva geniş eksitatör kavşak potansiyeller (EJPs) veya akımları (EJC'ler) izleyerek uyarılmış ve spontan sinaptik iletimi çalışma için kullanılmıştır. EJPs yaygın olarak intracellularly bir keskin cam mikro elektrot ile kaydedilir ve onlar sinapslarda belirli kas lif yapım tüm boutons de dahil olmak üzere tüm NMJ etkinliğini yansıtır.
Buna ek olarak, sınırlı sayıda sürüm site etkinliğini focally nöronal terminalleri veya sinaptik varicosities yakınındaki bir micropipette ipucu konumlandırma tarafından kaydedilir. Bu teknik aslında Katz ve Miledi2tarafından istihdam edildi ve başarıyla kurbağa3,4,5, fare6 dahil olmak üzere birkaç NMJ hazırlıklar istihdam Fokal hücre dışı kayıtları , 7 , 8, kabuklu9,10,11,12,13,14,15,16ve Drosophila17,18,19,20,21,22,23. Bu yaklaşım daha fazla elektrotlar24,25recoding macropatch en iyi duruma getirilmiş Dudel tarafından geliştirilmiştir. Dudel'ın uygulamasında bu teknik yakından gevşek-yama-kelepçe yöntemi26eşleştirdi.
Drosophila larva NMJ sinaptik boutons açıkça tanımladığı ve genetik olarak kodlanmış nöronal floresan Etiketler ( Tablo malzemelerigörmek) transgenik çizgilerle kolayca kullanılabilir. Bu avantajları bize EJC'ler ve seçili sinaptik bouton20,21,22mEJCs kaydetmek etkin. Burada, bu teknik ayrıntılı olarak açıklanmaktadır.

<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

Drosophila nöromüsküler kavşak (NMJ) glutamatergic sinaptik iletimi çalışmaya bir mükemmel model sistemidir. Biz odak macropatch kayıtları görüntülenmeyecektir boutons Drosophila larva NMJ, sinaptik akımlardan tekniğini tanımlamak. Bu teknik MİKROPİPETLER yanı sıra bileşik yüksek büyütme, uzun mesafe su daldırma amacı, farklı girişim kontrast (DIC) optik ve bir floresan mikroskop kayıt özelleştirilmiş imalat gerektirir Eki. Kayıt elektrot DIC optik, epi-floresan veya her ikisi ile görüntülenir seçilen bir sinaptik bouton üst kısmında konumlandırılmış. Bu teknik avantajı bu sınırlı sayıda site açıklaması sinaptik etkinliğini izlemeye olanak sağlamasıdır. Kayıt elektrot birkaç mikron çapında ve elektrot RIM dışında konumlandırılmış yayın siteleri kayıtlı akımları önemli ölçüde etkilemez. Kaydedilen sinaptik akımlar hızlı Kinetik var ve kolayca çözülebilir. Bu avantajlar özellikle mutasyona uğramış sinek hatları ile gelişmiş kendiliğinden ya da zaman uyumsuz sinirsel aktivite çalışmaları için önemlidir.

Odak macropatch kayıtları seçili sinaptik boutons (şekil 5) üzerinden izleme sinirsel aktivite etkinleştirin. Elektrot bir sinaptik bouton üst kısmında (şekil 5A, 1 sitesi) getirildiğinde kaydedilen mEJCs (şekil 5C, 1 sitesi) gürültü seviyesini önemli ölçüde aşan bir genlikleri var ve aşamaları (alt milisaniyelik mesafeden) yükselen keskin. Ne zaman kayıt elektrot uzak sinapti...

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

Drosophila larva nöromüsküler kavşak sinaptik akımlardan Fokal Macropatch kayıtları

Sinaptik akımları, Drosophila Üçüncü INSTAR larva nöromüsküler kavşak görüntülenmeyecektir sinaptik boutons üzerinden focally kaydedilebilir. Bu teknik, tek bir sinaptik bouton etkinliğini izleme sağlar.

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

Research

JoVE Journal

Neuroscience

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

6.0K Görüntüleme.  Wayne State University. Sinaptik akımları, Drosophila Üçüncü INSTAR larva nöromüsküler kavşak görüntülenmeyecektir sinaptik boutons üzerinden focally kaydedilebilir. Bu teknik, tek bir sinaptik bouton etkinliğini izleme sağlar.

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.

Motor-nöron Paired Patch Kelepçe Kayıtlar ve Zebra balığı Hedef İskelet Kası

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

Nörobilim

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.

Aktif Bölgeleri Diseksiyon ve Görüntüleme Drosophila Nöromüsküler Junction

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.

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. ...

Biyoloji

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 ...

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.

C. Elegans Nöromüsküler Kavşağı Diseksiyon ve kayıt

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.

Hazırlanması<em&gt; Drosophila</emIçin&gt; Orta Nöronlar<em&gt; In situ</em&gt; Patch Sıkma

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.

Elektrofizyolojik Kayıt Drosophila Embriyo

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.

Embriyonik Zebra balığı Mauthner Hücreler Patch Kelepçe Kayıtlar

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.

Drosophila larvalarında nöromüsküler kavşakların transmisyon elektron mikroskobu için numune hazırlama sürecinin optimize edilmesi

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.

Drosophila Nöromüsküler Kavşak (NMJ) Miktar Tayini: Sinaptik Morfoloji ve Fonksiyonu Değerlendirmek İçin Bir Yöntem

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