Yazar laboratuarda zebrafish kurmak onların erken çabalarına Peter Castro ve Matthew Dinday teşekkür etmek istiyorum. Bu çalışma Sağlık EUREKA hibe (# R01NS079214-01) Ulusal Sağlık Enstitüleri tarafından finanse edildi.

1. Yumurta Üretim ve Toplama <ol><li> Zebra balığı yetiştiriciliği daha önce 8 açıklanan standart prosedürleri izler. Kısaca, yetişkin zebrafish yerde bölücüler ile tanklar üreme kurulur. Oda ışıkları ertesi sabah geldiğinde, bölücüler tank ve balık rahatsız çiftleşme zamanı yaklaşık 20 ila 60 dakika izin üreme kaldırılır. </li><li> Yetiştirme tanklarından Yumurta bir süzgeç toplanır ve yumurta su ile durulanır. Yumurta sonra yumurta su ile bir Petri kabı aktarılır. Döllenmemiş yumurtalar ve enkaz bir transfer pipet kullanarak kaldırılır. </li><li> Küvözde toplanan yumurtalar (28-32 ° C) içeren Sıra Petri. Yumurta iki gün sonrası fertilizasyon (DPF) etrafında, yumurtadan sonra, bir transfer pipet ile koryon ve diğer pislikleri temizleyin. </li><li> İstediğiniz gün sonrası fertilizasyon (3 ila 8 DPF) oda sıcaklığında laboratuarlarında serbestçe yüzme larva ve yeri içeren Petri çıkarın. </li></ol><ol><li> Bir mikropipet çektirmenin kullanarak, 1.2 mm OD borosilikat cam aptalca macun rampalarda koyarak bir 150 mm Petri kabında ya da boş pipet kutusunda iki iğne ve mağaza içine kılcal çekin. İğneler önceden çekilebilir. Farklı OD cam kılcal mevcut amplifikatör headstage türüne bağlı olarak kullanılabilir. </li><li> Nalgene bir şırınga filtresi (4-mm) ve Mikrofil filaman (Warner Precision Instruments) takılı olan bir 1 ml şırınga kullanarak hücre dışı kayıt solüsyonu (2 M NaCl) ile Backload mikroelektrot. Kalan az veya hiç kabarcığı kalmayıncaya kadar iğne ucuna doğru bolus çalkalayın ya dokunun. </li></ol> 3. Agar İmmobilizasyon <ol><li> Yumurta suda% 1.2 düşük erime noktası agar taze bir çözüm hazırlayın. ~ 37 ° C&#39;de bir su banyosunda grubu içinde yer agar çözeltisi </li><li> Dondurucuya kayıt odasına ve yeri (5-10 dk) ile bir lamel hazırlayın. Kayıt odasına Matc gerekirh bu elektrofizyoloji teçhizat kullanılır. Biz Warner Instruments (Model RC-26GLP) bir düşük profilli açık elmas hamamı görüntüleme odası kullanın. </li><li> Pasteur veya transfer pipet, temiz bir Petri plaka yumurta su küçük bir damlacık yer biri larva zebrafish kullanma. Bu damlacık için, bir anestetik (% 0.02 tricaine) ve felç edici ajan (1 mg / ml α-bungarotoxin) ihtiva eden bir çözelti damla ilave edin. </li><li> Hareket kaybı (5-10 dk) için zebrafish izler. </li><li> Dondurucu lamel / kayıt odasına çıkarın. Stereomikroskopta evresine odasına yerleştirin. Bir transfer pipet kullanarak, damlacık ve lamel / kayıt odasına aktarım çözümü (balık) ile% 1.2 düşük erime noktası agaroz birkaç ml karıştırın. </li><li> Bir pipet veya ince künt iğne, balık dorsalinde agaroz jel yüzeyine maruz böylece stereomikroskop altında pozisyon larva kullanma. Sonra düz bir spatula transferi agar b kullanarak, sertleşmeye (5-10 dk) agaroz İzinBir elektrofizyoloji kulesi kurmak bir kayıt odasına kilitleyin. </li></ol> 3. Ekstrasellüler Alan Kaydı <ol><li> Kayıt odasına zebrafish kayıt ortamı 2-5 ml ekleyin. Ilaç değişiklikler yaklaşık 1 ml / dak &#39;lık bir hızla kayıt solüsyonu ile gerektiğinde, perfuse bölme ise. Kayıt çözelti No oksijen gereklidir. </li><li> , Üç boyutlu bir mikromanipülatör üzerine monte edilmiş olan amplifikatör headstage içine bir mikroelektrot (yaklaşık 1 mikron uç çapı, MΩ 2-7) takın. Mikromanipülatör hareketi ve ayar bir dizi için izin vermek için uygun bir konumda olup olmadığını kontrol edin. Sahneden yüksek mikroskop görünümü düzlem, ve sadece yukarıda ve biraz zebrafish kafası önünde bir noktaya daha düşük kaba ayarı kullanarak içine mikroelektrot ucu getir. </li><li> &quot;Akım-kelepçe&quot; modunda sıfır elektrodunda amplifikatör ile. </li><li> Sadece s dokunacak şekilde kaba ayarı düşük elektrot ucu kullanarakhafifçe ön beyin önünde zebrafish urface. </li><li> Bu zebrafish cildi delen, kadar ince ayar adımları kullanarak elektrot ucu ilerlemek. Önbeyin içine birkaç mikron yerleşmek ve sonra elektrot ilerlemek için izin ver. </li><li> Axoscope yazılımını kullanarak akım-klemp modunda elektriksel aktivite kaydedin. Veri 10 kHz örnekleme hızında elde edilir. </li></ol>

<ol>
	<li>Clark, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

Çıkar çatışması ilan etti.

Burada sunulan ekstraselüler kayıt yöntemi, beyin aktivitesinde çok hassas ve hızlı analizine olanak sağlar. Bu kayıtlar genellikle epilepsi 11 ve hastaların 12 kemirgen modellerinde anormal elektriksel deşarj (yani, nöbet) varlığını değerlendirmek için kullanılan elektroensefalografi (EEG) izleme ile benzerdir. Burada gösterildiği gibi ekstraselüler kayıtları, farmakolojik manipülasyonlar ile kombine edilebilir. Kayıtları Bu tip de genetik olarak modifiye edilmi?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Agaroz düşük erime </td><td> Fisher-Bilimsel </td><td> BP1360-100 </td><td> % 1,2 embriyo ortamda çözülür </td></tr><tr><td> Kayıt ortamı </td><td> Fisher-Bilimsel </td><td> BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 </td><td> 1 mM NaCI, 2.9 mM KCI, 10 mM HEPES, 1.2 mM MgCl2, 10 mM dekstroz, 2.1 mM CaCl2 1 N NaOH ile pH değeri yaklaşık olarak 7.3 ile </td></tr><tr><td> Tricaine </td><td> Argent Laboratuvarları </td><td> MS-222 </td><td> % 0.02 &#39; </td></tr><tr><td> α-bungarotoxin </td><td> Bioscience Tocris </td><td> 2133 </td><td> 1 mg / ml </td></tr><tr><td> Kılcal cam boru </td><td> Warner Instruments </td><td> G120TF-3 </td><td> MΩ 2 -7 bir direnç çekin </td></tr><tr><td> Yama kelepçe amplifikatör </td><td> Warner Instruments </td><td> PC-505B </td><td> Biz mevcut-klemp modunda bir Warner amplifikatör kullanmak; Kazanç 2K ayarlanır 2 mV / pA ve Bessel filtre ayarlanmıştır. Karşılaştırılabilir model üreticinin talimatlarına göre kullanılır. </td></tr><tr><td> Filtre / amplifikatör </td><td> Kuğu Teknoloji </td><td> FLA-01 </td><td> 10-20 ayarlanır Kazanç;; 1-2K ayarlanmış Kesim frekansı; Notch filtresi IN Biz Cygnus pre-amplifikatör kullanın. Karşılaştırılabilir model üreticinin talimatlarına göre kullanılır. </td></tr><tr><td> Akson A / D kurulu ve Axoscope yazılımı </td><td> Molecular Devices </td><td> Akson Digidata 1320A; Axoscope 8.2 </td><td> Veri boşluğu serbest edinimi modunu kullanarak Axoscope toplanır; 10 kHz örnekleme. Karşılaştırılabilir modelleri ve programları üreticinin talimatlarına göre kullanılır. </td></tr><tr><td> Yumurta su </td><td&gt; Anında Okyanus </td><td></td><td> 3 g Anında Okyanusu deniz tuzu, 10 ml deiyonize su içinde 2 mL% 0.1 &#39;lik metilen mavisi </td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

Epilepsi Amerika Birleşik Devletleri yaklaşık 3 milyon kişi kadar ve 50 milyon kişi dünya çapında etkiler. Spontan nöbetlerle oluşumu olarak tanımlanan, epilepsi beyin ya da genetik bir mutasyon bir hakaret sonucunda elde edilebilir. Hayvanlarda modeli nöbet çabaları öncelikle kullanılan kemirgenlerde hakaret (konvülsan ilaçlar, uyarılma ya da beyin hasarı) ve genetik manipülasyonlar (antisens demonte, homolog rekombinasyon veya transgenesis) almış. Zebra balığı kemirgen tabanlı epilepsi araştırma için değerli bir alternatif sunabilecek bir omurgalı model sistem 1-3 vardır. Zebra balığı omurgalı genetik veya geliştirme çalışmalarında yaygın olarak kullanılan, memeliler için genetik benzerlik yüksek dereceli ve bilinen insan tek gen epilepsi mutasyonların ~% 85 homologları ifade. Çünkü onların küçük boyutlu (4-6 mm uzunlukta) arasında zebrafish larva erken gelişim ve arra sırasında 100 ul gibi düşük sıvı hacmi muhafaza edilebilirçok oyuklu plakalar içinde yapabilirler. Reaktifler embriyolar ilaç uygulaması basitleştirilmesi ve test bileşiklerinin in vivo 4 tarama hızlı sağlayan, geliştiği çözeltiye doğrudan ilave edilebilir. Sentetik oligonükleotidler (morpholinos), mutagenez, çinko parmak nükleaz ve transgenik yaklaşımlar hızla 5-7 zebrafish gen devirme veya mutasyon oluşturmak için de kullanılabilir. Bu özellikler zebrafish çalışmalarda epilepsi gibi nörolojik hastalıkların çalışmada kemirgenler üzerinde görülmemiş bir istatistiksel güç analizi avantajı göze. Epilepsi araştırma için &quot;altın standart&quot; merkezi bir beyin yapısı (yani, nöbetler), verimli larva zebrafish beyin aktivitesini kaydetmek için bir yöntem menşeli anormal elektrik deşarjı izlemek ve analiz etmek için burada tanımlanmıştır. Bu yöntem geleneksel ekstraselüler kayıt teknikleri bir uyarlamasıdır ve sağlam zebrafish larvaları beyin aktivitesinin istikrarlı, uzun vadeli izleme için izin verir. Sbol kayıtları bir genetiği değiştirilmiş balık kaydedilen konvülsan ilaçlar ve spontan nöbetlerin banyo uygulaması ile oluşturulan akut nöbetler için gösterilmiştir.

Bir agar-gömülü zebrafish larvalarının ön beyin kaydedilen elektrografik nöbet gibi deşarj örnekleri Şekil 1 &#39;de gösterilmiştir. Veya 1 mM picrotoxin (B; 8 dpf); bu örneklerdeki Büyük genlikli çoklu başak patlama boşalma konvülsan ilacın banyo uygulaması, 40 mM pilokarpin (6 dpf&#39;e A) tarafından uyarılmış oldu. Bu kayıtlar içinde, immobilize ve agar-gömülü zebrafish sürekli 90 dk kadar izlenir. Balık kadar 24 saat için bu kayıt koşullarında canlı kalır. İla?...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

Larva zebrafish önbeyin ekstrasellüler alanın potansiyellerini kaydetmek için basit bir yöntem tarif edilmiştir. Yöntem, bir sağlam içerir<em&gt; In vivo</em&gt; Salt üzerinden aktivite nöbet gibi. Bu teknik, epilepsi ile ilişkili genlerin ya da konvülsan ilaçların verilmesi ile uyarılmış nöbetler taşıyan genetik olarak modifiye edilmiş zebrafish larvaları ile birlikte de kullanılabilir.

Larva Zebra balığı olarak önbeyin Elektrofizyolojik Kaydı

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

forebrain-electrophysiological-recording-in-larval-zebrafish

Research

JoVE Journal

Neuroscience

17.0K Görüntüleme.  University of California, San Francisco. Larva zebrafish önbeyin ekstrasellüler alanın potansiyellerini kaydetmek için basit bir yöntem tarif edilmiştir. Yöntem, bir sağlam içerir In vivo Salt üzerinden aktivite nöbet gibi. Bu teknik, epilepsi ile ilişkili genlerin ya da konvülsan ilaçların verilmesi ile uyarılmış nöbetler taşıyan genetik olarak modifiye edilmiş zebrafish larvaları ile birlikte de kullanılabilir.

Video: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Gelişmekte olan Zebra balığı önbeyin klonal olarak ilişkilidir Sinir Progenitör Hücreler Zaman atlamalı Canlı Görüntüleme

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Nörobilim

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Sağlam Yetişkin Zebra balığı Beyin Elektrofizyolojik Kayıt

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Elektrofizyolojik Kayıt<em&gt; Drosophila</em&gt; Labellar Sensilla Taste

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Larva Zebra balığı Omurilik Kesisi

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Eşzamanlı Elektrofizyolojik Kayıt ve kemirgen Beyindeki inhibitör Ajanların Mikro-enjeksiyon

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Elektrofizyolojik Kayıt<em&gt; Drosophila</em&gt; Düşük Volatilite Kokularına Karşı Trikoid Sensilla

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Larva zebra balığı sonradan içini kaplamak saç hücrelerinin vivo kalsiyum görüntüleme

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

Elektrofizyolojik Alan Kaydı ile Interlamellar Hipokampus CA1'de Uzun Süreli Sinaptik Plastisitenin Araştırılması

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Serbest Yüzme Balık hareketli Tetrodes tarafından Nöronların Kablosuz Elektrofizyolojik Kayıt

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...