TK G2205 (Bilim Teşvik Ulusal Bilim Vakfı (BAC için IOS-1.050.701), Ulusal Sağlık Enstitüleri (SM için NS54174 ve AML-W F30DC0111907.), Uehara Memorial Foundation ve Japon Derneği tarafından sağlanan finansman .) Biz hücre dışı aksonal kayıtların konuda verdiği destek ve rehberlik için Julian Meeks teşekkür ederim. Biz bir prototip kayıt odasına sağlamak için Carl Hopkins teşekkür ederim.

<ol>
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Çıkar çatışması ilan etti.

Bir kere hakim, bu teknik bir çok model sistemler in vivo kayıtlarında için ayrı akson dahil olmak üzere tespit nöronlar, hedef sağlayacaktır. Buna ek olarak, bu teknik bir güvenilir zorlu in vivo kayıt yöntemleri geleneksel sağlayan benzersiz anatomik özelliklere sahip nöronlardan başak çıkış kaydetmenize olanak verir. Biz mormyrid zayıf elektrik balık ELa &quot;küçük hücreler&quot; den kaydetmek için bu tekniği kullanmış olurlar. &quot;Küçük hücreler&quot; ayar özelliklerini incelemek için önceki girişimler zorlu kayıt koşullarına 11, 14 nedeniyle başarısız oldu. Benzer anatomik özellikleri çok farklı omurgalı işitsel ve electrosensory nöronlar 8-10 tek birim kayıtları elde etmek için engeller oluşturmak. Sistemimizde bu zorlukları aşmak için, &quot;küçük hücreler&quot; bu ELa tek hücrelerin olduğu gerçeğini yararlandı ELP proje. Böylece, ELP yerleştirilir boya retrograd taşıma &quot;küçük ELa Etiketleme sınırlarhücre &quot;somas ve akson. akson Floresan etiketleme erişilemez somas rağmen mümkün tespit hücrelerinden tek birim kayıtları yapma, etiketli aksonlar yanında hassas elektrot yerleştirme izin verdi. Biz çevredeki yutma sinaps 11 nedeniyle muhtemelen, somatik kayıtları çalıştı, ancak başarısız oldu , 17. Bununla birlikte, açıkça bu somatik etiketleme tekniği diğer hücre türleri ve diğer devreler somatik kayıtları hedeflemek için kullanılabilir düşündüren görünür hale geldi. in vivo olarak geriye doğru aktarıma yoluyla nöronların fluoresan etiketleme in vitro 19-21 hedeflenen kayıtları ilerletilmesi için kullanılmaktadır . Benzer bir teknik zebrabalıkları omurilik 22 motor nöronların in vivo kayıtları hedeflenen için kullanılmıştır. Çalışmalarımız, etiketleme ve kayıt hem de beyinde in vivo yapılmaktadır ki, bu yaklaşım için yeni bir genişleme gösterir. Bizim yöntemi gösteren in vivo bir retrograd t MSS alanların etiketlemeyarışçı benzer seçici projeksiyon nöronları ile diğer sağlam devrelerin çalışma genişletilebilir. Örneğin, memeli işitsel işleme, alt kollikulus (IC) birden fazla rhombencephalic yapılar 23 girişleri için önemli bir aktarma merkezi olarak hizmet vermektedir. IC içine enjeksiyondan seçici bu çekirdeklerin her birinden projeksiyon hücreleri etiketlemek olacaktır. Üstün colliculus (SC) vizyon 24 için benzer bir işlev görür. Omurilik preparatlar özellikle omurilik kolayca erişilebilir olduğu gibi, boya püskürtme kadar kayıt sitesinden oluşabilir, bu yöntem için uygun olan, ve daha detaylı bilgiler anatomik 25 elde etmek için seçme nöronların hücre içi kayıt ve doldurma ile kombine edilebilir. Son olarak, sistem-izleme karmaşık devre 26 eşleştirmek için merkezi sinir sistemi boyunca kullanılan iyi kurulmuş bir tekniktir. Bizim yöntemi w yapılmıştır bu çalışmalara işlevsel bilgi eklemek için kullanılabilirkedi görsel korteks 27. ith kalsiyum duyarlı göstergeler. Iyi kurulmuş olan cerrahi, güvenilir ve düzenli in vivo kayıtları 16 kör için kullanılan, minimal kanama ve hayvan ve hayatta kalmak için doku sağlamak için beynin yüzeyine zarar ile tamamlanması gerekir. Uygulama ile, cerrahi ve boya uygulama 30-45 dakika içinde tamamlanabilir. Biz başarıyla hazırlıkları% 67 in &quot;küçük hücreli&quot; akson etiketli. En hazırlıkları sadece 1 veya 2 görünür etiketli aksonlar var, ancak bazı olarak 8 var. Teşebbüs 119 etiketli birimlerinin, biz 12 preparatları (Tablo 2) üzerinden dağıtılan 26 birimlerden tek birim kayıtları elde. Böylece, veri% 28 genel başarı oranı için etiketli akson ile hazırlıkları 41% toplanmıştır. Boya uygulamasının kritik yönü derinliği etiketleme. Tungsten telinin Sığ ekleme yıkanmış w olan boya ile sonuçlanacaktıray. Penetrasyon çok derin ise, ancak, etiketli akson hedef görebilir olmayacaktır. Bundan başka, hücre için bazı mekanik hasar yeterince 25, 28 ele alınması boya için gerçekleşmelidir. Ancak, çok fazla zarar hücreleri öldürür. Biz ELP etiketlenmesine akson (Tablo 3) kayıt ile eşleştirilmiş ELa içine boya enjeksiyon yoluyla anterograd etiketleme dahil olmak üzere diğer boyalar ve diğer yöntemlerle (Tablo 2), ile etiketleme çalıştı. Biz etiketleme onlara tepkisiz hale somatik hasar hücrelerin sınırlı olduğu için ileriye etiketleme başarılı olmadığını hipotez. Buna ek olarak, pre-sinaptik terminalleri rahatsız olabilir. Buna karşılık, retrograd etiketleme bu endişeleri her iki en aza indirir. Miktarı ve boya uygulama yer çalışılmaktadır belli bir devre uygun olarak modifiye edilebilir. Maksimal etiketleme biz kaplanmış tungsten tellerin kullanılarak elde büyük boya konsantrasyonu oluşur. Ancak de olan aksonlar Etiketlemebu gelişmiş olduğu gibi ep projeksiyonlar, boya bir tungsten iğne yerinden çıkabilir. Bu durumda, basınçlı enjeksiyon daha uygun olacaktır. Boya alımı ve ulaşım 2 saat sonrası enjeksiyon ve 6 saat sonrası enjeksiyon gibi geç görünen ek etiketli akson gibi erken görünür olmak bizim hazırlık etiketli akson ile, hızlıdır. Böylece, etiketleme ve kayıt hayatta kalma cerrahi ile ilgili teknik sorunlar ortadan kaldırarak, tek bir gün içinde yapılabilir. Zamanlama boya nakil için gerekli olan mesafeye bağlı olarak, her uygulama için değişecektir. Diğer önemli yönü elektrot yerleştirilmesidir. Bu ucu tıkanmasını önlemek için etiketli akson siteye doku yakın girmek için önemlidir. Bu ELa gibi yoğun doku için, kayıt elektrot üzerinde uzun, ince bir sap çevre dokulara aşırı hareketi en aza indirir. Başarılı bir kayıt bir kayıt elde edilene kadar taze elektrotlar ile ilk denemede, tekrar üzerinde elde veya tiss değilseue akson artık görünür hangi noktaya bozulur. Bununla birlikte, hızlı bir şekilde ağartma fototoksisite neden maruz floresan, miktarını en aza indirmek için ve hücre 29-31 fizyolojik özelliklerini etkileyebilir akson yanındaki elektrot yerleştirmek için de önemlidir. Akson bir bölümünü başarıyla kayıt elektrot içine emilmekte sonra, kayıtlar birkaç saat boyunca elde edilebilir. Birimleri sürekli az 1 saat içinde kaybolur üzerinden kaymasını akson önlemek için küçük elektrot uçları yapmayı düşünün. Öte yandan, bir ucu çok küçük akson için tıkanma, düşük sinyal-gürültü, ya da hasara neden olabilir. Başak genlik ve ek emme ile bir birimin &#39;dönüş&#39; bir sürekli azalma ucu çok büyük bir göstergesidir. Çok fazla emme akson onarılamaz hasara neden olabilir. Bir çözüm, basınç yavaş yavaş sıfıra döner böylece hava hattı küçük bir sızıntı sağlamaktır. Hızla inci dengelemee basınç akson sınırdışı edebilir geçici bir akraba-dışa &#39;itme&#39; verilecektir. Bu teknik belirlenen projeksiyon nöronlardan hedeflenen kayıtları elde etmek için büyük bir avantaj sağlamasına rağmen boya enjeksiyon sitesinde tüm hücre tipleri tarafından alınması gibi, bu, yerel internöronlar ayırt için yararlı olmayacaktır. Teorik olarak, ayrı enjeksiyon siteleri ile birden fazla fluorophores kullanımı bu yöntem genişletilebilir izin verebilir. Örneğin, tek-veya çift-etiketleme karşılaştırma devresi 32, iki nokta, çift enjeksiyonları ardından projeksiyon nöronlarının ikinci internöron ayırt etmek için kullanılabilir. Benzer şekilde, retrograd izleyiciler zebra ispinoz Yüksek Vokal merkezi (HCV) 32 son zamanlarda yapıldı gibi iki foton görüntüleme gibi diğer gelişmiş görüntüleme teknikleri,, ile kombine edilebilir. Epifloresans mikroskop kullanırken Ayrıca, kayıt bölgeleri biz sadece ve 1 çözmek mümkün olduğu gibi, yüzeye yakın olanlar ile sınırlıdırmu, doku ilk 30 mikron içinde m yapılar. Ancak, bu derinliği gibi iki foton mikroskopi 33 veya objektif-birleştiğinde düzlemsel aydınlatma mikroskobu 34 gibi diğer mikroskopi teknikleri kullanımı yoluyla genişletilebilir. Nispeten erişilebilir olduğunu da dahil olmak üzere - bu model sistemler çeşitli birçok farklı devrelerinde tek nöronlardan kaydetmek için kullanılabilir, çünkü genel olarak, bu teknik, in vivo olarak nöral devrelerin çalışma önemli bir gelişmeyi temsil etmektedir.

<table border="1" cellspacing="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

Bu yöntemin genel amacı nöronların tanımlanmış bir nüfus tek ünite, kaydetmektir. Bireysel nöronlar in vivo elektrofizyolojik olarak nöral devrelerin doğal şartlar altında nasıl çalıştığını anlamak için önemlidir. Geleneksel olarak, bu kayıtları kaydedilen hücrenin kimliğini kaydın başlangıcında bilinmemektedir yani, &#39;kör&#39; yapılmıştır. Hücresel kimlik ardından hücre içi juxtacellular 1, 2 ya da gevşek yama boya 3 iyontoforez vasıtasıyla tespit edilebilir, ancak bu kayıtlar fonksiyonel farklılıklar hücre tipleri ile bölgelerindeki özel nöron için önceden hedeflenen edilemez. Floresan proteinleri görsel güdümlü tek hücreli elektrofizyoloji 4-6 izin veren bir hücre tipi özel bir şekilde ifade edilebilir. Ancak, bu genetik araçlar mevcut değildir kendisi için birçok model sistemler vardır. Hatta genetik kolay kullanılabilir sistemlerde, istenen promoter bilinmeyen olabilir veya genetik olarak homojen nöronların projeksiyon modelleri farklı olabilir. Benzer şekilde, viral vektörler projeksiyon nöronlarının 7 belirli alt etiketlemek için kullanılmaktadır, ancak bu yöntem kullanımı trans-sinaptik özgüllük toksisite ve yokluğu yüzünden kısıtlıdır. Böylece, in vivo olarak tanımlanan tek nöronlardan kaydetmek için özel ön-görselleştirme sunan ek teknikler ihtiyaç vardır. Hedef nöron Ön görüntüleme klasik tek hücre kayıtları genellikle 8-11 engelleyici zor olan kayıt için zorlayıcı koşulları için özellikle yararlıdır. Bu yazıda anlatılan roman tekniği böylece hedef elektrofizyolojik elde etmek için görsel bir ipucu sağlayan, hızlı ve seçici kendilerine özgü aksonal projeksiyonlar göre belirli bir beyin bölgesi içindeki hücrelerin belirli bir alt etiket tungsten iğne kullanarak bir floresan boya retrograd taşıma uygulandı kullanır sağlam bir devre withi tespit nöronlardanna MSS omurgalı. Bizim yöntemin en önemli yeni bir gelişme, bir sigara genetik olarak erişilebilir bir model sistemde özel hücre tipleri hedef floresan etiketleme kullanılmasıdır. Zayıf elektrik balık hayvanlar 12 davranıyor, uyanık nöral devreleri eğitim için mükemmel bir model sistem bulunmaktadır. Biz zayıf elektrik mormyrid balık ön exterolateral çekirdeğinde &quot;küçük hücreler&quot; (ELa) tarafından duyusal işleme incelemek için bu teknik kullanılmaktadır. &quot;Küçük hücreler&quot; presinaptik sivri 13 gelişi zamanlarda submillisecond farklılıkları tespit etmek için önemli bir zaman karşılaştırma nöronlar olduğu öne sürülmüştür. Ancak, bu tür yoğun miyelin, içine çeken sinaps ve küçük hücre gövdeleri gibi anatomik özellikleri son derece zor geleneksel yöntemlerle 11, 14 kullanılarak bu hücrelerden kayıt yaptık. Burada güvenilir, sağlam kayıtları ve karakteristiklerini sağlayan, bizim yeni bir yöntem seçerek hazırlıkları 28% bu hücrelerin etiketler göstermektedirelectrosensory uyarıya yanıtların zasyon.

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Floresan boya retrograd taşıma anatomik projeksiyona dayalı nöronların bir alt-nüfus etiketler. Etiketli akson görsel olarak hedeflenebilir<em&gt; In vivo</em&gt;, Belirlenen aksonlardan ekstraselüler bir kayıt izin verir. Bu teknik nöronlar genetik manipülasyon yoluyla etiketli ya da izole etmek zor olamaz zaman &#39;kör&#39; kullanarak kayıt kolaylaştırır<em&gt; In vivo</em&gt; Yaklaşımlar.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

retrograde-fluorescent-labeling-allows-for-targeted-extracellular

Research

JoVE Journal

Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

19.8K Görüntüleme.  Washington University in St. Louis. Floresan boya retrograd taşıma anatomik projeksiyona dayalı nöronların bir alt-nüfus etiketler. Etiketli akson görsel olarak hedeflenebilir In vivo, Belirlenen aksonlardan ekstraselüler bir kayıt izin verir. Bu teknik nöronlar genetik manipülasyon yoluyla etiketli ya da izole etmek zor olamaz zaman 'kör' kullanarak kayıt kolaylaştırır In vivo Yaklaşımlar.

Video: Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

Çift Somatik Kayıtlar Gonadotropin-Releasing Hipotalamik Dilimleri Yeşil Floresan Protein (GFP) Belirlenmiş Hormon (GnRH) Nöronlar

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

Nörobilim

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Tek birim In vivo</emSıçan, optik kiazma> Kayıtlar

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

Tek Birim Faaliyet ve elektrokortikografik Sinyalleri Kayıt için Kronik Sinir Elektrotların Cerrahi İmplantasyonu

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Belirlenen Nöronlar ve Beslenme Davranışı Kalsiyum sinyaller aynı anda kayıt Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Sinirler, yolları ve Floresan Boyalar ile Spinal Roots Retrograd Yükleniyor

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Transgenik Balık Sağlam Beyin Tespit Edilen nöronlar Elektrik Aktivitesi Kayıt

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Hücre içi Kaydı, Duyu Alan Haritalama ve Sülük de Kültüre Belirlenen Nöronlar Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Floresan Boyalar Yetişkin Zebra balığı Retina Ganglion Hücreleri retrograd Etiketleme

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A Guide<em&gt; In vivo</emOptogenetically Belirlenen Kortikal inhibitör dan&gt; Tek birim Kayıt

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Bozulmamış neuroepithelium Fare Koku Duyu Nöronlar Delikli Patch-kelepçe Kayıt: Bir Belirlenen Odorant Reseptör ifade Nöronlar Fonksiyonel Analiz

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...