Bu çalışma, Kanada Sağlık Araştırmaları (CIHR) Hibe Wayne S. Sossin Enstitüleri tarafından desteklenmiştir. Carole A. Farah de la Recherche en Santé du Québec (FRSQ) ve bir Conrad Harrington dostluk Viyana bir doktora sonrası dostluk alıcı. Yazarlar Joanna buji, Margaret Hastings ve Margaret Labban grafik genel yardım için video çekimi ve Madeline Richmond Havuzu ile ilgili yardım için teşekkür etmek istiyorum.

1. Hazırlık ve Sf9 Hücre Monolayer Kültürler Bakım <ol><li> Bir doku kültürü kaputu Sf9 hücre kültürü ve bakım yapılır. </li><li> Sf9 hücreleri Spodoptera frugiperda yumurtalık hücreleri (Sf21 hücre) türetilmiştir ve Invitrogen Grace medya donmuş hücreleri olarak satın alınabilir. </li><li> Yeri 8 mL takviye Grace böcek, orta (1X),% 30 fetal sığır serumu (FBS) eğimli bir boyun ile 75 cm 2 hücre kültürü şişesi eklenmiştir. </li><li> 37 ° C su banyosunda hızla (yaklaşık 1-2 dakika), ileri ve geri tüp hareket Sf9 hücreler dondurulmuş tüp çözülme. </li><li> Hücreler eşit olarak dağıtmak için elle hafifçe Sf9 hücreler 75 cm 2 hücre kültürü şişesi ve taş plaka transfer. </li><li> Hücreleri bağlı 27 ° C kadar 1-2 saat inkübe edin. </li><li> Eski orta çıkarın ve% 30 FBS freshGrace böcek orta 10 ml ile değiştirin. </li><li> Konfluent hücreleri 27 ° C kadar inkübe edin. </li><li> Tek tabaka kültürleri korumak için, eski orta Sf9 hücrelerinin konfluent balon kaldırmak ve 5 ml% 10 FBS Grace böcek orta ekleyin. </li><li> Balonun tarafında Sf9 hücreleri ayırmak için birkaç kez hafifçe dokunun. </li><li> Pipet, bir kez hafifçe aşağı pipetleme sırasında balon duvar püskürtme, yukarı ve aşağı 10 ml pipet ve bir pipet medya kullanımı. 15 ml steril bir polysterene tüp hücreleri aktarın. </li><li> El ile yavaşça 75 cm 2 yeni hücre kültürü şişesi ve taş plaka% 10 FBS (log fazı büyüme sağlamak için seyreltme 01:05) ile Grace böcek orta 8 mL, Sf9 hücre süspansiyonu 2 mL ekleyin . </li><li> Konfluent hücreleri 27 ° C kadar inkübe edin. </li></ol> 2. Sf9 Hücreler floresan Takip edilen PKC İfadesi <ol><li> Canlı görüntüleme deney, kültür 35 mm Mattek cam alt kültür disheswith cam yüzey 14 mm ve 0,16 - 0,19 mm lamel kalınlığı bir Sf9 hücre. </li><li> Floresan proteinleri ile etiketlenen PKC aşağıda ayrıntılı değişiklikler ile üreticinin Cellfectin II transfeksiyon reaktif aşağıdaki tavsiye Sf9 hücreleri kullanılarak ifade edilir. </li><li> 1. Gün: hücrelerin kaplama önce, en az 30 dakika süreyle% 10 FBS Grace böcek orta her Mattek çanak tedavi. </li><li> Hemasitometre kullanarak adım 11 (üstte) Sf9 hücreleri sayın. </li><li> Plaka 0.05 x 10 6 hücre toplam hacmi 150 mcL her Mattek cam lamel. Hücrelerin oda sıcaklığında 30 dakika boyunca takmak için izin verin. </li><li> Grace hücreleri koparmasını önlemek için yavaş yavaş bir anda her çanak 1 ml% 10 FBS ile takviye 2 mL ekleyin. </li><li> 27 ° C&#39;de bir gece inkübe edin. </li><li> Bu noktadan itibaren, plazmid DNA ile Sf9 hücre transfeksiyon için üretici firmanın tavsiye kullanın. </li><li> 2. Gün: Cellfectin II transfeksiyon reaktif kullanarak floresan etiketli PKC Transfect Sf9 hücreleri. Biz genellikle gelişmiş Yeşil Floresan Protein (eGFP) ve monomerik kırmızı floresan proteini (MRFP) (plazmid inşaat için daha önce 2,5 tarif edilmiştir) ile etiketlenen bu sistem PKC eş dile getirdiler. İfade bir defada bir floresan protein% 70-80 hakkında ifade hücreler 72 saat sonrası transfeksiyon verir. Co-iki floresan proteinleri ifade transfeksiyon verimliliği yaklaşık% 30 co-ifade hücreleri azalır. EGFP etiketli PKC ve MRFP etiketli PKC co-ifade edilirken, optimum protein ekspresyonu için MRFP etiketli protein daha plazmid DNA 2x kullanın. </li><li> Gerçekleştirin canlı görüntüleme 48-72 saat sonrası transfeksiyon (optimal protein ekspresyonu, 72 saat bekleyin). </li></ol> 3. Aplysia Duyu Nöronlar floresan Takip edilen PKC Açıklanması <ol><li> Zhao ve meslektaşları 4 video makalede açıklanan Aplysia nöronal hücre kültürlerinin hazırlanması olmuştur. </li><li> Floresan proteinleri ile etiketlenen PKC Aplysia duyusal nöronlar aşağıda ayrıntılı mikroenjeksiyon yordamı kullanarak ifade edilir. </li><li> Distile su içinde% 1 Hızlı Yeşil (FCF) stok solüsyonu hazırlayın, bir şırınga ve 28 mm şırınga filtresi (0.20 mikron) filtresi. -20 ° C&#39;de kısım ve mağaza </li><li> Duyu nöronlarının Mikroenjeksiyon nöronlar izole sonra günde 1 veya 2 günde ancak 2 gün bekledikten lamel daha iyi hücre bağlılık sağlar olabilir. </li><li> Duyu nöronlarının izolasyon sonra, günde 2 Hızlı Yeşil bir kısım Çözülme ve bir şırınga ve 28 mm şırınga filtresi (0.20 mikron) kullanarak tekrar filtre. </li><li> Distile su içinde% 0.5 Hızlı Yeşil içeren plazmid DNA bir çözüm hazırlayın. Mcg / ml 0.4 gibi yüksek bir plazmid DNA konsantrasyonları kullanılabilir ama bu değerlerin üstünde gitmek için tavsiye edilmez. </li><li> Filtre 1 ml şırınga ve 4 mm&#39;lik bir şırınga filtresi (0.20 mikron) kullanarak plazmid DNA / Hızlı Yeşil çözüm. </li><li> Bir mikrofon kullanarak 15 dakika boyunca 16.110 xg&#39;de plazmid DNA / Hızlı Yeşil çözüm Santrifüjrocentrifuge. </li><li> Mikroelektrot çektirmenin kullanarak nöronlar mikroenjeksiyon (Sutter Flaming / Brown Mikropipet Çektirme, Model P-97) keskin elektrotlar hazırlayın. 1 mm dış çapı, iç çapı 0.75 mm ve 100 mm uzunluğunda içinde filament (Dünya Hassas Aletler TW100F-4) ile ince cidarlı cam kapilerleri kullanarak cam bir kutu filament kullanarak, Mikroelektronlar çekin. Sutter elektrot yemek kitabı mikroenjeksiyon elektrotlar çekme hakkında daha fazla bilgi için bakınız. </li><li> Microloader pipet ucu (Eppendorf CA32950-050) kullanarak plazmid DNA / Hızlı Yeşil çözüm 2 mcL her elektrot doldurun. </li><li> Mikroenjeksiyon istasyonu Aplysia nöronal kültürlerin içeren Mattek cam alt kültür kaplarına aktarın. </li><li> Mikroenjeksiyon istasyonu aşağıdakilerden oluşur: bir ev yapımı bir titreşim izolasyonu tablosu (Kinetik Sistemleri titreşim izolasyonu iş istasyonu), dış halojen aydınlatma ile stereo kapsam mikromanipülatör (Sutter Huxley Duvar Tipi Mikromanipülatör kültür yemekleri tutmak için büyük cam aşamasında olan) üç eksen kaba ve ultra-ince konumlandırma sağlar, pnömatik pompa (Dünya Hassas Aletler PV820 Pnömatik Pico-Pompa) bağlı bir mikroelektrot sahibi. Pnömatik pompa basıncı Giriş sıkıştırılmış bir Azot tankına bağlı. </li><li> Mikroelektrot mikroelektrot tutucu içine yerleştirin. </li><li> Stereo-mikroskop altında görüntülenen, hücre çekirdeği içine mikropipet ucuna takın. </li><li> Azot kısa basınç darbeleri sunun (10 ila 100 ms süresi, 20 lb / 2) çekirdeğin düzgün olarak yeşil olana kadar. </li><li> Hücreleri 4 saat oda sıcaklığında inkübe edin ve 4 ° C kullanana kadar az tutmak. Enjeksiyon sonrası 24 saat optimum PKC translokasyonu için canlı görüntüleme deney 20 gerçekleştirin. </li></ol> 4. Sf9 Hücreler Yaşayan ve Aplysia Duyu Nöronlar PKC Translokasyon Görselleştirme <ol><li> Görüntüleme için, biz bir Axiovert 200 ile Zeiss LSM510 ters konfokal mikroskop kullanıyoruz. </li><li> Aplysia duyusal nöronlar x40 immersiyon yağı hedefi ile görüntülü ise Sf9 hücreleri X63 immersiyon yağı hedefi imaged: Görüntüleme protokolü Sf9 hücreler ve Aplysia duyusal nöronlar için aşağıdaki hariç aynıdır. Aplysia duyusal nöronlar, 18-20 o C civarında muhafaza oda sıcaklığında görüntülü ise Sf9 hücrelerin bakımı, 26-27 o C sıcaklık kontrollü bir odasında görüntülü </li><li> % 50 lazer çıktı eGFP protein ve MRFP protein ile görüntü etiketledi PKC HeNe lazer (543nm azından uyarma) etiketli görüntü PKC 30 mW Argon lazer (488nm az eksitasyon). Argon ve HeNe lazer çizgileri% 4 oranında azaltılmış ve görüntüleme ve iğne deliği önce sırasıyla% 50 iletim çıkışı bir şekilde ayarlanır. </li><li> Kültür çanak görüntüleme sahnede stabilize olması ve hareket veya titreşim önlemek için önemlidir. </li><li> Yavaşça bırakarak bir tabak içinde 1 ml toplam hacmi 10 mL pipet kullanarak çanak 1 ml kültür ortamı çıkarın. </li><li> Çekirdeğin görülebilir hücrelerinin orta bölümünde fotoğraflar çekin. </li><li> Her 30 saniyede bir resim alarak 10-20 konfokal görüntüleri bir zaman serisi ayarlayın. </li><li> Zaman serisi başlayın. </li><li> 2 resim (30 saniye noktadan sonra) alındıktan sonra, yavaşça ilacın 1 ml (2X konsantrasyon) P1000 bir pipet kullanarak hücrelerin üstüne bırak bırak ekleyin. Ilaç yaklaşık 30 saniye boyunca sürekli olarak eklenir. </li><li> Bazı ilaçlar, diğerleri sadece en iyi 5-10 dakika tedavi sonrası (Film 1 ve Movie 2) yavaş bir translokasyon neden while (60 saniye noktada) hemen sonra ilaç tedavisi görülebilir hızlı bir translokasyona neden olur. </li><li> Ilaç yıkanıp gerekiyorsa, çanağı bir tarafta bir pipet yardımıyla hafifçe aşağı yıkama tamponu pipetleme ve diğer tarafta medya pipeting yıkama yapmak için 2 x 10 ml pipetler kullanabilirsiniz. </li><li> Yıkama etkili olmuştur ve ilacın etkisi geri dönüşümlü olup olmadığını, KZÇ sitoplazmada 30-60 saniye içinde geri döner. </li><li> Film LSM bir dosya olarak kaydedin. </li><li> LSM dosyaları açmak ve görüntüleri öncesi ve sonrası ilaç tedavisi ölçmek için NIH Image J yazılımı kullanın. Niceleme başka bir yerde 1 tarif edilmiştir. </li></ol>

<ol>
	<li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons.">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li><li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &. a. m. p. ;. a. m. p., Klein, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2+-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation.">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li><li>Farah, C. A., Weatherill, D., Dunn, T. W. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia.">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J Vis Exp. , (2009).</li><li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia.">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li></ol>

Biz Sf9 hücreleri ve Aplysia duyu nöronları gerçek zamanlı olarak floresan etiketli PKC görüntü translokasyon bir teknik tanımlanmıştır. Kültür ve onları transfecting oldukça düz ileri olduğundan Sf9 hücreleri görüntü PKC translokasyonu basit bir sisteme de sağlar. Aksine, Aplysia duyu nöronlarının mikroenjeksiyon master için biraz zaman alır; birkaç ay. Bu tekniği ile ilgili güçlükler elektrot tıkanma içerir. Enjeksiyon çözüm Filtreleme ve santrifüj genellikle ya...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sf9 frozen cells</td>
<td>Spodoptera frugiperda ovarian cells</td>
<td>Invitrogen</td>
<td>B825-01</td>
<td>&nbsp;</td>
<tr>
<td>Grace&#x2019;s Insect Medium, Supplemented (1X), liquid</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11605-094</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Corning 75cm2 canted neck cell culture flask</td>
<td>Tool</td>
<td>Corning</td>
<td>430720</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>CanSera</td>
<td>CS-C08-500</td>
<td>Supplement Grace's media with FBS prior to use</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309604</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309602</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431229</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431212</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Glass Bottom Dish</td>
<td>Tool</td>
<td>Mattek</td>
<td>P35G-1.5-14-C</td>
<td>Use to culture Sf9 cells prior to transfection and imaging</td>
</tr>
<tr>
<td>Cellfectin II Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10362-100</td>
<td>Used to express fluorescently tagged PKCs in Sf9 cells</td>
</tr>
<tr>
<td>Fast Green FCF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F-7252</td>
<td>Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons</td>
</tr>
<tr>
<td>Thin wall glass capillaries</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>Use to make microelectrodes for microinjection</td>
</tr>
<tr>
<td>Microloader pipette tip</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>CA32950-050</td>
<td>Autoclave before using to fill up the microelectrodes for microinjection</td>
</tr>
<tr>
<td>PV820 Pneumatic PicoPump </td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>Part of microinjection station</td>
</tr>
<tr>
<td>Microelectrode holder</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH6S</td>
<td>Use to hold microelectrode </td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Sutter</td>
<td>MP-85</td>
<td>Use to position microelectrode in the three-axis</td>
</tr>		</tbody>
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live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

Protein kinaz Cs (PKC) gibi hücre büyüme ve öğrenme ve hafıza gibi çok çeşitli hücre süreçlerini düzenleyen merkezi bir rol oynayan treonin kinazlar serin. Omurgalıların PKC izoformlarını dört anılan aileler bulunmaktadır: klasik PKC (α, βI, βII ve γ), yeni tip I PKC (ε η), yeni tip II PKC (δ ve θ) ve atipik PKC (ζ ve ι .) Klasik PKC Ca 2 ile aktive olan roman PKC DAG aktif, ancak Ca 2 +-bağımsız iken + ve diacylclycerol (DAG). Atipik PKC + ne DAG Ca 2 ne tarafından aktive edilir. Aplysia californica, bellek oluşumunu incelemek için model sistem olmak üzere üç sinir sistemine özgü PKC izoformlarını her ana sınıfı, geleneksel PKC Apl Ben, roman türü PKC Apl II ve atipik PKC Apl III vardır . PKC lipid aktive kinazlar ve böylece ekstrasellüler sinyaller yanıt klasik ve yeni PKC aktivasyonu sık sık, plazma zarı sitoplazmaya PKC translokasyonu ile korele olmuştur. Bu nedenle, canlı hücreler gerçek zamanlı PKC translokasyonu görüntülenmesi PKC aktivasyonuna yol açan sinyal iletim yolları nedenlerine açıklık için paha biçilmez bir araç haline gelmiştir. Örneğin, bu tekniğin farklı PKC izoformlarını sinaptik plastisite farklı tip arabuluculuk için farklı koşullar altında yerini değiştirmek ve II. PKC Apl bu serotonin (5HT) aktivasyonu için hem DAG ve phosphatidic asit (PA) üretim gerektirir kurmak için bize izin verdi translokasyon 1-2. Önemlisi, aynı nöron görselleştirmek için yeteneği sürekli olarak bize, örneğin, en ince ayrıntısına kadar 3 PKC yanıt duyarsızlaştırma ölçmek için izin verdi . Bu video Sf9 hücre kültürlerinin hazırlanmasında her adımı gösteren, Aplysia duyusal nöronlar kültürleri yanıt olarak floresan etiketli PKC ifade Sf9 hücreleri ve Aplysia duyusal nöronlar ve PKC translokasyonu canlı görüntüleme, başka bir video 4 üncü maddesinde tarif edilmiştir lazer tarama mikroskopisi kullanarak farklı aktivatörleri.

Watch this Scientific Journal Video about Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons at JoVE.com

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Bu video, floresan etiketli PKC kullanarak canlı hücreler PKC translokasyonu görselleştirme göstermektedir.

Canlı görüntüleme Sf9 Hücreler ve PKC Translokasyon Aplysia Duyu Nöronlar

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

live-imaging-pkc-translocation-sf9-cells-aplysia-sensory

Research

JoVE Journal

Neuroscience

11.9K Görüntüleme.  McGill University. Bu video, floresan etiketli PKC kullanarak canlı hücreler PKC translokasyonu görselleştirme göstermektedir.

Video: Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

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 ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Nöromodülasyon ve Mitokondrial Ulaştırma: Uzun Süreleri içinde Hipokampal Nöronlar Canlı Görüntüleme

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Koku Duyu Nöronlar lentivirüs aracılı Genetik Manipülasyon ve Görselleştirme In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Koku Duyu Nöronlar Cilia Kafesli Bileşiklerin Flash Fotoliz

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Ekstraselüler bir kas Motor Pool için motor nöronlar belirlenmesi<em&gt; Aplysia&#39;nın californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Duyusal nöronlar izolasyonu<em&gt; Aplysia californica</em&gt; Glutamaterjik Akımlar Patch Kelepçe Kayıtlar için

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Bir Yöntem Hedef ve Fare İzole Havayolu-innerve Duyu Nöronlar için

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...