This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1.. Cerrahi Aparatı Construct Not: esnasında ve işlem, damla anestezi çözelti etil 3-aminobenzoat metansülfonat (MS-222, ya da Tricaine) balığın ağız yoluyla 1 damla / sn &#39;lik bir hız ile bir buçuk konsantrasyonunun (% 0.015) ile sonra balık canlı kalmasını sağlamak için, Şekil 1 &#39;de gösterilen bir homebuilt damlama sistemi kullanılarak. <ol><li> Şekil 1A gösterildiği gibi bir kutu yapmak (uzunluk x genişlik yüksekliği 5 cm, 10 cm, 28 cm), orta (uzunluk genişlik 9 cm, yüksekliği 4 cm olduğunu, 6 cm) bir sünger koymak ve bir açın Üzerinde boşluk. </li><li> Daha sonra balık ağız içine sokulacaktır ışık sertleştirme bağ (Şekil 1B) ile iğnenin ucunda düz bir küresel yüzey olun. Iğne yüzeyinin hasar görmesini balığın ağzını önlemek için yumuşak olduğundan emin olun. </li><li> MS-222 (sol bölme, uzunluk 6 cm olan yarı konsantrasyonu ekleme, genişlik 10 cm, yükseklik 8 cm) ve Şekil 1C &#39;de gösterildiği gibi rezervuar salin (sağ odası, uzunluk yüksekliği 8 cm, genişliği 10 cm olan, 4 cm)&#39; dir. Not: Taze MS-222 önerilir. </li><li> 2 dakika boyunca% 0.03 MS-222 balık anestezisi. </li><li> Süngerin boşluğundaki balık yerleştirin ve göğüs yüzgeç arkasında ve sırasıyla Kloak gözenek, 11 yanına yerleştirilen iki çift pim ile balık sabitleyin. </li><li> Şekil 1C &#39;de gösterildiği gibi, bir T tipi tüp ile ağız balığın, MS-222, ve yetiştirme su bağlayın. </li><li> MS-222 bağlı tüp açın. </li></ol> Sağ TECTUM 2. Retrograd Etiket RGCs NOT: Ön başına, hiçbir etanol kalmasını sağlayarak,% 70-75 etanol ile tüm cihazları dezenfekte. <ol><li> Rossing: hafif sclerectome ile bütün kafatası üzerinde cilt kaldırmak (kafatası dorsal görünüşüdür Şekil 2A ve 2B&#39;de gösterilmiştir <li> Takas: tuzlu su ile kafatası yıkayın ve sonra kulak yıkama ampul ile kurulayın. </li><li> İlk korozyon: 10 saniye boyunca yanığı ile kafatası zarar. </li><li> Takas: tamamen pamuklu çubukla pürüzlendirici silin ve daha sonra tuzlu su ile yıkayın ve kurulayın. </li><li> Koruma: Aşırı korozyona doğru TECTUM dışında diğer alanlarda kafatası korumak için, ışıkla sertleşen bağ ile bu alanları kapsayacak ve daha sonra taşınabilir aydınlatıcı ile mavi ışığa maruz kalma ile onu tedavi. </li><li> Komple korozyon: 2 dakika için tam bir aşındırma için tekrar doğru TECTUM merkezi alanında pürüzlendirici koydu. Not: Büyük balık kafatasları kalsifiye olduğundan, korozyon daha uzun sürer. </li><li> Takas: tamamen pamuklu çubukla pürüzlendirici silin ve daha sonra tuzlu su ile yıkayın ve tekrar kurutun. </li><li> Kafatası açılış: dikkatle sağ TECTUM (Şekil 2D açığa forseps ile malacic kafatası (Şekil 2C) kaldırmak </strong&gt;). </li><li> Takas: gelfoam adet delikten mukus ve kan taşmayı silin ve kanama durana kadar tuzlu su ile yıkayın. </li><li> Boya yerleştirme: tüm sağ TECTUM üzerine boya (doku-etiketleme yapıştır) koymak ve gelfoam ile örtün. </li><li> Ayrılan: aynı hayvandan steril bir ölçek ile deliği kapsamaktadır. </li><li> Tartının üzerine yer ışık kür bağ ve daha sonra 40 saniye için tekrar mavi ışık ile tedavi: Sızdırmazlık. </li><li> Revival: bağının yüzeyini yıkamak ve tuzlu su ile hayvan canlandırmak. </li><li> Hemşirelik ve ıslah: 5 gün boyunca 28.5 ° C embriyo orta (ZFIN) olarak balık tutun ve 2 kez / gün besleme. Kimin boya deneyde 5 gün boyunca TECTUM kalmak için başarısız balık dahil etmeyin. Not: Embriyo orta (EM) çözümü balık hayatta kalmak için hayati önem taşıyan ve sıcaklık DiI etiketleme için önemli bir faktördür. </li></ol> 3.. Wholemounting Retina ve Görüntüleme <ol><li> Retina önce 2 saat için karanlık bir alanda balık yerleştirin olanlemount. </li><li> Buzlu su ile balık anestezisi. </li><li> Kornea marjı bir delik delinerek ve Venus makas ile kornea kaldırarak göz fincan olun. </li><li> Lens çıkarın ve sonra buz gibi soğuk 1x PBS ile retinanın merkezini yıkayın. </li><li> Herhangi bir pigment kalana kadar, retina ve buz gibi soğuk 1 x PBS ile sklera arasındaki boşluğu yıkayın. </li><li> Diskte optik sinir kesti. </li><li> Yukarı RGC katmanı tutmak ve bir self-made cam kaşık (Şekil 3A ve 3B) ile retina tutun. Bir asma teli aynı zamanda retinayı transfer etmek için kullanılabilir. </li><li> Yukarı RGC tabakası (Şekil 3C) ile bir slayt buzlu% 30 gliserin, bir damla retina koydu. </li><li> Gliserin çıkarın ve sonra dört parçaya (Şekil 3D) içine retina kesmeden önce cam slayt retina düzleştirmek. RGC tabakası ile gelişeceğini retina tutarak yukarı retina düzleştirme için yararlı olacaktır. </li><li> T buzlu% 50 gliserin damlao pigment parçaları silsin retina. </li><li> Gliserin çıkarın ve sonra retina üzerinde buzlu% 75 gliserin 20 ul bırakın. </li><li> 1.8 cm x 1.8 cm kare lamel ile örnek örtün. </li><li> Tırnak cilası ile lamel mühür. Retinanın anında görüntüleme önerilir. </li><li> Sigara aralıklı resim dp72 CCD (her görüntü 1360 x 1024 piksel) ile 20X nesne mercek altında bütün retina (NA 0,70 =). Her alan üç farklı odak vardır. </li><li> Yazılım &quot;alanında genişletilmiş derinlik&quot; aracı ile her alanın derinliğini genişletin. </li><li> Photomerge fonksiyonu ile sanal retina birleştirin. </li><li> RGCs ortalama yoğunluğu (Şekil 4A) analiz etmek için retinanın her yönde 680 x 512 piksel üç alan (gerçek alan 0.092 mm 2, 264 mikron ile 350 mikron) seçin. </li><li> Ölçümlerin fonksiyonu ile alan elde - görüntüde bir çokgen özellik oluşturmakyazılım. </li><li> Denklemle RGCs toplam sayısını hesaplayın &quot;RGCs sayısı = kader x alanı&quot; 9. </li></ol>

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We have nothing to disclose.

RGCs retrograd etiketleme memeli hayvanlarda RGCs hayatta araştırma önemlidir, ancak zebrabalıkları kullanılmamıştır. Alternatif yöntemler, HE 7, boyama ve antikor boyama 8, RGCs sayısını saymak için altın standart ve etiketlenmiş tüm RGCs ile transjenik olmayan ama 12, 13, inşa edilmemiştir. Bu videoda, biz yetişkin zebrafish retinada etiket RGCs retrograd bir yöntemi tanıtmak. Akson yaklaşık% 1 koku ampul veya diğer alanların 14 çıkıntı olsa da, bu yöntem, hemen hemen tüm retinada RGCs etiket olabilir. Zebra balığı neşter kesi 3 ya da matkap 5, 15 yetişkin zebrafish kafatasını açmadan, fiziksel yaralanma ayı çok kırılgan olduğu gibi ölüme neden olabilir. Yanığı ile kafatası yumuşatma beyin hasarı en aza indirebilirsiniz. Hafif bir kür bağı ile delik sızdırmazlık ve mavi ışık altında sabitlemekEn çok balık ameliyattan sonra hayatta. Bu sadece sıçan ve diğer memeli hayvanlar 5 ile karşılaştırıldığında daha uygun ve etkili olduğu, uzman operasyon kapsamında ameliyatı gerçekleştirmek için 12-15 dakika sürer. DII dokuya etiket yapıştırma lipofilik bir boyadır. DII kristaller ya da konsantre edilmiş çözeltilerin mikroenjeksiyon ile karşılaştırıldığında, bu iki ve yüzeyin altında aksonlar etiketleme, dokuya boyanın penetrasyonu artırır. Ayrıca, floresan uzun bir süre 10 devam edebilir. Öte yandan, bu boya pasif taşıma yolu ile emilir, bu nedenle tam bir etiketleme için, en az 5 gün için TECTUM içindeki boya bırakmak gerekli olmasıdır. Zebra balığı görsel yenilenme mükemmel bir model organizmadır. Optik sinir ezmek sonra 7 gün, hemen hemen tüm RGCs TECTUM için yeniden akson, ancak RGCs sadece yarısı optik sinir kesme modelinde 9 yeniden. Bu yöntem RGC araştırma için ideal bir yolduroptik sinir ekstra yaralanmaları önlemek olabilir optik sinir ezmek, sonra s hayatta kalma ve yenilenme. Biz yetişkin Zebra balığı ve bir wholemount retinada RGCs sayısını ölçen başarıyla retrograd etiketleme RGCs için tam bir protokol sundu. Ayrıca, hafif bir sertleşme bağ kullanarak, bu yöntemin daha etkili hale zebrafish hayatta kalma oranını artırmak.

Yetişkin zebrabalıkları saymak optik sinir hasarı sonrası 1 aksonlar yeniden güçlü bir yeteneği, uygun bir yöntemi var gibi bütün RGCs RGCs yaşam ve yenilenme 2 değerlendirilmesi esastır. Memeli ve akvaryum balığı 3&#39;te retrograd etiketleme RGCs yöntemlere dayalı - 5, biz yetişkin zebrabalıkları TECTUM gelen RGCs etiketlemek yöntemini inşa. Yetişkin Zebra balığı için, iki kritik teknik endişeler dikkat edilmelidir: yetişkin zebrafish kafatası 6 çok küçük; onlar su ortamında yaşıyoruz. Burada, 5 sondaj ile ilişkili tehlikeleri en aza indirir yanığı ile kafatası tedavi. Sonra, biz ameliyattan sonra hayvan yaşamını geliştirir ışık kür bağı ile delik mühür. Daha önce, diğer bazı teknikler dolaylı yollarla RGCs saymak kabul edildi. HE retina bölümleri boyama RGCL 7 hücrelerinin her türlü etiketler. Bütün r Antikor etiketlemeBöyle adacık-1 gibi etina, ayrıca amacrine hücreleri 8. etiketleyebilirsiniz. Optik sinir güdük retrograd etiket retinada tüm RGCs etiketleyebilirsiniz olsa da optik sinir ekstra yaralanmasına neden olduğundan, bu ezilme modelinde kabul edilemez. TECTUM gelen retrograd etiket yararlanarak, optik sinir ezilme RGCs yaşam ve yenilenme araştırdım. Sonuçlar hemen hemen tüm RGCs hayatta ve RGCs% 90 üzerinde ezilme modelinde 9 ilk hafta TECTUM rejenere olduğunu göstermektedir. Başarıyla RGCs etiket için, DII macun birçok ticari boyalarda 10 ile karşılaştırılmasından sonra seçildi. İlk olarak, özellikle in vivo doku etiketleme için tasarlanmıştır. İkinci olarak, bu su içinde yaygın olmayan bir lipofilik boyadır. Ayrıca, bu floresan o RGCs hayatta kalma araştırma için mükemmel bir aday yapar uzun süre devam edebilir.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Gluma Etch 35 Gel</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:152px;">Blue LED</td>
			<td style="width:152px;">Shenruo Medical Equipment Co.</td>
			<td style="width:152px;">Power Blue Light Curing Unit</td>
			<td style="width:152px;">China</td>
		</tr>
	</tbody>
</table>

retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

Olarak retrograd etiketleme retina ganglion hücrelerinin (RGCs) siteleri ölüyor RGCs somata izole olabilir, bu RGCs hayatta kalma ve rejenerasyon deneylerde RGCs sayma için altın standart haline gelmiştir. Birçok çalışma, optik sinir yaralanmasından sonra RGCs hayatta aratmak için memeli hayvanlarda yapılmıştır. Bazı alternatif yöntemler retina ganglion hücre tabakası (RGCL) hücre sayıları saymak olsa Ancak, yetişkin zebrabalıkları RGCs retrograd etiketleme henüz rapor edilmemiştir. Yetişkin zebrabalıkları kafatasının küçük boyutu ve kafatası üzerinde sondaj sonrası ölüm riski yüksek göz önüne alındığında, biz asit gravür yardımıyla kafatası açık ve önemli ölçüde hayatta kalma oranını getirebileceği bir ışık kür bağ ile delik mühür. 5 gün için boyalar emici sonra, hemen hemen tüm RGCs etiketlenir. Bu yöntem, optik sinir transect gerekmez gibi, yetişkin zebrabalıkları optik sinir ezmek sonra RGCs hayatta araştırma yeri doldurulamaz. Burada, biz tanıtmakBu yöntem, adım adım ve temsili sonuçları sağlar.

Şekiller 4B-D gösterdiği gibi, DII + hücrelerinin sayısı in RGCL DAPI + hücrelerinin üçte ikisi. Normal bir retina olarak, bir bütün retina (Şekil 4E) bir montaj resmi DII + RGCs orta bölgesinde RGCs kendi hedef olarak yeniden gibi tüm retina üzerinde olan, ancak bir rejenere retina (Şekil 4F), dağıtılır olduğunu göstermektedir İlk hafta, onlar etiketli olamazdı. <img alt="Şekil 1" fo:content-width="5in" src="/files/ftp_upload/50987/50987fig1highres.jpg" width="500" /> Şekil 1. RGC retrograd etiketlenmesi için kullanılan cerrahi cihaz. (A) (a) balık düzeltmek için bir sünger kullanın. Çalışma platformu ve infüzyon tüpü, metal iğne (b) üzerinden bağlanır (uzunluk x genişlik yükseklik 5 cm olan 10 cm olan, 28 cm.) Metal iğne fazla su boşluğun (c) &#39;de depolanır. (B) bir yakın çekim. İğnenin baş hafif kür bağ (d) ile sarılır. (C) rezervuarlar MS-222 saklamak için kullanılan (e, boy yüksekliği 8 cm, genişliği 10 cm, 6 cm) ve tuz (f, uzunluğu 4 olan cm, genişlik sırasıyla) yüksekliği 8 cm, 10 cm. Enfüzyon boru (g) ​​rezervuarı ve metal iğne bağlanır. <img alt="Şekil 2," fo:content-width="5in" src="/files/ftp_upload/50987/50987fig2highres.jpg" width="500" /> Şekil 2.. Yetişkin zebrafish kafatasının dorsal görünümü. Rossing önce (A) Sağlam kafatası. Sarı noktalı çizgi TECTUM işaretleri; kırmızı noktalı çizgi telencephalon işaretleri; ve yeşil noktalı çizgi beyincik işaretler. (B) Kafatası çıkarıldı deri sonra. (C) yanığı ile korozyona sonra, kafatası malacic ve bir içbükey alanı forseps (beyaz *) ile işaret edilir. (D) Sağ TECTUM maruz kafatası kaldırıldıktan sonra. Ölçek 500 &amp; # olduğu956, m. <img alt="Şekil 3," fo:content-width="5in" src="/files/ftp_upload/50987/50987fig3highres.jpg" width="500" /> Şekil 3.. Retina wholemounting Anahtar adımlar. Retinayı karıştırıyordu için (A) Homebuilt cam kaşık, alt görüntü bütün görünümünü gösterir. (B) (noktalı çizgi ile işaretlenmiş) &quot;kaşık&quot; ile retina tutun. (C) buz-soğuk% 30 gliserin içine retina taşıyın. ( D) 4 çeyrek içine retina kesin; bir ok yönünü gösterir. Ölçek çubukları (A) 1 mm; (BD) 500 mikron. <img alt="Şekil 4," fo:content-width="5in" src="/files/ftp_upload/50987/50987fig4highres.jpg" width="500" /> F ŞEKIL 4. Sanal retinada sayma RGCs örnekleme. (A) retina şematik görünüşüdür dört alan (N, D, V, T alan) sahiptir ve bunların her birisibeyaz kutu içinde gösterilir üç alanlar. Optik disk genellikle ventral-zamansal yönlendirme (siyah nokta) yer almaktadır dikkat edin. (B) DiI RGCs etiketli. RGCs ve çekirdeğin (C) DAPI etiketli çekirdeği. (D) Birleştirildi resim. Bütün retina (E) Montaj resim Normal Zebrafish. (F), optik sinir hasarı sonrası rejenere retina gösteren Montaj görüntü. RGCs ilk haftasında kendi hedeflerine rejenere değil gibi retinanın merkezi alanı, etiketli değildir. Kısaltmalar: N = burun, D = dorsal, V = ventral, T = zamansal. Ölçek çubukları: 30 mikron (BD); 500 um (E, F).

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

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

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 ...

retrograde-labeling-retinal-ganglion-cells-adult-zebrafish-with

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.4K Görüntüleme.  University of Science and Technology of China. We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Zebra balığı Beyin Etiketleme ve Görüntüleme Hücreleri

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Nörobilim

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Genetik Etiketli Retina Ganglion Hücreleri Işık Tepkisi Record İzole Retina Hazırlık

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Gerilim duyarlı Boyalar ile Yengeç Stomatogastric Ganglion Nöronlar Optik Görüntüleme

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

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

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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 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. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Kültür Yetişkin Dorsal Root Ganglion Nöronlar ile Axon Rejenerasyon Genetik Çalışma

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Fare Retina Ganglion Hücreleri Synaptic ve Yapay iletkenlikleri Dinamik Kelepçe Uygulama

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Retinal Ganglion Hücrelerinin Tanımlanmış Alt Kümelerinin Tek Hücreli RNA-Seqi

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Optik Koherens tomografi: Fare retina Ganglion hücreleri Vivo içinde görüntüleme

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...