Bu çalışma, R. Brewster (1R01GM085290-01A1) verilen bir NIH hibe ile desteklendi.

1.Microinjection <ol><li> Sulandırınız plazmid kodlama membran hedefli Yeşil Floresan Protein (mGFP Richard Harland nezaket) 40 ng / ml &#39;lik bir konsantrasyon. DNA Qiagen Maxi Hazırlık Seti kullanarak saflaştırılmış plazmid (Richard Harland mGFP/PCS2 +, nezaket) doğrusallaştırma tarafından hazırlanmıştır. Çözümü için fenol kırmızısı (1 / 10 toplam hacmi seyreltilmiş) eklenmesi çözüm görselleştirmek için tercih edilir. Buz üzerinde DNA çözüm tutun. </li><li> Kademeli bir slaytta iğne hizalayarak ve yavaşça temiz bir jilet kullanarak çapı 1 mikrona kadar iğne vurma, stereomikroskop altında, enjeksiyon iğnesi (Cat No. NA-GD-1 Narishinge Bilimsel, 90mm cam kılcal damarlar) kalibre bıçak (uygun büyüklükte bir ipucu elde etmek için). </li><li> Bilinen bir hacmi (2nl) enjekte etmek için iğne kalibre için, mikroenjeksiyon aparatı iğne bağlamak (PCI 100 mikroenjektör; Harvard Apparatus) ve ön bir parça yerleştirilir fenol kırmızısı ve su içeren bir çözüm 0.5μl iğne doldurmak parafin. Çözüm, bir ayak pedalı kullanılarak reçete edilebilir. 7 KPa basınç ayarlama ve çözüm 0.5μl dağıtmak için 250 pedallar alır, böylece (10msec 30msec) zaman değişebilir. </li><li> Nemlendirilmiş bir 100 mm Petri kabında balmumu bir şerit kalibre iğne dikkatlice yerleştirin. Çanak nemlendirmektir, kenarlarda ıslak Kimwipes (iğne çözüm buharlaşmasını önler) koyun. </li><li> Mikropipet iğne sonunda (kalibre değildi sonu) içine DNA 0.5-1μl yüklemek için kullanın. </li><li> Çözüm iğne ucu ulaştığında, mikroenjeksiyon aparat yüklemek için kullanılan iğnenin ucunu. Ayak pedalı ile yaklaşık 2 nl / enjeksiyon sunmak için mikroenjektör ayarları yapın. </li><li> Embriyo Orta (1.0 ml Hank Kullanıcı Stok # 1, 0.1 ml Hank Kullanıcı Stok # 2, 1.0 ml Hank Kullanıcı Stok # 4, 95.9 ml GKD 2 O, 1.0 ml Hank Kullanıcı Stok # 5 dolu bir Petri çanak ortasında Sonraki, pozisyon zebrafish embriyolar 1.0 ml taze Hank Stock # 6, pH 7,2 - 10 damla 1 M NaOH hakkında kullanın). </li><li> Embriyoların istenilen aşamaya geliştirilen sonra, yavaşça ince forseps ile koryon kavrama ve enjeksiyon için hedeflenen bölgeye iğne bulunurdu. Geniş mGFP ifade 1cell aşamada yumurta sarısı ya da sitoplazma içine DNA enjekte edilir. Küçük bir bölgeye mGFP ifade kısıtlamak, tek bir hücrenin sitoplazmasında içine 4 (veya daha fazla) hücreleri aşamasında DNA enjekte etmek. </li><li> Mikroenjeksiyon cihazların ayak pedalına basın ve yaklaşık 2 nl teslim çözüm (fenol kırmızı ek nedeniyle görünür) düzgün bir şekilde enjekte edilir sağlamak. </li><li> Embriyo iğne dikkatlice çıkarın. </li><li> Petri kabındaki bütün embriyolar için yineleyin. </li><li> Embriyolar 28.5 ° C istenilen aşamaya geliştirmek için izin verin. </li><li> Yavaşça ince forseps (embriyolar embriyo hayatta kalma oranını artıran bir cam Petri kabındaki dechorionating, 24hpf daha genç iseniz) kullanarak koryon sıyrılarak Dechorionate embriyolar. </li><li> Petri kabı cam bir pipet kullanarak boş koryon çıkarın. </li></ol> 2. Görüntüleme mGFP etiketli vibratome bölümleri <ol><li> 1X fosfat tampon solüsyonu (PBS) sabitleştirici (% 4 paraformaldehid içine embriyolar taşımak için bir cam pipet kullanın. 4 ° C gecede embriyolar Fix. </li><li> 1X PBS embriyoların 3 kez her biri için 5 dakika yıkayınız. </li><li> Petri kabı ve güzel forseps kullanarak embriyoların yerleştirin, yumurta sarısı embriyonun zarar vermeden çıkarın. Mümkün olduğunca çok sarısı çıkarın. </li><li> H 2 O% 4 düşük erime agaroz hazırlayın </li><li> Erimiş agaroz, plastik bir kesit kalıp içine yerleştirin. </li><li> Petri kabı enjekte embriyo kaldırmaya ve ince forseps agaroz yer ve yönlendirmek için kullanın. Embriyo kalıp sonuna doğru yerleştirilmelidir. </li><li> Agaroz sertleştikten sonra agaroz blok, kalıptan çıkarmak için, düz bir yüzey aracı, bir spatula gibi kullanın. </li><li> Hatta böylece bir jilet ile agaroz bloğunun alt ucundan kesin. </li><li> Vibratome sahne süper yapıştırıcı bir damla ekleyin ve agaroz bloğu (ustura ile tesviye blok yan tutkal bakmalıdır) yerleştirin. Birden fazla embriyoların kesit için, sahnede birkaç agaroz gömülü dokulardan monte edin. </li><li> (Vibratome A.Ş.), sahne vibratome vibratome takın. </li><li> (Kalınlık-50μm hız-3 ortak ayarları) vibratome parametreleri ayarlayın. Eğer birden fazla embriyoların kesit, sahnede agaroz bloklar hizalayın ve (aynı embriyo ilgili bölümler bir araya kalacağını gibi) bıçak agaroz bloklar tüm genişliği ile tüm embriyo geçer ama o sağlamak. </li><li> Vibratome aşamasında 1X PBS dökün. </li><li> Bölümü. </li><li> Embriyolar sectio.ned, vibratome aşamasından agaroz blok (ler) kaldırmak. </li><li> Her bölüm, agaroz blok (ler) in arka kesim tarafından dikkatle ayırın. </li><li> 1X PBS dolu çok iyi çanak (farklı embriyolar karşılık bölümleri için farklı kuyu kullanmak) istenilen bölümleri ve yer seçmek için ince forseps kullanın. </li><li> Her embriyo için tekrarlayın. </li><li> Antikor etiketleme ile devam mGFP görüntüleme (bölümler hazırlık, bir veya iki gün içinde görüntülü eğer bu adım gerekli olmayabilir) geliştirmek için olsa 23 19 adım. </li><li> Non-spesifik bağlama engellemek için 1X PBS engelleme çözümü ile oda sıcaklığında 30 dakika için bölümler davranın. </li><li> Birincil antikor (tavşan-GFP, Invitrogen Cat No A11122) 4, 5 saat oda sıcaklığında veya gece için bir çalkalayıcı engelleme çözümü @ 1:200 seyreltilmiş ° C ile inkübe bölümler </li><li> % 1, BSA/1X PBS /% 1 DMSO çözüm dört kez (10 dk, 15 dk, 30 dk ve 60 dk, her) yıkayın. </li><li> 4 5 saat oda sıcaklığında veya gece için engelleme çözümü uygun ikincil antikor ile bölümleri davranın ° C. </li><li> % 1, BSA/1X PBS /% 1 DMSO çözüm dört kez (10 dk, 15 dk, 30 dk ve 60 dk, her) yıkayın. </li><li> Çekirdeklerin leke 30 dakika; DAPI (Invitrogen Cat No D1306 50ml 1X PBS içinde% 0.1 DAPI) bölümleri davranın. </li><li> Her 5 dakika için 1X PBS içinde üç kez yıkayın. </li><li> Mount slaytta. </li><li> Konfokal mikroskobi kullanarak görüntü. </li></ol> 3. Canlı görüntüleme <ol><li> Bunlar istenen gelişimsel aşamaya ulaşmak Dechorionate canlı, enjekte edilen embriyolar 30 dakika önce. 15 dakika için sedatif çözüm embriyolar (0.01% Tricaine Embriyo yılında Orta) yerleştirin. </li><li> (Parça No P35G-o-20-C Mattek) 35mm cam alt kültür tabağına% 1 agaroz bir damla yerleştirin. </li><li> Agaroz katılaşmasını sağlayın. </li><li> 3 delik agaroz (mikropipet ipuçları gibi diğer nesnelerle delik açmak için kullanılan olabilir) yapmak için ateşte Isı kılcal tüp. Hiçbir kalıntı agaroz delik altında olduğuna emin olun. </li><li> Embriyo Orta Petri kabı doldurun. </li><li> Delik embriyo bir cam pipet kullanın. </li><li> Daha sonra, şark alanı (dorsal baş) görüntülü böylece ince forseps kullanılarak embriyo cam ile temas halinde. </li><li> Dikkatlice mikroskop aşamasında (buharlaşmasını önlemek için Petri kabı kapağı üzerinde yer) Petri kabı taşımacılık. </li><li> Görüntüleme ederken, embriyoların 28.5 ° C olduğunu olun </li></ol> 4. Sonuçlar Burada mozaik ifade kullanarak zebrafish nöral tüp görüntü tek hücre için basit bir yaklaşımdır açıklar. Zebrafish embriyo optik şeffaflık rağmen, sinir hücrelerinin görselleştirme vibratome kullanılarak elde edilen kesitler büyük ölçüde gelişmiş olduğunu bulduk. Bu bölümler konfokal mikroskop kullanarak farklı odak düzlemleri belirli bir hücre, hücresel uzantısı görüntüleme için izin kalın (50 mm). Sonuçlar bu yöntemi kullanarak başka bir yerde 2 yayınlandı, ama bir WT embriyo (Şekil 1) ve N-kaderin (N-cad) nöral tüp hücreleri temsilcisi görüntüleri mutant (Şekil 2) verilmektedir. İkincisi, mutantlar (Şekil 2) orta hatta doğru düzgün bir şekilde yönlendirmek için başarısız N-cad nöral plaka lateral bölgelerde hücrelerin ortaya koymaktadır. Bu mozaik etiketleme aksine, beta-katenin gibi genel bir hücre yüzey işaretleyici ile neuroepithelium immün tek tek hücrelerin morfolojisi (Şekil 3) görüntülenebilmekte izin vermez. Canlı embriyo zaman atlamalı görüntüleme neurulation sırasında yer alan hücresel dinamiklerinin daha iyi anlaşılmasını sağlayan, sabit örneklerinin mozaik etiketleme tamamlanmaktadır. Movie 1 WT hücreleri aktif mediale odaklı membran çıkıntılar uzanan orta hatta doğru göç ettiğini ortaya çıkardı. <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="Şekil 1" /> WT zebrafish embriyo nöral tüp Şekil 1. mGFP ifade. <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="Şekil 2" /> Şekil 2 N-kaderin zebrafish embriyo nöral tüp mGFP ifade. <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="Şekil 3" /> Neuroepithelium Şekil 3-katenin immün boyama.

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Practical approach series. , (2002).</li><li>Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+is+required+for+the+polarized+cell+behaviors+that+drive+neurulation+in+the+zebrafish.">N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.</a> Development. 133, 3895-3905 (2006).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Motility+and+Actin+Cytoskeleton+of+Individual+Neurons+and+Neural+Crest+Cells+in+Zebrafish+Embryos.">Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos.</a> J Vis Exp. , (2010).</li></ol>

Sonuç olarak, etiketleme teknikleri zebrafish embriyo morfogenetik süreçleri tek bir hücre analizi için izin olarak tanımlanmıştır. Bu protokolün temel vurgu nöral tüp kontrolü altındaki her yerde bir organizatörü mGFP kullanarak görüntüleme etiketli hücreleri için yöntemler. Andersen ve arkadaşları tarafından yeni bir kağıt bu geçici ifade test okuyucuların ek uygulamalar için başvurmalıdır. 3.

A correction was made to <a href="http://www.jove.com/index/Details.stp?ID=1976">Labeling and Imaging Cells in the Zebrafish Hindbrain</a>. There was an error in the authors affiliations. The authors have been corrected to:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster1 1Department of Biological Sciences, University of Maryland, Baltimore County 2Center for Neuroscience, Children's National Medical Center
instead of:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster2 1Center for Neuroscience, Children's National Medical Center 2Department of Biological Sciences, University of Maryland Baltimore County

A correction to the author affiliations has been made for the article Labeling and Imaging Cells in the Zebrafish Hindbrain.

Erratum: Labeling and Imaging Cells in the Zebrafish Hindbrain

Çözümler <ul><li> Hank Stock # 1: 8.0 g NaCl, 0,4 g KCl 100 ml dd H 2 O </li><li> Hank Stock # 2: 0,358 g Na 2 HPO 4 Susuz, 100 ml GKD 2 O 0.60 g KH 2 PO 4 </li><li> Hank Stock # 4: 50 ml GKD 2 O 0.72 g CaCl 2 </li><li> Hank Stock # 5: 50 ml dd H 2 O 1.23 g MgSO 4 x7H 2 O </li><li> 1 X Fosfat Tamponu:% 0.8 NaCl,% 0,02 KCl 0,02 M PO4, pH 7,3 </li><li> Engelleme Çözüm:% 2 normal keçi serumu,% 1 sığır serum albumini (BSA),% 1 dimethysulfoxide (DMSO) </li></ul>

labeling and imaging cells in the zebrafish hindbrain

Anahtar erken omurgalı embriyo şekli morfogenetik süreçleri anlamak için yüksek çözünürlükte görüntü hücrelerinin yeteneği. Zebra balığı embriyosu, enjeksiyon, mozaik ifade plazmid DNA sonuçlarının tek hücreler veya küçük hücre kümeleri görünüm için izin<sup&gt; 1</sup&gt;. Biz her yerde bir organizatörü kontrol altında plazmid DNA kodlaması membran hedefli Yeşil Floresan Protein (mGFP) enjeksiyon, neurulation geçiren görüntüleme hücreleri için nasıl kullanılabileceğini açıklar. Bu protokol Orta bölümleri ve aynı zamanda gerçek zamanlı olarak yüksek çözünürlükte görüntüleme etiketli hücreleri için metodoloji. Bu protokol, genç zebrafish embriyolara mGFP DNA enjeksiyon gerektirir. Embriyolar sonra vibratome kesit, antikor konfokal mikroskop ile etiketleme ve görüntüleme için işlenir. Alternatif olarak, mGFP ifade canlı embriyolarının zaman atlamalı konfokal mikroskopi kullanılarak görüntülenebilir. Biz daha önce nöral tüp oluşumu zebrafish embriyoların Beyin bölgedeki hücresel sürücü davranışlarını analiz etmek için bu basit yaklaşım kullanıldı.<sup&gt; 2</sup&gt;. Canlı görüntüleme neurulation sırasında yer alan hücresel dinamiklerinin daha iyi anlaşılmasını sağlayan bu yaklaşım ise sabit hazırlıklar tamamlanır hücre şekil ve nöral tüp organizasyon daha önce benzeri görülmemiş bir görünüm için izin verdi.

Watch this Scientific Journal Video about Labeling and Imaging Cells in the Zebrafish Hindbrain at JoVE.com

Labeling and Imaging Cells in the Zebrafish Hindbrain

Erken embriyo şekli morfogenetik süreçleri anlamanın anahtarı, yüksek çözünürlükte görüntü hücrelerinin yeteneği. Biz burada tüm zebrafish embriyolar membran hedefli Yeşil Floresan Protein hücrelerin tek hücreler veya küçük kümeler etiketleme için kullanılan bir tekniktir açıklar.

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 ...

labeling-and-imaging-cells-in-the-zebrafish-hindbrain

Research

JoVE Journal

Neuroscience

11.2K Görüntüleme.  University of Maryland, Baltimore County. Erken embriyo şekli morfogenetik süreçleri anlamanın anahtarı, yüksek çözünürlükte görüntü hücrelerinin yeteneği. Biz burada tüm zebrafish embriyolar membran hedefli Yeşil Floresan Protein hücrelerin tek hücreler veya küçük kümeler etiketleme için kullanılan bir tekniktir açıklar.

Video: Labeling and Imaging Cells in the Zebrafish Hindbrain

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

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

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

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

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

Nörobilim

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Civciv Embriyo aksonal Yörüngeler ve Synaptic Hedefler Trace için Beyin elektroporasyon

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Merkez Sinir Sistemi Tek Hücreler Etiketleme<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

Embriyonik Neurogenesis çalışmak için bir Model olarak fare arka beyin

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

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

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

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

Larva zebra balığı sonradan içini kaplamak saç hücrelerinin vivo kalsiyum görüntüleme

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

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Primer Nöral Kök ve Progenitor Hücrelerinin Hücre Yüzey Belirteçlerinin Sialoglycan'ın Metabolik Etiketlemesi ile Belirlenmesi

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Doğal Polifenol Curcumin Kullanarak Beyin Dokusunda Amiloid Plakların Etiketlenmesi ve Görüntülenmesi

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Kafatası ve Deri Temizleme ile Uyanık Zebra Balığı Larvaları ve Yavrularında Tam Aktif Beyin Dokusunun In vivo Görüntülemesi

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...