Name: organotypic slice cultures to study oligodendrocyte dynamics and myelination
Uploaded: 2014-08-25T15:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

NOT: Tüm hayvan prosedürleri yönergeleri izleyerek ve Connecticut Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. NOT: YFP muhabiri 20 (JAX # 006148) çizgiler sırasıyla kullanıldı: NG2cre 18 bünye (JAX # 008533) ve NG2creER 16 uyarılabilir bu deneylerde (JAX # 008538) için transgenik fareler 19 (JAX # 003920) veya gtRosa26 / EG Z&#39;ye geçti Görüntü NG2 hücreleri ve soylarına. Görüntü olgun oligodendrositlere, PLPDsRed t...

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Merkezi sinir sisteminde etkin Myelinasyon nöronal iletişim ve aksonal hayatta 22 için gereklidir. 25 - En beyin bölgelerinde 16,23 içinde yerleşik nüfusu korurken NG2 hücreleri sürekli yetişkinlik içine Myelinating oligodendrositleri oluşturmak. Bu hücrelerin farklılaşmasını düzenleyen bazı genetik ve moleküler mekanizmalar tarif edilmiştir ama çok keşfedilmeyi beklemektedir. Organotipik dilim kültürleri nedeniyle anatomik bölge, hücre dışı ortama kolay man...

4 - merkezi sinir sistemi Organoytpic dilim kültürlerinin semiintact sistem 1 &#39;de nöron ve glial hücre biyolojisi çalışmak için çok yararlı olduğu kanıtlanmıştır. 9 - Bu kültürler, kabul ve bu hücre dışı ortamın bir manipülasyon ve tekrarlamalı uzun-süreli canlı hücre görüntüleme için kolay erişim ve elektrofizyolojik kayıtları 5, birincil hücre kültürleri ayrışmış birçok yararlarını korumak için nispeten basittir. Buna ek olarak, dilim kültürler, 3 boyutlu doku hücre mimarisini, bölgesel sinir bağlantısına sahip, ve en önemli hücre tiplerinin sistemde bulunmaktadır. Bu özellikler hücresel ve çevresel etkileşimleri ile tek hücre davranış ve fizyolojisini araştırmak için bu Kültürlerin benzersiz ve kullanışlı bir sistem yapmak. NG2 hücrelerinin çoğalmasını ve m oluşturmaya devam memelilerin merkezi sinir sistemindeki glial hücreler bir nüfusembriyonik ve postnatal gelişim 10 sırasında oligodendrositleri yelinating. Bunlar yaygın olarak ayrışmış primer hücre kültürlerinde incelenmiştir ve floresan proteinleri NG2, hücre spesifik ifadesi ile transgenik fare hatları son gelişmeler, akut dilim preparasyonlarında in vivo kaderi haritalama ve elektrofizyolojik kayıtları kolaylaştırmıştır. Hatta bu çalışmalar ile, küçük NG2 hücre çoğalması ve olgodendrosit farklılaşmasının zamansal dinamikleri hakkında bilinmektedir. Ayrışık hücre kültürü geniş farmakolojik ve genetik manipülasyon göreli tesis için kullanılır, ancak, bunun hücresel mikro-bağlamını sağlamak tercih edilir, özellikle, beynin farklı bölgelerinde bu hücrelerin fonksiyonel farklılıklar sorgulamak için uygun değildir. Dilim kültürleri, hücre farmakolojik manipülasyonlar mükellef ve olgodendrosit myelinleşmeyi 11,12 araştırmak için kullanılır olmuştur basit bir alternatif sağlamak(LPC) lisolesitin veya antikor farmakolojik tedavinin 15 üzeri demiyelinizasyonu 13,14 ve remiyelinizasyon indüklenmesini sonra ular yanıt. Bir yöntem araştırmak ve canlı görüntüleme ve sabit doku (veya Postfiksasyon) ön beyin ve beyincik hem de alınan organotipik dilim kültürlerde NG2 hücre çoğalması ve farklılaşması olgodendrosit analizini tarif gerçekleştirmek için. Bu bölünme sonrası 16 tek NG2 hücrelerinin hücre kaderine incelemek için ve büyüme faktörü ile uyarılan NG2 hücre çoğalması 17 bölge ve yaşa bağlı farka için kullanılabilir güçlü bir yöntemdir. Bu, nispeten basit teknik ayrıca doğuştan ve / veya çevresel, bu glial hücre fizyolojisi ve nöronal aktivite ya da mielin hasarına cevabı düzenleyen mekanizmaların hücre araştırılması yaygın erişilebilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Invitrogen</td>
			<td>14175</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td>25mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>1mg/L</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>0.4mM</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Hyclone</td>
			<td>SH30074.03</td>
			<td>25%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>124mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>3.004mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>26mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>2.0mM</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>0.075mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</td>
		</tr>
		<tr>
			<td>4-hydroxy tamoxifen (4OHT)</td>
			<td>Sigma-Aldrich</td>
			<td>H7904</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EM Sciences</td>
			<td>19200</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L-6027</td>
			<td>0.1M</td>
		</tr>
		<tr>
			<td>Sodium Metaperiodate</td>
			<td>Sigma-Aldrich</td>
			<td>S-1878</td>
			<td>0.01M</td>
		</tr>
		<tr>
			<td>Rabbit anti NG2 antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>AB5320</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mouse anti CC1 antibody</td>
			<td>Calbiochem (Millipore)</td>
			<td>OP80</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Mouse anti Ki67 antibody</td>
			<td>Vision Biosystems</td>
			<td>NCL-Ki67-MM1</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Mouse anti NeuN antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Species specific secondary antibodies</td>
			<td>Jackson Immunoresearch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td></td>
			<td></td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture membrane inserts 0.45&#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filters</td>
			<td>Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2 gas mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile six well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting (hippocampal) or weighing spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manual or automatic tissue chopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm and 60mm sterile Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microcope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2 ifade eden hücreler (polydendrocytes oligodendrosit öncü hücreleri), merkezi sinir sisteminde, dördüncü bir glial hücre içerisinde yer alırlar. Embriyonik ve postnatal gelişim sırasında aktif çoğalırlar ve Myelinating oligodendrositleri oluşturmak. Bu hücreler sık ​​primer ayrışmış kültürler, nöron cocultures okudu ve sabit dokuda edilmiştir. Kullanımı uygun yeni transgenik fare soyları kültür sistemleri ön beyin ve beyincik hem gri ve beyaz madde bölgelerde oligodendrosit soy hücrelerinin çoğalması ve farklılaşması araştırmak için kullanılabilir dilim. Dilim kültürleri erken doğum sonrası farelerde hazırlanan ve 1 aya kadar kültür içinde tutulur. Bu dilimler hücresel davranış ve etkileşimleri araştırmak için kültür dönemi boyunca birden fazla kez görüntülenmiş olabilir. Bu yöntem NG2 hücre bölünmesi görselleştirme ve ayrıntılı analizini sağlarken olgodendrosit farklılaşma giden adımlar bölgede-bağlıdır veriyorent NG2 hücre ve olgodendrosit fonksiyonel heterojenite. Bu ise, in vivo olarak bulunan andıran bir hücresel ortamda zaman bu hücreleri etkileyen iç ve dış sinyalleri araştırmak için kullanılan bir güçlü bir tekniktir.

Örnek veri örnekleri aşağıda verilmektedir P8 NG2cre bir ön beyin ve beyincik hem de dilim kültürleri kullanılarak elde edilmiştir ki: zeg, NG2creER: YFP ve transgenik fare hatları PLPDsRed. NG2 hücreleri ön beyin ve beyincik dilimleri (Şekil 1B-D, video 1) ve bu hücrelerin fenotip gün boyunca görüntülü olabilir tespit edilerek ve NG2 ve CC1 antikorları (Şekil 1E) ile immünboyama edilebilir. Canlı görüntülemeye ek olarak, hücre çoğalması aynı zamanda Ki67...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotipik Dilim Kültürler Oligodendrosit Dinamikler ve miyelinasyonun incelemek için

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Nöronal Sakatlık incelenmesi için Organotipik Hipokampal Dilim Kültür Modeli

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Nörobilim

Organotypic Hippocampal Slice Cultures

Organotipik Hipokampal Dilim Kültürler

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotipik Serebellar Kültürler: Apoptotik Zorluklar ve Tayin

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Neokorteks bir Organotipik Dilim Hazırlık Tüm Hücre Kaydı

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Organotipik Kültürleri nöronal Çığ Çok elektrot Dizi Kayıtlar

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Post-natal murin Dokular Co-kültürler Myelinating zenginleştirilmiş oligodendrosit Kültürleri ve oligodendrosit / Nöron türetilmesi

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Embriyonik ventral orta beyin Organotipik Dilim Kültürler: dopaminerjik Nöronal Gelişim Eğitim Bir Sistem In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Organotipik Dilim Kültürleri Purkinje Hücre Dendritik Morfoloji Analizi

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Içinde Neurovascular Yaptırma Analizi Entorhino-hipokampal Organotipik Dilim Kültürler

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...