Name: two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation
Uploaded: 2014-05-12T22:00:00
Description: Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

Biz grafik gösterim için James Perna teşekkür ederim. Bu çalışma YZ Ruh Sağlığı Ulusal Enstitüsü hibe tarafından desteklenmiştir

Onay cerrahi ve görüntüleme araştırmanın başlamasından önce eve kurumlardan elde edilmesi gerekmektedir. Bu yazıda anlatılan deneyler University of California, Santa Cruz Kurumsal Hayvan Bakımı ve Kullanımı Komitesi kurallara ve yönetmeliklere uygun olarak yapıldı. 1. Cerrahi <ol><li> Tüm cerrahi aletler otoklav ve iyice ameliyat öncesi% 70 alkol ile çalışma alanını sterilize. </li><li> KX anestetik çözeltisi (200 mg / kg ketamin ve 20 mg / kg ksilazin) intraperi...

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Başarılı bir inceltilmiş kafatası hazırlık elde etmek için, bu protokolde birkaç adım çok önemlidir. 1) kafatası kalınlığı. Kafatası kemiği, iki yüksek yoğunluklu kompakt kemik tabakaları ve düşük yoğunluklu süngersi kemiğin bir orta tabaka ile, bir sandviç yapıya sahiptir. Yüksek hızlı mikro delme kompakt kemik ve süngersi kemiğin dış katmanlarını çıkarılması için uygun olmakla birlikte, mikrocerrahi bıçak kompakt kemik iç tabaka inceltme için idealdir. Gelişim sırasın...

Memeli korteks duyusal algı ve hareket kontrolü soyut bilgi işleme ve biliş, birçok beyin fonksiyonları katılır. Çeşitli kortikal işlevleri nöronların farklı iletişim ve bireysel sinaps bilgi alışverişi oluşur farklı sinirsel devreler, üzerine inşa. Sinapsların yapısı ve işlevi sürekli deneyimleri ve patolojiler yanıt modifiye ediliyor. Olgun Beyinde, sinaptik plastisite oluşumu ve fonksiyonel bir nöral bakımında önemli bir rol oynayan, kuvvet değişiklikler ve sinaps ekleme / kaldırma hem şeklini alır. Dendritik dikenler, memeli beyninde uyarıcı sinapsların çoğunluğunun postsinaptik bileşenleridir. Sürekli ciro ve dikenler morfolojik değişimler sinaptik bağlantıları 1-7 modifikasyon iyi bir göstergesi olarak hizmet inanılmaktadır. Iki-fotonlu lazer tarama mikroskopi kalın, opak hazırlıkları ve sağlam 8 beyin canlı görüntüleme için uygun hale düşük fototoksisite aracılığıyla derin nüfuz sunuyor. Floresan etiketleme ile birlikte, iki-foton görüntüleme yaşayan beyin içine gözetleme ve yüksek uzaysal ve zamansal çözünürlüğe sahip bireysel sinapsların yapısal reorganizasyon takip için güçlü bir araç sağlar. Çeşitli yöntemler, canlı görüntüleme 9-13 için fareler hazırlamak için kullanılmıştır. Burada, biz fare kortekste postsinaptik dendritik dikenler yapısal plastisite araştırmak için in vivo iki foton görüntüleme bir inceltilmiş-kafatası hazırlanmasını açıklar. Bu yaklaşımı kullanarak, bizim yeni çalışmalar, floresanla işaretlenmiş alt-nöronal ve in vivo etiketleme teknikleri hızlı bir gelişme ile transgenik hayvanların artan durumu ile öğrenme, motor beceri tepki olarak dendritik omurga değişikliklerin bir dinamik resim tasvir var, burada açıklanan benzer prosedürler de uygulanabilir araştırılmakte başka hücre tipleri ve kortikal bölgelerinde, diğer manipülasyonlar ile birlikte, hem de 16-23 hastalık modellerinde kullanılmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="1253">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

Memeli kortekste, nöronlar son derece karmaşık ağları ve sinaps bilgi alışverişi oluşturur. Sinaptik gücü, hem de sinaps ekleme / kaldırma değişiklikleri, nöronal plastisite yapısal temel sağlayan, bir deneyim bağımlı bir şekilde meydana gelir. Kortekste en uyarıcı sinapsların postsinaptik bileşenleri olarak, dendritik dikenler sinaps iyi bir vekil olarak kabul edilir. Fare genetik ve floresan etiketleme teknikleri, bireysel nöronlar ve bunların sinaptik yapılarının alarak avantajları sağlam beyinde etiketli olabilir. Burada in vivo zamanla floresan etiketli postsinaptik dendritik dikenler takip için iki-foton lazer tarama mikroskobu kullanarak bir Transkranial görüntüleme protokolü tanıtmak. Bu protokol sağlam kafatası tutar ve menenjlerin maruz kalma ve korteks tarafından kaynaklanan inflamatuar etkileri ortadan kaldıran inceltilmiş-kafatası hazırlık, kullanır. Bu nedenle, görsel su sonra hemen elde edilebilirrgery gerçekleştirilir. Deneysel prosedür saat yıla kadar değişen çeşitli zaman aralıklarında tekrar tekrar yapılabilir. Bu preparatın Bu uygulama aynı zamanda, fizyolojik ve patolojik koşullar altında, değişik bölge ve kortikal katmanları, hem de diğer hücre türlerini araştırmak için genişletilebilir.

YFP-H hattı farelerde 25&#39;de, sarı floresan protein kortekstekinden yüzeysel tabakalarına apikal dendrit çıkıntı tabaka V piramidal nöronlar, bir alt grubunda ifade eder. Inceltilmiş-kafatası hazırlık, floresan etiketli dendritik kesimleri hkr saatten ay arasında değişen çeşitli görüntüleme aralıklarında iki foton mikroskop altında görüntülenebilir. Burada bireysel dikenleri yanı sıra filopodia açıkça dendrit boyunca görselleştirilebilen bir 1 aylık fare, motor kortekste ...

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

İki-Foton<em&gt; In vivo</em&gt; Bir inceltilmiş-kafatası Hazırlık kullanarak Fare Cortex dendritik dikenler Görüntüleme

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Research

JoVE Journal

Neuroscience

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

Fare Visual Cortex İnceltilen kafatası Hazırlık Kronik Görüntüleme

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Nörobilim

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

Fare Omurilik İki foton Mikroskop kullanarak in vivo Görüntüleme

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Endojen NADH Floresans iki-foton Imaging tarafından Fare Serebral Korteks olarak Microregional Hipoksi Algılama

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

Fare Beyin Uzun vadeli Görüntüleme için Parlak ve Takviyeli inceltilmiş-kafatası Pencere

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Için inceltilmiş-kafatası Kortikal Pencere Tekniği<em&gt; In Vivo</em&gt; Optik Koherens Tomografi Görüntüleme

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Kortikal Nöronlar In Vivo İki foton Of Görüntüleme Deneyimi bağımlı Moleküler Değişiklikler

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Fare Omurilik Hücresel Dynamics İki foton Görüntüleme

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Eşzamanlı İki foton<em&gt; İn Vivo</em&gt; Fare Retrosplenial kortekste sinaptik Girişlerin Görüntüleme ve postsinaptik Hedefler

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Vivo Yenidoğan farelerde kortikal nöronların iki fotonlu görüntüleme

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...