Name: time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation
Uploaded: 2017-07-25T12:30:00
Description: Watch this Scientific Journal Video about Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation at JoVE.com

Yararlı tartışmalar için Jacqueline Andratschke, Elena Werle, Sachi Takenaka ve Matthias Toberer&#39;a mükemmel teknik yardım ve Victor Tarabykin&#39;e teşekkür ediyoruz. Bu çalışma Deutsche Forschungsgamesinschaft&#39;ın SB&#39;ye (BR-2215) verilmesi ile desteklendi.

Tüm deneysel prosedürler Hayvan Refahı Komitesi (Regierungspräsidium Tübingen) tarafından onaylanmış ve Alman Hayvan Refahı Yasası ve 2010/63 / AB AB Direktifi uyarınca gerçekleştirilmiştir. 1. Utero Elektroporasyonunda <ol><li> Mikroenjeksiyon iğneleri <ol><li> Kutu filamentli (2.5 mm x 2.5 mm) bir mikropipet çekici ve aşağıdaki program ile borosilikat cam kapillerleri (dış çap: 1.0 mm, iç çap: 0.58 mm, uzunluk: 100 mm) çekerek mikroenjek...

<ol>
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Radyal göç, neokorteks gelişiminde önemli bir süreçtir. Bu sürecin farklı adımlarını etkileyen genlerdeki mutasyonlar, lissensefali ve beyaz cevher heterotopisi gibi ağır kortikal malformasyonlara neden olabilir 1 , 2 . Geçtiğimiz günlerde genç göç eden kortikal projeksiyon nöronlarında ifade edilen Bcl11a&#39;nın radyal göçte rol oynadığını gösterdik. Aktif nöronlarda Bcl11a&#39;nın genetik olarak silinmesinin ku...

Neokorteks bilişsel, duygusal ve sensorimotor işlevlerin başlıca sitesidir. Beynin yüzeyine paralel altı yatay tabakadan oluşur. Gelişme sırasında, dorsal telencefalonun yan duvarındaki progenitör hücreler, radyal olarak pial yüzeyine doğru göç eden ve katmana spesifik bir nöronal kimlik edinen projeksiyon nöronlarına neden olur. Ventriküler / subventriküler bölgelerde (VZ / SVZ) üretildikten sonra, bu nöronlar geçici olarak çok kutuplu olurlar ve göçlerini yavaşlatırlar. Orta bölgede (IZ) kısa bir süre kaldıktan sonra, iki kutuplu morfolojiye geçerler, radyal glial iskele üzerine bağlanırlar ve radyal olarak göç ederek korteks plakasına (CP) geçerler. Nihai hedef yansıtmalarına ulaştıklarında nöronlar radyal glial süreçlerden ayrılır ve katmana özgü kimlik kazanır. Nöronal migrasyonun farklı adımlarını etkileyen genlerdeki mutasyonlar, lissen gibi şiddetli kortikal malformasyona neden olabilirEfali veya beyaz cevher heterotopisi 1 , 2 . Enerjide elektroporasyon, kemirgen embriyolarının 3 , 4 gelişmekte olan beyinde sinir öncü hücreleri transfekte etmek için hızlı ve güçlü bir tekniktir. Bu teknikle, nöronların geliştirilmesindeki işlevlerini incelemek için ilgilenilen genleri knockdown ve / veya aşırı ifade etmek mümkündür. Bu yöntem özellikle morfolojik ayrıntıların tanımlanmasına ve radyal göç sürecinin moleküler mekanizmalarının karakterize edilmesine yardımcı olmuştur ( 5 , 6 , 7 , 8 , 9) . Radyal olarak göç eden nöronlar, hücre şekli, göç hızı ve göç yönü gibi zaman içerisinde doğrudan ve sürekli gözlem gerektiren dinamik değişiklikler yaparlar. Organotipik dilim kültürüElektroporasyona uğramış beyinlerin tekrar ve zaman atlamalı konfokal görüntülemesi zaman içinde göç eden nöronları doğrudan gözlemlemesine izin verir. Bu kombine yaklaşımı kullanarak, elektroporasyona uğramış beyinlerin sabit doku kesitlerinde araştırılamayan göç eden nöronların farklı özelliklerini analiz etmek mümkündür. Geçenlerde kortikal gelişim 10 sırasında transkripsiyon faktörü B hücreli KLL / lenfoma 11a (Bcl11a) rolünü incelemek için Electroporated beyinlerinin dilim kültürlerde nöronları göç zaman atlamalı konfokal görüntüleme uyguladı. Bcl11a, genç göç eden kortikal nöronlarda eksprese edilir ve fonksiyonlarını incelemek için bir koşullu mutant Bcl11a aleli ( Bcl11a flox ) 11 kullanılır . Bcl11a flox / flox beyinlerinin kortikal progenitörlerine Cre optik proteini (GFP) ile birlikte Cre rekombinazın elektroporasyonu, sadece birkaç hücrenin bir mutasyona uğramış olduğu bir mozaik mutant durum yaratmamızı sağladı.Aksi takdirde vahşi tipli arka plan. Bcl11a&#39;nın hücre özerk işlevlerini tek hücre düzeyinde incelemek bu şekilde mümkündü. Bcl11a mutant nöronlarının, hız profillerinde düşük hız, kaymalar ve ayrıca göç sırasında rasgele benzeri oryantasyon değişiklikleri sergilediğini bulduk 10 . Anlatılan protokolde, fare beyinlerinin başarılı elektroporasyon ve dilim kültür hazırlığı 12 için bir iş akışı ve kortikal dilim kültürlerinin zaman kazanım konfokal görüntülemesi açıklanmaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="771">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">isoflurane</td>
			<td style="width:171px;">Abbott Laboratories&#160;</td>
			<td style="width:111px;">506949</td>
			<td style="width:209px;">Forene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">6-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:111px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">12-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:111px;">351143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">non-absorbable surgical suture</td>
			<td style="width:171px;">Ethicon</td>
			<td style="width:111px;">K890H</td>
			<td>3/8 circle, 13 mm, taper point</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Micro Adson Forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11018-12</td>
			<td>serrated, length: 12 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fine scissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">14063-09</td>
			<td>angled to side, length: 9 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Mathieu Needle Holder</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">12510-14</td>
			<td>tungsten carbide, length: 14 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fine tipped forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11370-40</td>
			<td>straight, 11 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Vannas T&#252;bingen Spring Scissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">15005-08</td>
			<td>angled up, 9.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">ring forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11103-09</td>
			<td>OD: 3mm, ID, 2.2 mm, length: 9 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">HBSS (10X)</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">14180046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">L-Glutamine</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">horse serum</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">26050088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BME</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">41010026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">borosilicate glass capillaries</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:111px;">30-0016</td>
			<td>1.0 OD x 0.58 ID x 100 L mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">anesthsesia system</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">72-6471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">anesthetizing chamber</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">34-0460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fluosorber filter canister</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">34-0415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">low melting point agarose</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:111px;">16520100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">vibrating blade microtome</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">VT1200 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fluorescence stereo microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">M205 FA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">stereo microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">M125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">inverted fluorescence tissue culture microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">DM IL LED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">confocal laser scanning microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">TCS SP5II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">hybrid detector</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">HyD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">objective, 40x/0.60 NA</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">11506201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">microscope temperature control system</td>
			<td style="width:171px;">Life Imaging Services</td>
			<td style="width:111px;">Cube, Brick &#38; Box</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">cell culture insert</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:111px;">PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">microgrinder</td>
			<td style="width:171px;">Narishige</td>
			<td style="width:111px;">EG-45</td>
			<td>use 38&#176; angle for beveling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">microinjector</td>
			<td style="width:171px;">Parker Hannifin&#160;</td>
			<td>052-0500-900</td>
			<td style="width:209px;">Picospritzer III</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">carprofen</td>
			<td style="width:171px;">Pfizer Animal Health</td>
			<td style="width:111px;">NDC 61106-8507</td>
			<td>Rimadyl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">emdedding mold</td>
			<td style="width:171px;">Polysciences</td>
			<td style="width:111px;">18986-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">endotoxin-free plasmid maxi kit</td>
			<td style="width:171px;">Qiagen</td>
			<td style="width:111px;">12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fast green</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">F7252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">laminin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">poly-L-lysine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">P5899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">HEPES</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">D-glucose</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">C7902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">magensium sulfate</td>
			<td>Sigma</td>
			<td style="width:111px;">M2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">sodium bicarbonate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">S6297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">square wave electroporator</td>
			<td style="width:171px;">Sonidel</td>
			<td style="width:111px;">CUY21EDIT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">tweezers with 5 mm platinum disk electrodes</td>
			<td style="width:171px;">Sonidel</td>
			<td style="width:111px;">CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">micropipette puller</td>
			<td style="width:171px;">Sutter Instrument</td>
			<td style="width:111px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">box filament</td>
			<td style="width:171px;">Sutter Instrument</td>
			<td style="width:111px;">FB255B</td>
			<td>2.5 mm x 2.5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">micro-spoon spatula</td>
			<td style="width:171px;">VWR</td>
			<td style="width:111px;">231-0191</td>
			<td>185 mm x 5 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">glass bottom dish, 50 mm</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:111px;">FD5040-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation

Uterus elektroporasyonu, gelişmekte olan fare embriyolarının serebral korteksindeki radyal göç sürecini incelemek için hızlı ve güçlü bir yaklaşımdır. Radyal göçün farklı adımlarını tarif etmede ve bu işlemi kontrol eden moleküler mekanizmaları karakterize etmede yardımcı oldu. Göç eden nöronları doğrudan ve dinamik olarak analiz etmek için zamanla izlenmesi gerekiyor. Bu protokol , utero elektroporasyon ile organotipik dilim kültürü ve radikal göç eden kortikal nöronların doğrudan muayene ve dinamik analizine imkan veren zaman atlamalı konfokal görüntüleme yöntemlerini bir araya getiren bir iş akışı tanımlamaktadır. Ayrıca, göç hızı, hız profili ve radyal yönlendirme değişiklikleri gibi göç eden nöronların ayrıntılı karakterizasyonu da mümkündür. Bu yöntem, kurtarma deneylerinin yanı sıra fonksiyon kaybı ve kazancı ile radyal olarak göç eden kortikal nöronlarda ilgi çekici genlerin işlevsel analizlerini gerçekleştirmek için kolayca adapte edilebilir. HızlandırılmışGöç eden nöronların görüntülenmesi, ortaya çıktıktan sonra, nöronal migrasyon bozukluklarının fare modellerinde serebral korteks gelişimini incelemek için etkili bir araç olan en gelişmiş tekniktir.

Daha önce, utero elektroporasyon ile Bcl11a&#39;nın genetik silinmesinin, geç doğan üst katman projeksiyon nöronlarının radyal göçünü 10 engellediğini göstermiştik. Kre-IRES-GFP ihtiva eden bir DNA plazmid vektörü Elektroporasyon verimli flox / flox beyinleri 11 koşullu Bcl11a içinde Bcl11a silindi. Elektroporasyondan üç gün sonra E14.5 elektropo...

Watch this Scientific Journal Video about Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation at JoVE.com

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Bu protokol, radyal olarak göç eden kortikal nöronların doğrudan gözlenmesi için talimatlar sağlar. N utero elektroporasyonda, organotipik dilim kültürü ve zaman atlamalı konfokal görüntüleme, göç eden nöronlardaki ilgi genlerinin aşırı ekspresyonunun veya downregülasyonunun etkilerini doğrudan ve dinamik olarak incelemek ve gelişme sırasındaki farklılaşmalarını analiz etmek için birleştirilir.

Embriyonik Fare Beyni Organotipik Dilim Kültüründe Nöronların Geçiş Sürekli Konfokal Görüntüleme Kullanımı<em&gt; Utero&#39;da</em&gt; Elektroporasyon

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

The overall goal of this procedure is to directly observe radially migrating neurons in organotypic slice culture prepared from electroporated embryonic brain by time-lapse confocal microscopy. This method can help study key aspects of neocortex development. It allows investigation of the molecular mechanisms involved in neuron polarization and the radial migration of neurons to their final position within the brain.The main advantage of this technique is the dynamic properties of migrating neurons can be analyzed including speed profiles, average migration speed as well changes of migratory direction. Begin by placing a properly anesthetized pregnant mouse in the supine position on a warming plate. Check for the absence of the pedal reflex.And then, carefully cover the eyes with petroleum jelly to prevent them from drying. Next, gently spread out the limbs and fix them to the warming plate with surgical tape. Sterilize the abdomen by swabbing with 70%ethanol followed by iodine solution, then place sterile gauze, in which a cut has been made for the abdominal incision, over the abdomen.Moisten the gauze with bacteriostatic sodium chloride solution. After reassessing the development of surgical anesthesia, by loss of the pedal reflex, use serrated micro Adson forceps and angled fine scissors to cut the skin approximately 1.5 centimeters along the midline of the abdomen. Then, cut the underlying muscle along the linea alba.Grasp the uterine horn between embryos with ring forceps and gently place it onto the moistened gauze without perturbing the placentas or supply vessels. Then, carefully position an embryo to give a clear view of the lateral ventricle which presents as a crescent-shaped shadow parallel to the sagittal sinus. The injection site lies in the middle of a line between the pigmented eye and the confluence of sinuses where the sagittal sinus branches to two transfer sinuses.Once identified, push the microinjection needle through the uterine wall and into the lateral ventricle. Next, use a microinjector operated with a foot pedal to inject one to two microliters of DNA solution with five to 10 pulses. Successful injection can be monitored by the colored DNA solution which should fill the ventricular system.Then, place tweezers-type electrodes so that the positive terminal is on the same side as the injected ventricle and the negative terminal is on the opposite side of the injected ventricle below the ear of the embryo's head. Moisten the electroporation site with a few drops of bacteriostatic sodium chloride solution and apply five electrical current pulses of 50-millisecond duration with 950-millisecond intervals. Bubbles at the negative electrode indicate electrical current.60 to 90 milliamperes are usually sufficient for successful electroporation. Lower currents will not efficiently transfect neurons whereas higher currents may lead to embryonic death. After repeating the procedure for every embryo of the first uterine horn, gently place it back into the abdominal cavity.After suturing the muscle layer and skin, carefully disinfect with iodine solution and gently remove the petroleum jelly from the eyes using cellulose swabs. Place the mouse under an infrared lamp until it wakes up. After euthanizing the pregnant female using an approved method, remove the uterus containing the embryos and place it into a 10-centimeter Petri dish with ice-cold complete HBSS.From this point onward, keep all solutions, embryos and brains on ice. While working under a stereomicroscope, use fine-tipped forceps and a pair of Vannas Tubingen spring scissors to separate each embryo from the uterus. Transfer the embryos to another Petri dish containing ice-cold complete HBSS.Make an incision at the level of the brainstem and cut along the sagittal midline. Peel away the skin and cartilage covering the brain. Then, cut off the brainstem right behind the hemispheres and remove the brain from the skull.Transfer the brain with a micro spoon spatula to a 12-well plate containing ice-cold complete HBSS. Collect all brains from the litter in the 12-well plate. Then, use a fluorescent stereomicroscope to screen the brains in the 12-well plate for brightness of florescence as well as the size of the electroporated region.Select two to four brains with bright fluorescent signals and the presumptive somatosensory cortex for further processing. Then, pour molten 3%low-melting-point agarose solution into a peel-away disposable embedding mold. Use a micro spoon spatula to remove the brain from the 12-well plate and carefully drain excess complete HBSS from around the brain using fine tissue paper.Place the brain gently into the agarose solution. Then, use a 20-gauge needle to push it to the bottom of the mold and carefully adjust its position. For coronal sections, orient the brain with the olfactory bulbs pointing upwards.Keep the mold on ice until the agarose solution is solidified then proceed to sectioning the brain. Use a clean razor blade to trim away excess agarose around the brain leaving approximately one millimeter of agarose on all sides except for the olfactory bulbs where two to three millimeters of agarose should be left. After applying a small amount of cyanoacrylate glue to a specimen, fix the trimmed agarose block with the olfactory bulbs pointing downwards.Transfer the specimen stage to the slicing chamber of a vibrating blade microtome containing ice-cold complete HBSS and position the brain with its dorsal side towards the blade. Cut 250 micron-thick brain slices containing the electroporated region with a low to moderate amplitude and a very slow sectioning speed. Usually four to six slices with bright fluorescent signal are obtained from each successfully electroporated brain.Grasp the agarose rim of one section with fine-tipped forceps and pull the slice onto a bent micro spoon spatula. In this manner, carefully transfer the brain slices to a six-well plate containing ice-cold complete HBSS. Moisten laminin-coated membrane inserts with 100 microliters of complete HBSS.Then, carefully transfer the slices onto the membranes using the bent micro spoon spatula and forceps. Gently push the slice from the spatula onto the membrane, then position using forceps. Continue to transfer the remaining slices.Up to five slices can be placed on one membrane insert. Remove excess complete HBSS with a pipette. Then, with the help of forceps, carefully place the membrane inserts with the slices into a six-well plate containing 1.8 milliliters of sliced culture medium.Select a brain slice for imaging by viewing the slices through an inverted microscope. Choose a slice with bright single neurons in the upper SVZ migrating radially towards the pile surface of the slice. The presence of fine fluorescent processes of radial glial cells that span the entire conjugate plate indicates an intact radial glial scaffold which is used by migrating conjugate neurons.Transfer the membrane insert with a selected slice into a 50-millimeter-diameter glass-bottomed dish containing two milliliters of slice culture medium. Place the dish into the climate chamber of the confocal microscope. Set the resolution to 512 by 512 pixels.Increase the scan speed from 400 hertz to 700 hertz to increase the frame rate from about 1.4 to about 2.5 frames per second. Use no more than two times averaging. Define a Z-stack through the electroporated region with the step size of 1.5 microns.Start the time-lapse series by taking a Z-stack every 30 minutes. The settings described allow for sufficient resolution and brightness of the image while keeping photo damage low during acquisition. This movie shows E16.5 cortical neurons migrating from the ventricular subventrical zones to the cortical plate.In the Bcl11a floxed conditional transgenic after electroporation of the IRES-GFP control plasmid on embryonic day 14.5. The movie is comprised of 108 frames at the rate of five frames per second. The scale bar represents 100 microns.Electroporation of a DNA plasmid vector containing Cre-IRES-GFP affected cortical neuron migration in conditional Bcl11a floxed brains. As seen here, very few neurons move past the intermediate zone to reach the cortical plate. This movie shows the polarization of cortical neurons from a GFP-labeled control on the left compared to neurons from a Bcl11a mutant on the right.These animated traces of representative control in Bcl11a mutant neurons were obtained from time-lapse series of E16.5 slice cultures with one-hour-interval resolution. Again, data from the control brain is shown on the left and data from the Cre-IRES-GFP electroporated brain is shown on the right. As seen from this collection of representative traces from control and Bcl11a mutant slice cultures, the Bcl11a mutant neurons frequently undergo repetitive phases of reduced migration speed and randomly changed orientation as marked by the red arrowheads.Speed profiles can be calculated from traces of migrating neurons. As seen here, a greater proportion of Bcl11a mutant neurons have slower migration speeds compared to controls. The deviation angle may be calculated from the trace data.As seen from this histogram, the Bcl11a mutant neurons represented by the black bars exhibit greater angles of deviation compared to controls. Once mastered, this technique can be done in less than two hours each for the in utero electroporation and the preparation of organotypic brain slice culture. This procedure can easily be adapted to perform fractional analysis of genes of interest in radially migrating cortical neurons by gain and loss of function as well as vascular experiments.After watching this video, you should have a good understanding of how to directly observe radially migrating neurons in organotypic slice culture prepared from electroporated embryonic brains by time-lapse confocal microscopy.

Research

JoVE Journal

Neuroscience

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

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

Nörobilim

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Doğum Sonrası Beyin Nöronal Göç Yüksek Çözünürlüklü Zaman Aşımı Görüntüleme için bir Organotipik Dilim Testi

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Verimli kullanarak birden çok CNS Topraklar&#39;da Gen Teslimat In Utero Elektroporasyon

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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 ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Fare Akut Beyin Dilimleri Neuroblast Göç vivo Doğum Sonrası Elektroporasyonla ve Time-lapse Görüntüleme

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Eski Utero Elektroporasyon ve embriyonik fare beyni canlı görüntüleme, GABAergic Interneurons geçiş için Organotypic dilim kültürleri

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...