We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1.. Aksonal Etiketleme <ol><li> Tırmanma lifler yüksek molekül ağırlıklı dekstranlar veya floresan protein 26-29 sentezlenmesini uyaran plasmid / virüslere karşı konjuge organik boyalar ya da enjekte edilmesi ile etiketlenebilir. Bu protokolde, organik boya Dekstran Alexa Fluor 488 tırmanma elyaflar ve etiket (Şekil 1), serebellar korteks Onları görselleştirmek için alt oliva enjekte edilir. Burada açıklanan tüm prosedürler İtalyan Sağlık Bakanlığı tarafından onaylanmıştır. </li><li> Bir mikropipet çektirmesi çekerek cam kapiller tüp hazırlayın. Dış çapı yaklaşık 30 um olan kadar makasla cam kılcal tüpün ucu kesin. </li><li> Dekstran-bağlı boya 1-2 ul kılcal yerleştirin. </li><li> Başlamadan önce, bir otoklav içinde, tüm cerrahi aletler sterilize edin. İşlem sırasında, bir cam boncuk sterilizatör kullanılarak kontaminasyon riskini azaltır. </li><li> Fareyi (9-12 anestezisiketamin (90 mg / kg) ve ksilazin (9 mg / kg) intraperitonal enjeksiyonu ile hafta). Hayvan tam kuyruk ve / veya ayak tutam kullanılarak sedasyon emin olun. Anestezi altında iken kuruluğunu önlemek için gözleri veteriner merhem kullanın. </li><li> Bir jilet veya tüy dökücü krem ​​ile ya boyun üzerinde tüyleri tıraş. Etanol% 70 ile dönüşümlü betadin, iki kez cerrahi yerin silinmesi. </li><li> Stereotaksik tutucuya fare yerleştirin. Bir ısıtma pedi ile hayvan sıcak tutun. </li><li> Baş sıkıca tutulur şekilde hafifçe yanındaki kulaklara kemik üzerinde earbars itin. Gövde ile 140 ° açı oluşturacak şekilde baş aşağı çevirin. Istenen yönlendirme yavaşça stereotaktik tutucuya burun tutucu çubuğun yüksekliğini değiştirerek elde edilebilir. </li><li> Kulak arasında ciltte lidokain bazı damla uygulayın. Oksiputa altındaki cilt üzerinde yaklaşık 1 cm&#39;lik dikey bir kesi gerçekleştirmek için forseps veya bir tıraş bıçağı kullanma. </li><li> Yavaşça künt cımbız ile deri altı doku ve kasları ayrıDiseksiyon mikroskobu altında. Yatay kas demeti aşağı doğru itin ve foramen magnum üzerinde dura ortaya koymak için iki dikey kaslar fasiküller ayrı tutun. </li><li> Çok dikkatli kullanılması, beyin sapı ortaya çıkarmak için bir şırınga iğnesi ya da çok keskin forseps ile dura teşrih. Dura kesilmiş edildiğinde beyin-omurilik sıvısı küçük bir miktar dökülebilir. </li><li> Stereotaktik mikromanipülatör, dikeyden kapiler 45 ° &#39;lik tutucuyu döndürün. Serebellar korteks kuyruk kenarı ve ilk servikal omur arasındaki orta noktada, orta hatta kılcal yerleştirin. Yüzeyden 2 mm derinlikte pipet yerleştirin. </li><li> 10-15 dakika boyunca bir boya 0.5-1.5 ul hacmi sunun. Bir mikroenjeksiyon dağıtma sistemi, kılcal içindeki sıvının kontrollü basınç dağıtımını sağlar: (Pulse basıncı = 20 psi; darbe süresi = 3-4 msn, darbe doğum = 12 Hz arasında frekansı). </li><li> Daha sonra, 15 dakika boyunca yerinde kılcal bırak slowly (2-3 dk) çıkarın. İncelikli kasları yeniden ayarlamak ve üzeri cilt sütür. De işlem boyunca bazı tuzlu su uygulayarak sulu kasları tutun. </li><li> Su kaybını önlemek için, deri altına 0.5 ml% 0.9 NaCl enjekte edilir. Anesteziden tam iyileşme kadar ısıtılmış bir kafeste hayvan tutun. </li><li> Deri altına ameliyattan sonra 2-3 gün süreyle günde Carprofen (5 mg / kg) uygulanması. </li></ol> Serebellar Korteks 2. Optik Pencere Not: boya (bkz. Şekil 1) CFS terminallere serebellar korteks için alt olivary çekirdekten yukarıya 2 hafta içinde taşınmaktadır. Seyrek etiketli CFS aşağıdaki gibi gerçekleştirilebilir sürekli kafatası pencerenin altında görüntülenmiştir olabilir: <ol><li> Tüm cerrahi aletler sterilize. </li><li> Yukarıda tarif edildiği gibi fare anestezisi. Hayvan kontrol etmek için ayak tutam kullanmak tam sedasyon. </li><li> Sk silin, baş yukarıda saçlarını tıraş% 70 alkol ve betadin alternatif bira ile ve göz merhemi uygulanır. Bir stereotaksik çerçeveye fare yerleştirin ve sıkıca kafa tutmak için kulak çubukları yerleştirin. </li><li> Deri altına, kraniyal pencere yerinde beyin ve olası iltihaplanma şişme önlemek deksametason (0.2 mg / kg) ve Carprofen (5 mg / kg) uygulanması. </li><li> Kulak arasında gözlere yukarıda, kafatası üzerinde ciltte lidokain bir damla ve bir kanat kesti. Bir neşter ile kazıma önce periost üzerine tekrar lidokain çözümü uygulayın. </li><li> Beyincik Yukarıdaki kafatası ince bir yarım daire bölgeye diş matkap kullanın. Kafatası derinliği yarım daire çevresi boyunca hemen hemen sabit böylece penceresi genellikle, orta ve arka dikişin doğru yerleştirilir. Bu çok kalın bir bölgesi üzerinde lambda dikiş ve tekrar delme çok yakın bir pencere konumlandırma ödemlerin beyin ve oluşumu aşırı ısınmaya neden olabilir. </li><li> Hemostatik Uygulakanama durumunda kafatası sünger. </li><li> Bir jilet cımbız yardımı ile iki yarısı bir coverglass kesin. Coverglass kemik adası biraz daha büyüktür ve cımbız ile kemik kapağı çıkarmadan önce benzer bir şekle sahip olduğunu kontrol edin; değilse, coverglass yeniden şekillendirmek veya başka bir tane seçin. Kemik çizerken cımbız ile dura mater dokunmayın. Dikkatle dura üzerine coverglass uzandı. NOT: dura üzerinde, camın tüm yüzey yapışma uzun süreli beyin pencerelerin önceden bildiren edilir. </li><li> Art arda tuzlu su çözeltisi ile durulama ve pamuk sopalarla kurutulmasıyla cam altında hava kabarcıklarını çıkarın. Bu da camını yerleştirdikten sonra küçük kılcal damarlar dışarı dökülen olabilecek bazı kalıntı kan kaldırarak yardımcı olacaktır. Akrilik yapıştırıcı ve diş çimentosu 30 oluşan bir karışım ile pencereyi kapatın. </li><li> Tam anestezi kurtarıldı kadar bir kurtarma odasında bir ısıtma pedi üzerinde hayvan tutun. Re etmeyintamamen iyileşti kadar evde kafes içinde fareler açmak; Onlar sternal yatma korumak için yeterli bilinç kazanmış kadar onları yalnız bırakmayın. </li><li> Deri altına ameliyattan sonra 2-3 gün süreyle günde Carprofen (5 mg / kg) uygulanması. </li></ol> 3.. Vivo Multi-foton Lazer Axotomy <ol><li> Ilk görüntüleme oturumu gerçekleştirmeden önce ameliyat 2-7 gün bekleyin. Sapphire lazer kaynağı (120 fs genişlik bakliyat, 90 MHz tekrarlama oranı) ilk galvanometrik aynalar, bir çift tarafından taranan ve daha sonra bir su daldırma 20X tarafından numune üzerine odaklanmıştır: Burada kullanılan iki foton mikroskop yerleşik özel bir Ti ile sağlanır objektif (NA 0.95, WD 2 mm). Bir kapalı devre piezoelektrik aşamalı hedefi 400 mikron kadar eksenel değiştirmeleri gerçekleştirir. Photomultiplier tüpler floresan sinyalini toplar. </li><li> Kafatası penceresi objecti paralel bırakır, böylece hayvan anestezisi, daha sonra stereotaktik bir tutacak içerisine baş pozisyonuodak düzlemi ettik. </li><li> TPF kranial pencerenin altında korteks görüntüleme ile keşfedin. Parlak olanlar arasında hasarlı akson seçin. </li><li> Z-yığını Edinme hedef akson bir 3D rekonstrüksiyon elde etmek için (genellikle görüş alanı 512 x 512 piksel, 2 mikron z-ekseni adımında, 100 x 100 mikron 2). Görüntülü hacmi içinde, odak düzlemi çoğunlukla paralel uzanan bir akson dalında lezyon site seçin. Bu yönelim hızla başarılı bir diseksiyon akıllı olan morfolojik değişiklikler, puan sağlar. </li><li> Bir CF uzak bir bölümüne seçilen noktasında dozu yüksek bir enerji ile ışın tedavisi. Bu Labview yazılım aracılığıyla yapılır: (~ 4 bir satır tarama yapmak galvanometrik ayna konumunu ayarlamak, görüntüleme (objektif lens sonra ölçülen ≈ 300 mW) için kullanılan güç daha 5-10x daha fazla lazer gücünü arttırmak - 10 lezyon sitede mikron) ve 400 için lazer kapağını açın - 600 msn. Çizgi tarama oryantasyonCF düzlemine dik transeksiyonu tamamen akson için. Parlak noktalar (örn.., Varisler) aydınlatarak genellikle akson diseksiyon yardımcı olur. Lazer dalga boyu axotomy için görüntüleme (920 nm) için kullanılan ile aynıdır. </li><li> Genellikle ışınlanmış alanında akson floresans geçici ışıkla ağartma nedeniyle azalır. Işınlama ablasyon iyi bir performans sağlamak için edilmiş sonra aynı bölgenin birkaç dakikalık bir yığın elde edin. Bölge şişmeye başladı ve akson boncuk benzeri yapılar oluşmuş ise, nöron başarıyla disseke edildi. Bu durumda, birkaç saat içinde (dk birkaç on 24 saat için) akson distal kısmı dejenere ve tamamen kaybolur. </li><li> Ne yazık ki, bir tam axotomy üretmek için gerekli enerji dozu her zaman tahmin edilebilir değildir. Işınlama tek sonucu, foto ağartıcı ise, hızlı bir şekilde difüzyon floresan boya nedeniyle geri olmalı ve ışınlama etkili tekrar tekrar edilmelidirly akson teşrih. Ayrıca durumda şişlik belirtileri 5 dahilinde görünmüyor - artan Yeni taramalar ile tekrar deneyin, (. Benzer lazer dendrotomy için tarif edilmiştir ne Allegra Mascaro ark 23 bakınız) 20 dakika, ya da şişme kurtarma takip ediyor süresi ve / veya lazer gücü bekletilmiştir. </li><li> Vivo görüntüleme iki-foton ile lazer axotomy takip günlerde akson nihai yeniden Monitör (Şekil 2 ve 3). Vasküler desen şu günlerde disseke akson almak için referans model olarak kullanılır. </li><li> Deri altına Carprofen (5 mg / kg) Deney süresinin tamamı boyunca her görüntüleme oturumu yönetmek. </li></ol>

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S., Sugiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entire+trajectory+of+single+climbing+and+mossy+fibers+in+the+cerebellar+nuclei+and+cortex.">The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex.</a> Prog Brain Res. 124, 173-186 (2000).</li><li>Strata, P., Tempia, F., Zagrebelsky, M., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+trophic+interactions+between+climbing+fibres+and+Purkinje+cells+in+the+rat+cerebellum.">Reciprocal trophic interactions between climbing fibres and Purkinje cells in the rat cerebellum.</a> Prog Brain Res. 114, 263-282 (1997).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. (12), (2008).</li></ol>

We have nothing to disclose.

Bu protokol, bir floresan boya ile alt zeytinin nöronlar etiket gösterilmiştir. Daha sonra, serebellar korteks üzerinde bir kafatası pencere yapmak için bir yöntem tarif edilmektedir. Bu teknik olivocerebellar nöronlar, tırmanma liflerin terminal kısmı için optik erişim sağlamaktadır. Ne yazık ki, etiketleme ve kraniotomi cerrahi hem de sonucu (genellikle 3 farelerin üzerinden 1 etiketli ve 1 out of 3 kafatası pencereleri 1-2 hafta sonra açık kalır) bile vasıflı operatörler elinde oldukça düş?...

Mekanik yaralanma, zehirli hakaret ya da nörodejeneratif hastalıklara bağlı aksonal transseksiyon genellikle hücre gövdesi 10-13 ayrılır akson distal kısmının dejenerasyonu tarafından takip edilir. Birkaç istisna dışında 2,7,14,15 ile, yetişkin hayvanların MSS kopmuş aksonlar genellikle büyütme programı 16 aktive edemiyoruz. Küçük, hücresel düzeyde dejeneratif olayların gerçek-zamanlı dinamikleri hakkında bilinmektedir. Nöronal hasarı sınırlama ve sinir hücrelerinin yeniden büyümesini teşvik için yeni stratejilerin geliştirilmesi singularly yaralı nöronal hücreleri ölür ve yeniden hangi mekanizmasını açıklığa kavuşturulması, bir ilk adım olarak, gerektirir. Bu çalışma, en doğrudan in vivo tek bir nöronun dinamiklerinin görüntülenmesi tarafından ele alınmaktadır. Bir foton flüoresan görüntüleme teknikleri görünür ışık yoğun bir saçılma ile sınırlı olsa da, iki foton uyarma li derin kortikal katmanları ulaşırsubsellüler çözünürlük 3,4,17 ile fareler ettik. Floresan proteinleri selektif nöron 18-20 alt grupları olarak ifade edildiği transjenik farelerin yararlanarak, TPF mikroskopi vivo 21,22 gelişme sırasında sinaptik plastisite ve aksonal uzama keşif uygulanmıştır. Tek başına hasar görmüş sinir hücrelerinin T o yeteneği için yaralanma özellikle ilgi akson hedefleyen yaralanma modeli ile iki foton görüntüleme ile vivo izleme bağlantısı ile incelenebilir sonra tekrar kavuştu. Femtosecond palslarının çoklu foton emme tek dendrit ya da tek dikenler 5,23 bozmak için kullanılmıştır. Ayrıca, bu yaralanma paradigma temas dendrit 6 aksatmadan tek akson dalları kesme sağlar. Kez hasar, serebellar tırmanma lifler (CFS) aksonları yeniden belirli nöronal nüfusu izin özellikleri diseksiyon kapsamında yararlı bir model si vardırnce onlar bile yetişkin hayvanlarda 24,25 yaralanmadan sonra olağanüstü plastik özelliklerini korurlar. Son zamanlarda, CFS uzun süreli görüntüleme bu aksonlar lazer axotomy 6 takip günlerde regrowing yeteneğine sahip olduğunu göstermiştir. Bu protokol anterograde izlemesi ile olivocerebellar nöronlar ve bunların akson uzamasını etiketlemek için nasıl açıklar. Ilgi konusu nöronlar floresan etiketli sonra, bir kafatası pencerenin altında hafta veya ay boyunca rasgele zaman noktalarında tekrar tekrar izlenebilir. In vivo lazer axotomy tarafından tek akson dalları incelemek için prosedürü daha sonra açıklanacaktır. Burada sunulan teknikler in vivo aksonal yeniden mekanizması yeni bakış açıları ve nöronal dejenerasyonu sınırlamak ve akson büyümesini teşvik etmek için tedavi stratejilerinin geliştirilmesine yardımcı olabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser nanosurgery of cerebellar axons in vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Bu protokol tek nöronların aksonal etiketleme, in vivo görüntüleme ve lazer axotomy nasıl gerçekleştirileceği açıklanmıştır. Deneyin çizelgesi, Şekil 1 &#39;de gösterilmiştir. Alexa Fluor 488 Dekstran ile etiketlenmiş ve in vivo iki foton mikroskobu ile kafatası pencerenin altında görüntülendi CFS bir örnek, Şekil 2&#39;de verilmiştir. Önce 6,27 bildirdiği gibi, artan dalları birkaç gün gözlem sü...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

Lazer nanodissection bağlanmış iki foton görüntüleme, hücre içi çözünürlük ile merkezi sinir sisteminin dejeneratif ve rejeneratif işlemlerini incelemek için yararlı araçlardır. Bu protokol, etiket görüntü ve in vivo serebellar korteks bir tırmanma lifleri incelemek için gösterilmiştir.

Serebellar Aksonlar Lazer Nanosurgery<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K Görüntüleme.  University of Florence.  Lazer nanodissection bağlanmış iki foton görüntüleme, hücre içi çözünürlük ile merkezi sinir sisteminin dejeneratif ve rejeneratif işlemlerini incelemek için yararlı araçlardır. Bu protokol, etiket görüntü ve in vivo serebellar korteks bir tırmanma lifleri incelemek için gösterilmiştir. 

Video: Laser Nanosurgery of Cerebellar Axons In Vivo

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila Larvaların segmental Aksonlar içinde Sinaptik veziküller in vivo Görselleştirme

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Nörobilim

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

C in vivo Lazer Axotomy elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Ultrasonik Kan-beyin bariyeri bozulması ve Manganez kontrastlı MRG kullanma İşlevsel beyin görüntüleme

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Serebellar Granül Nöronlar Genetik Manipülasyon<em&gt; In Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Nöronal morfoloji ve Göç Eğitim için

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Konfokal Makroskopi kullanarak Postnatal Serebellar Granül Hücre Göç Ex Vivo Görüntüleme

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Eşzamanlı<em&gt; Ex vivo</emİki retina&gt; Fonksiyonel Test tarafından<em&gt; In vivo</em&gt; Elektroretinogram Sistemi

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modelleme nöronal ölüm ve fare birincil serebellar granül nöronlar dejenerasyon

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Vivo Multimodal görüntüleme ve fare lazer kaynaklı Choroidal neovaskülarizasyon Model analizi

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Myelinated akson in Situ üzerinde spektral Reflectometric mikroskobu

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Erişkin Serebellar Dilimlerde Uzun Süreli Depresyon İndüksiyonunun Değerlendirilmesi

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...