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) intraperitonal (IP) enjeksiyon ile fare anestezisi fare vücut ağırlığına göre Not:. KX doz sağlık durumu suşu, yaşa göre ayarlanmış edilebilir ve Farelerin. Optimum dozun belirlenmesi için veteriner danışmanlık da tavsiye edilir. </li><li> Farenin ayak basarak ve anestezi durumunu izlemek için refleksif bir tepki için kontrol ederek periyodik bir ayak tutam testini gerçekleştirin.Fare tamamen ameliyat başlamadan önce anestezi emin olun Not:. Uzun görüntüleme oturumları sırasında, gerekirse ek KX yönetmek, periyodik fare anestezi durumunu kontrol edin. </li><li> Ameliyat sırasında vücut ısısını korumak için bir ısıtma pedi üzerinde fare yerleştirin. </li><li> Kafa derisi maruz bir jilet kullanarak fare başını tıraş. </li><li> Alkol pedleri ve betadine alternatif ile cildi silerek traş alanı sterilize edin. Herhangi bir kalıntı saç kupürleri çıkarın. </li><li> Yavaşça bu nedenle deney sırasında dehidratasyon neden olduğu kalıcı hasarı önlemede, hem gözleri yağlamak için göz merhemi uygulanır. </li><li> Kafa derisinin orta hat boyunca düz bir kesi yapmak ve kafatasının kenarlarına doğru yanal cildi taşıyın. </li><li> Kafatasına bağlı bağ doku çıkarın. </li></ol> 2.. Inceltilmiş-kafatası Hazırlık <ol><li> Ster dayalı görüntüleme bölgeyi tespit. eotaxic koordinatlar Not: büyük kan damarları, blok ışık penetrasyonunu önlemek ve görüntülü yapıları bulanıklık çalışın. Kafatası steril tuzlu su ile nemli olduğunda damarsal iyi görülmektedir. </li><li> Kafatasının (0.5-1.0 mm çapında) ince bir dairesel bölgeye yüksek hızlı mikro matkap kullanın. Yerine kafatası karşı tutarak ve bastırarak yerine, kafatası yüzeyi matkap paralel hareket. Matkap dış kompakt kemik tabakası ve orta süngerimsi kemik tabakası hem kadar kaldırılır Not:. Aşırı ısınma nedeniyle zarar görmesini önlemek için, matkap ile kafatası arasındaki uzun süreli temasından kaçının. </li><li> Matkap-inceltilmiş bölgenin merkezinde bir mikrocerrahi bıçak ile iç kompakt kemik tabakası inceltme devam edin. Bir eşit yaklaşık bir kalınlığı olan küçük bir bölge (200-300 mikron çapında) inceltilmiş kadar kafatası karşı basmadan kafatası kazımak için yaklaşık 45 ° &#39;lik bir açı ile mikro bıçak tutunly 20-30 mikron elde edilir. . Nedeniyle kafatasının bir oto-floresan için, kalınlığı iki foton mikroskop altında Not kafatasının üst ve alt yüzey arasındaki mesafeyi tarama ile ölçülebilir: en az 20 um bir kalınlığı kafatası iyi bir görüntü kalitesi sağlar, ancak, daha sonraki yeniden görüntüleme oturumları incelme süreci zorlaştırır çünkü bu tavsiye edilmez. Aşırı inceltme önlemek için, kafatası kalınlığı (Tartışma) ameliyat sırasında periyodik olarak kontrol edilmesi gerekmektedir. </li></ol> 3. Immobilizasyonu <ol><li> Dikkatle kemik enkaz kaldırmak. </li><li> Otoklavlanmış olan steril kafa plakası, merkezi açıklığın her bir kenarında siyanoakrilat yapıştırıcı bir damla (bkz. Şekil 1) koyun. . Açıklığın ortasına Not bulunan inceltilmiş bölge ile kafatası sıkıca kafa plakası tutun: Tutkal, inceltilmiş r karışması durumundakazara egion, mikrocerrahi bıçak ile dikkatlice çıkarın. </li><li> Yavaşça kafa plakasının merkezi açıklığın kenarlarına kafatası taraftan deri çekin. </li><li> Kafa plakası de kafatası bağlı olduğu kadar, yaklaşık 10 dakika bekleyin. </li><li> (Bkz. Şekil 1) tutma plakasının iki yan bloklar üzerinde kafa plaka koyun ve ardından tutma plaka üzerinde fare hareketsiz kafa plakasının kenarları üzerinde vidaları sıkın Not:. Arasında deri ya da bıyık olduğundan emin olun vidaları sıkmadan önce kafa plaka ve blokları. Hayvanın geri hafifçe dokundu zaman iyi bir hareketsiz preparat mikroskop altında, kafatası gözlenebilir bir hareket göstermelidir. </li><li> . Polimerleşmemiş tutkal kaldırmak için tuzlu su ile maruz kafatası durulayın Not: polimerleşmemiş tutkal görüntüleri buğulu ve zarar hedeflerini mikroskop gibi, kalan tutkal kaldırmak için önemlidir &lt;./ Li&gt; </ol> 4. Görüntüleme <ol><li> (Bkz. Şekil 2) sonraki görüntüleme oturumlarda görüntülü bölgeyi taşınmaya kullanılan Haritası 1, gibi inceltilmiş bölge ile maruz kafatası damar bir fotoğraf çekin. </li><li> Görüntüleme mikroskop altında fare yerleştirin. Epifloresans kullanarak 10X hava objektif altında inceltilmiş alan bulmak ve bakış merkezine ince alanı taşımak. </li><li> Kafatasının üst tuzlu bir damla ekleyin ve steril su ile durulanır edilmiş 60X objektif, geçin. Bireysel dendritik dikenler açıkça dendritler boyunca görüntülenmiştir bir bölge seçin. Belirlemek ve epifluorescent görünümü ve damarsal fotoğraf arasındaki damarsal karşılaştırarak Haritada 1 ilgili bölgeyi etiket. </li><li> Fluorophores göre Ayarlama iki-fotonlu lazer dalga boyu. Örneğin, YFP için 920 nm; GFP için 890 nm; DsRed ve tdTomato 24 için 1,000 nm. </li><li> 2 ile görüntü yığınlarını Edinme60X objektif kullanılarak z-ekseni boyunca um adımlar. Bu görüntü yığını yaklaşık 200 mikron x 200 mikron alanı (512 x 512 piksel) kapsar ve (bkz. Şekil 2) sonraki görüntüleme oturumları sırasında tehcir için 2 Haritası olarak kullanılır. </li><li> 3X dijital zoom kullanarak 2 Haritası içinde dokuz görüntü yığınlarını edinin. . Her bir görüntü yığını z ekseni boyunca not 0,7 um adımlarla 70 um x 70 um (512 x 512 piksel) yaklaşık bir alanı kaplamaktadır: fototoksisite en aza indirmek için örnekler hızda ölçüldüğünde lazerin yoğunluğu 40 mW altında olmalıdır. </li></ol> 5. Kurtarma <ol><li> Görüntüleme ardından, yavaşça kafatası kafa plaka ayırmak. </li><li> İyice kafatası ve tüm kalan tutkal kaldırmak için cilt temizlemek Not:. Ciltte herhangi bir kalan tutkal tahrişe neden ve kafatası üzerinde kalan tutkal kafatasının erozyon ve neden olurken cilt iyileşme yavaşlarngiogenesis yeni yetişen kemik tabakasında, daha sonraki tehcir ve yeniden görüntüleme zorlaştırır. </li><li> Kafatası ve tuzlu birkaç kez cildi durulayın. </li><li> Steril cerrahi sütür ile kafa derisi dikin. </li><li> Ayrı bir kafes içinde bir ısıtma pedi üzerinde hayvan tutun. Post-operatif ağrı azaltmak için deri altına buprenorfin analjezik (0.1 mg / kg) yönetmek. Tam iyileşme sonra eve kafesine hayvan dönün. Kesi iyileşmiş ve dikişler kadar (günde en az bir kez kontrol edin) yakından hayvan izleyin. Gerekirse buprenorfin analjezik sonraki enjeksiyonları yönetmek. </li></ol> 6. Tekrar görüntü <ol><li> Tekrarlayın 1,1-1,8 adımları. </li><li> Tekrar 3,1-3,6 adımları </li><li> 1 Harita için damar desen ve 2 Harita için dendritik şube desen karşılaştırarak daha önce görüntülenmiş bölgeyi bulun. </li><li> Tekrar görüntü alma 1 hafta içinde gerçekleştirilirse, inceltilmiş yeniden üstüne yeni çıkan kemik ince tabakayı kaldırmak için adım 2.3 tekrarlayınmikrocerrahi bıçak kullanarak dür. Tekrar görüntü alma fazla 1 hafta sonra gerçekleştirilirse, Adım 2.2 ve 2.3 hem de tekrar Not:. Yeni çıkan kemik tabaka azaltılmış resim kalitesine neden orijinal kompakt kemik tabakası ile karşılaştırıldığında, daha az yoğunlaştırılmış bir yapı oluşur. Bu nedenle, aynı kaliteyi elde etmek için, bir önceki görüntüleme oturumu biraz daha ince kafatası için gereklidir. </li><li> Daha önce iki foton mikroskop altında görüntü yığınlarını çekilen eşleşme görüntü yığınlarını elde etmek için konumunu ve yönünü ayarlayın. </li><li> Adım 4.6 gibi görüntü kazanır. </li><li> Tekrar görüntüleme sonra 5,1-5,5 adımları. </li></ol>

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

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 ...

two-photon-vivo-imaging-dendritic-spines-mouse-cortex-using-thinned

Research

JoVE Journal

Neuroscience

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

17.8K Görüntüleme.  University of California, Santa Cruz. 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.

Video: 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

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain 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 using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

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

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

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

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

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

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 this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

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

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

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

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

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

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 of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

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

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Glutamat ve GABA hızlı Micro-iyontoforez: Synaptic Entegrasyon Araştırma yararlı bir araç

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the 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

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

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

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

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