Yazarlar mali destek için teknik yardım, CIMO Vakfı ve FGSN için Neurotar Ltd teşekkür etmek istiyorum.

Hayvan denekleri prosedürleri Ulusal Hayvan Deney Kurulu, Finlandiya tarafından onaylanmıştır. Görüntüleme için 1. Hayvan hazırlanması <ol><li> Intraperitional ketamin (IP) enjeksiyon ile (vücut ağırlığı başına 0.08 mg) ve ksilazin (vücut ağırlığı başına 0.01 mg), bir fare anestezisi. Arka ayak tutam refleks ile anestezi edin. </li><li> Göz dehidratasyon gözleri korumak için (Viscotears) damlası halinde hayvanın gözleri daldırın. </li><li> Hipotermiyi önlemek için 37 ° C &#39;de bir ısıtma pedi (Supertech) üzerine fare koyun. </li><li> % 70 etanol ile görüntüleme için belirlenmiş ayak pedi temizleyin. </li><li> Deri ve kapak cam arasındaki bağlantı için daldırma ayak tabanından deri üzerinde bir damla su ekleyin. </li><li> Kapak sınıfı ile metal bir halka altında konumlandırılmış arka pençe altında plastik ambalaj malzemesi koyun. </li><li> Cilt düzleştirmek için plastik ambalaj malzemesinin kalınlığını ayarlayın. </li><li> Kapak glas bir damla su koyunkapak cam ve hedefi arasında daldırma için s. </li></ol> 2. Metal Fiksator Yüzük Hazırlık <ol><li> Cilt stabilizasyonu için metal fiksatörün kullanmaktadır. Biz bir metal halka (Neurotar Ltd, Finlandiya nezaket) Topluluk Tasarım-korumalı iki kanat fiksatör kullanın. Superglue şırınga doldurun halkası üzerindeki iğne boyunca superglue küçük bir damla koymak ve yavaşça Halka yüzeyi boyunca yeknesak bir şekilde kaplanmasını tutkalı yaymak. Tutkal aşırı miktarda görüş alanını azaltmak ve daha sonra görüntüleme engelleyebilecek, çünkü sadece homojen kaplama için gerekli olan tutkal az miktarda koymak için çok önemlidir. NOT: (altından) ve bir metal çubuk (bir kapak olarak) standart bir mikroskop cam slayt Alternatif kombinasyonu cildi ve düzleştirmek için tertibat 14 uygun optik bağlantısının sağlanması için kullanılabilir. İki ataç cam ve metal çubuk, yüzeyi arasındaki pençe düzeltmek için kullanılabilir (Şekil 4 </strong&gt;). </li><li> Mikroskop lamı kapak (5 mm çapında, Elektron Mikroskobu Science) ile halka örtün. </li><li> Motorlu mikroskop sahnede montajlanmış olduğu sert metal bir çubuğa halka fiksatör vidalayın. </li></ol> 3. Görüntüleme Prosedürü <ol><li> Thy1-YFP lH farelerin kullanılması durumunda, sinir liflerini görselleştirmek için floresan lamba spektrumları mavi kısmını seçmek. Epifloresans modunda ilgi sinir bulun ve ona odaklanın. </li><li> Boyuna görüntüleme için bir nokta koordinatlarını yazın. Bir referans noktası olarak karpal pedi kullanın. Aşağıdaki görüntüleme oturumları sırasında, ilk karpal pedi algılar ve daha sonra aynı memba büyük sinir liflerini bulmak ve kaydedilen koordinatlarını kullanarak geri dönün. Bu referans, aynı sistemi korumak için metal çubuğun dik aynı yönde ayak pedi yerleştirmek için gereklidir. </li><li> Iki foton modunda açın. Thy1-YFP lH fareleri için, bir lazer ışınının dalga boyu 950 nm kullanım için uygundursinir liflerini görselleştirmek için. </li><li> (450 520&#39;den YFP etiketli sinirlerin görüntüleme için 550 nm ve 580 YFP floresans ve saç otofloresanm saptanması için 630 nm, ikinci harmonik oluşumu saptanması için 480 nm e) seçeneğini lazer dalga boyu kanalları ve emisyon ile başlar fotohasar ve ağartma önlemek için düşük lazer gücü (% 3-5). Photomultiplier tüpleri üzerinde tipik voltaj görüntüleme bu tür için 600-700 V. </li><li> (1-10 istediğiniz çözünürlüğü (512 x 512 veya hızlı olaylar ölçümleri için 640 x 640 x 800 veya morfolojik görüntüleme için 1.024 x 1.024 800), eksenel adımı (1-3 mikron), numune kalınlığı ve zaman aralıklarını ayarlayın dak). Pozlama süresi, bir piksel için 1 milisaniye düzenindedir. </li><li> İlk yazılım görüntüleme hacminin üst ve alt sınırları işaretleyin. </li><li> Lezyonun önce başvuru için referans görüntü yığınını edinin. Gerektiğinde, birkaç dakika boyunca taban çizgisini kaydetmek mümkündür. </li><liGerektiğinde&gt; arka arkaya (en azından her 10-15 dk) hayvan solunum hızını ve refleksleri kontrol anestezik ek bir miktarda uygulayın. </li><li> Bir doku ped ile temiz görüntülenmesi ve tamamen uyanık kadar 36 ° C &#39;de bir yeniden kazanma kutusuna fare yerleştirilir. Not: Genellikle, lazer tarafından kışkırtılan lezyonu ile birleşmiş bir görüntüleme oturumunun süresi (aşağıya bakınız) 1 saat aşmaz. Biz hayvan derinden her 10-15 dakikada anestezi doğrulayarak öneririz; Bu kendini görüntüleme engel olmamalıdır ama deneysel prosedür planlanmaktadır ise düşünülmelidir. Tek bir ketamin / xylazyne doz etkisinin süresi bu 25-30 dakika sonra, bir doz ½ ¼ ek uygulamak için tavsiye, 40 dakika yaklaşık 30 ° C&#39;dir. NOT: deneyler tek bir hayvan için görüntüleme oturumları frekans yeniden olumsuz etkilerini önlemek için 1 dönem 2 başına gün aşmaması planlıyor zaman yanı düşünülmelidirpetitive anestezi. </li></ol> 4. Lazer Lezyon <ol><li> Referans görüntü yığınını aldıktan sonra, mikro lezyonu yapmak için ağartma protokolünü kullanır. </li><li> % 100 lazer gücünü artırın. </li><li> Çevrede (100-1,000x standart görüntüleme durumunda daha fazla) bölgesinden başına 100-1.000 msn pozlama süresini artırın. </li><li> Lezyon için alan Anahat. </li><li> Ağartma açarak lezyon olun. </li><li> Geri normal görüntüleme moduna geçmek ve zaman atlamalı kayıtları devam. </li></ol> 5. Veri İşleme ve Analizi <ol><li> ImageJ 8 spektral Karışmama eklentisi kullanarak unmixing ile (Joachim Walter, Devam <a href="http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html">http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html</a> ). </li><li> Görüntü yığını açın ve kanal bölünmüş. </li><li> Arka plan ölçümleri için sinirlere veya saç serbest alanını bulun. Bir bölge o koyuno bölgede f ilgi. </li><li> Dikkatlice sinirler açıkça ayırt edilebilir saç parçası özetlemektedir. </li><li> Bu saçtan ayrı görüntü kısmında sinir özetlemektedir. </li><li> Matrisi Karışmama kaydedin. </li><li> Kül yığını için ölçülen karışmama matrisi uygulayın. </li><li> * .tif Formatında yeni işlenmiş görüntü yığınlarını kaydedin. </li></ol>

<ol>
	<li>Lumpkin, E. A., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sensory+transduction+in+the+skin.">Mechanisms of sensory transduction in the skin.</a> Nature. 445, 858-865 (2007).</li><li>Kennedy, W. R., Wendelschafer-Crabb, G., Johnson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+epidermal+nerves+in+diabetic+neuropathy.">Quantitation of epidermal nerves in diabetic neuropathy.</a> Neurology. 47, 1042-1048 (1996).</li><li>Wu, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morton's+interdigital+neuroma:+a+clinical+review+of+its+etiology,+treatment,+and+results.">Morton's interdigital neuroma: a clinical review of its etiology, treatment, and results.</a> J. Foot Ankle Surg. 35, 112-119 (1996).</li><li>Lauria, G., Lombardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy:+a+new+tool+for+diagnosing+peripheral+neuropathy.">Skin biopsy: a new tool for diagnosing peripheral neuropathy.</a> BMJ. 334, 1159-1162 (2007).</li><li>Cheng, C., Guo, G. F., Martinez, J. A., Singh, V., Zochodne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+plasticity+of+axons+within+a+cutaneous+milieu.">Dynamic plasticity of axons within a cutaneous milieu.</a> J. Neurosci. 30, 14735-14744 (2010).</li><li>Wang, B., Zinselmeyer, B. H., McDole, J. R., Gieselman, P. A., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Imaging+of+Leukocyte+Homing+and+Migration+in+vivo.">Non-invasive Imaging of Leukocyte Homing and Migration in vivo.</a> J Vis Exp. (46), e2062 (2010).</li><li>Sacconi, L., O'Connor, R. P., Jasaitis, A., Masi, A., Buffelli, M., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multiphoton+nanosurgery+of+cortical+neurons.">In vivo multiphoton nanosurgery of cortical neurons.</a> J Biomed Opt. 12, 050502 (2007).</li><li>Robinson, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+injury+to+peripheral+nerves.">Traumatic injury to peripheral nerves.</a> Muscle Nerve. 23, 863-873 (2000).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Marinkovic, P., Reuter, M. S., Brill, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+transfer+into+the+dorsal+root+ganglion+of+adult+rats.">Lentiviral gene transfer into the dorsal root ganglion of adult rats.</a> Mol Pain. 7, 63 (2011).</li><li>Yuryev, M., Khiroug, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+longitudinal+investigation+of+individual+nerve+endings+in+the+skin+of+anesthetized+mice+using+in+vivo+two-photon+microscopy.">Dynamic longitudinal investigation of individual nerve endings in the skin of anesthetized mice using in vivo two-photon microscopy.</a> J Biomed Opt. 17, 046007 (2012).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Bu video protokolü biz tek bir sinir uçlarının non-invaziv uzunlamasına iki foton görüntüleme yöntemini göstermektedir. Cilt innervasyon dinamikleri, sedef hastalığı ve periferik nöropati 2, gibi hastalıkların, ve travmatik yaralanmalar 9 etkilenir. İki foton görüntüleme kollajen matris içinde sinir lifleri yapıların detaylı analiz sağlar. Transgenik raportör farelerin kullanılması sinir liflerinin boyama ile ilgili problemleri önler. Thy1-m...

A correction was made to <a href="/video/51045">In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin</a>.
Leonard Khiroug was removed as an author.

Erratum: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Kutanöz sinir uçlarının farklı patofızyolojik altında dinamik değişikliklere uğrarlar. Sinir lifleri periferik nöropati 2 veya Morton nöroma 3 gibi hastalıkların seyrinde dejenerasyon ve rejenerasyon veya yeniden yapılanma sürecinde gidebilirsiniz. Travmatik yaralanma sonra, deri sinir uçlarının dinamiklerinin önemli bir parçası hasarlı alanın reinnervasyon olduğunu. Ancak, sinir uçlarının soruşturma ortak bir yaklaşım devam eden süreçler 4 gerçek-zamanlı bilgi eksikliği, ex vivo histolojik kesit olduğunu. Genetik olarak kodlanmış floresan markör kullanılarak, bu nedenle yapısal değişikliklerle ilgili zengin ve önemli ölçüde daha fazla ayrıntılı bilgi elde edilmesi, canlı hayvanların derisinde sinir uçlarını izlemek mümkündür. Deri altı sinir uçlarının araştırılması geleneksel fluoresan mikroskobu, ancak, cilt dokusunun güçlü ışık saçılım güçlü kalite zayıflatmaktadır kullanarak mümkündürVerilerin 5 satın almıştır. Multiphoton mikroskopi nedeniyle sadece objektif odak noktasından floresan emisyonu uyarma ışık fotonlarının enerjisi olmayan doğrusal toplamına kuvvetle saçılma dokularda yüksek çözünürlüklü görüntüler elde edilmesine izin verir. Bu etki, deri dokuları 6 ölçüm için sağlam bir penetrasyon derinliği artışı ve sinyal-gürültü oranının bir gelişmeye yol açar. Görüntüleme için kullanılan aynı lazer kullanarak, bu sinir liflerinin 7 seçici diseksiyon üretilmesi mümkündür. Aşağıdaki protokolde seçici lazer lezyonu ticari olarak temin multiphoton mikroskop sistemi kullanılarak raportör ile birlikte transjenik farelerde in vivo olarak deri altı sinir uçlarının uzunlamasına görüntüleme yöntemini göstermektedir.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
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			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
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in vivo two-photon microscopy of single nerve endings in skin

Deride sinir uçları, nöropatik ağrı 2 gibi 1 algılamak olarak hem de patolojik süreçlerde fizyolojik süreçlerde görev alır. Onların yakın-yüzey konumlandırma sağlam hayvan canlı cilt sinir uçlarının mikroskopik görüntüleme kolaylaştırır. Multiphoton mikroskopisi ile cilt dokusunun güçlü ışık saçılma sorunun üstesinden gelmek iyi görüntü elde etmek mümkündür. (Çevre duyu nöronları dahil olmak üzere) nöronlarda Thy-1 promotörünün kontrolü altında eksprese EYFP Raportör transgenik fareler ile birkaç aya kadar ya da yaşam boyu uzun bir süre boyunca tek tek sinir uçlarının uzunlamasına çalışmalar için uygundur. Ayrıca, görüntüleme için aynı femto saniye lazer kullanarak, bu sinir lifi yeniden çalışmalar için sinir lifleri yüksek ölçüde seçici lezyonlar üretmek mümkündür. Burada, in vivo görüntüleme uzunlamasına multiphoton için basit ve güvenilir bir protokolü vefare deri sinir uçları lazer tabanlı mikro.

Mümkündür Tarif edilen teknik kullanılarak, lezyon sonrası aynı elyaf izlemek ve hasarlı sinir uçları (Şekil 1) bozunmasını incelemek. 120-150 um&#39;lik bir kalınlığa sahip yığın elde edilmesi görüş alanı içinde bütün lif tutmak için birkaç gün boyunca tekrar görüntüleme için genellikle uygundur. Plastik ambalaj malzemesi cilt düzleştirmek için ayarlandığı zaman, genellikle lezyon öz üretilebilir, böylece kolajen tabakalar görüntü...

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Dinamik boyuna görüntüleme ve muhabiri transgenik farelerin sinir uçlarının selektif lazer lezyon için protokol sunulmuştur.

Cilt Tek Sinir Endings Vivo İki-Foton Mikrokopisi

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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Research

JoVE Journal

Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

10.7K Görüntüleme.  University of Helsinki.  Dinamik boyuna görüntüleme ve muhabiri transgenik farelerin sinir uçlarının selektif lazer lezyon için protokol sunulmuştur. 

Video: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

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 ...

Nörobilim

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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 ...

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 ...

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

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 ...

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.

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

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

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

HIV kaynaklı Nöroinflamasyondan bir Fare Modeli İki foton Mikroskop kullanarak Serebral Vasküler Mimarlık Niceleme

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

Saç Folikül hançer Endings Yaşam Sinir Terminal Etiketleme Optik İzleme<em&gt; Ex Vivo</em&gt; Fare Kulak Cilt

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Nöronal Dendrites beyin dilimleri içinde iki fotonlu kalsiyum görüntüleme

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo Yenidoğan farelerde kortikal nöronların iki fotonlu görüntüleme

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