Biz kendi teknik yardım için Yue (Pauline) Hu, Rachel Sinit teşekkür etmek istiyorum. Çalışma NIH hibe (PN2 EY016525) tarafından ve Down Sendromu Araştırma ve Tedavi Vakfı ve Larry L. Hillblom Vakfı fon tarafından desteklenmektedir.

Cerrahi ve hayvan prosedürleri Bakımı NIH Kılavuzu&#39;na ve Laboratuvar Hayvanları Kullanımı göre kesinlikle yapılır. Hayvanlarının kullanıldığı bütün deneyler UCSD Kurumsal Hayvan Bakımı ve Kullanımı Komitesi tarafından onaylanmıştır. 1. Plazmid Klonlama, mono-biyotinlenmiş BDNF sentezlenmesi ve saflaştırılması (mBtBDNF) NOT: HEK293FT hücrelerde 10 pcDNA3.1 vektörü ve birlikte açığa içine önceden proBDNFavi ve BirA cDNA Construct. MBtBDNF olgun ve biyolojik olarak aktif monobiotinylated sinir büyüme faktörü (mBtNGF) üretilmesi için 10, daha önce yayınlanmış bir metoda göre Ni-NTA boncuklar kullanılarak saflaştınlır. Mikroakışkan Odaları 2. Hazırlık Mikroakışkan nöron kültür cihazı akışkan nöron hücre gövdeleri aksonlar izole edilmesini mümkün kılar. Sağ her diseksiyon önce taze kaplı lamelleri ile odaları birleştirin. Kullanılan mikroakışkan odalarıBu protokolde ticari (malzeme ve ekipmanın tabloya bakınız) satın alınır. Handwash ve 5-6x kadar yeniden piyasadan satın odaları. <ol><li> El yıkama% 1 Alconox içinde mikroakışkan odaları. 30 dakika her biri için Milli-Q su ile üç kez durulanır. % 70 etanol içinde tekrar el yıkama odaları. </li><li> Laminer akış kaputu Parafilmlerin üzerinde kurumaya odaları dışarı yatıyordu. Yayar ve UV altında 20 dakika boyunca odacıkların her iki tarafını da sterilize edin. Mağaza oda sıcaklığında Parafilm ile mühürlenmiş steril bir 15 cm çanak odaları sterilize. </li><li> Cam bir kap içinde 24 x 40 mm 1 numaralı cam lamelleri yerleştirin. Gece boyunca, bir döndürücü üzerinde% 35 hidroklorik asit lamelleri bekletin. Ertesi gün, 30 dakika su ile her biri için lamelleri üç kez yıkayın. </li><li> % 100 etanol içine Her lamel daldırma ve bir Bunsen beki üzerinde yanan tarafından lamelleri tek tek sterilize edin. Steril bir petri tabağında muhafaza edilir ve kuru lamelleri kaplaması elde edilinceye kadar oda sıcaklığında saklayın. </li><li> Ou Lay15 cm kültür çanak lamelleri t. Kaplama% 0.01 poli-L-Lisin (PLL), 0.7 ml ile her lamel ve oda sıcaklığında kaputu inkübe edin. </li><li> 1 saat sonra, lamelleri steril su ile üç kez durulanır. 6 cm kültür kabına vakum ve yerde her lamel ile kuru lamelleri. </li><li> Mikroakışkan bölmesini monte etmek için, Mikroyivlerin temas etmemek için tedbir ile PLL kaplanmış bir örtücü camın üzerine altındaki microgroove yüzü bölmenin. Yavaşça odası sıkıca kapatılmıştır sağlamak için bir pipet ile odasını basılı. </li></ol> Chambers 3. Nöronal Kültür Diseksiyon ve Levha <ol><li> % 1 pen / strep, 10 mM HEPES, 2 ml tahlil tamponuna (HBSS, herhangi bir kalsiyum, magnezyum ihtiva eden herhangi bir 15 ml konik bir tüp içinde kesildi iki E17-E18 sıçan hipokampları (bir beyin) yerleştirin. Dokular, 5 ml ile 3 kere yıkayın diseksiyonu mümkün olduğunca diseksiyon tamponu çıkarın. her zaman tampon ve taze di 900 ulssection tamponu. </li><li> , Doku sindirmek 1x çalışma konsantrasyonunu yapmak için diseksiyon tamponuna 10x tripsin (% 2.5) 100 ul ekleyin. Bir 37 ° C su banyosu içinde konik tüp yerleştirin. Sindirim 10 dakika sonra, 1 mg / ml civarında nihai bir konsantrasyona kadar, 10 mg / ml Dnaz I, 100 ul ilave edin. </li><li> Bir yangın cilalanmış Pasteur cam pipet kullanarak, hafifçe aşağı ve yukarı pipetlenerek 5-10x dokuyu Karışım. Sağ toz haline getirildikten sonra, (% 10 FBS, 2 mM Glutamax,% 2 ile B27 Neuro temelli) ortam maddesi 2 ml tripsin söndürün. </li><li> Dibe oturmaya dokuların birikintileri girmesi için 5 dakika için başlık örnek bırakın. Dikkatlice pelet hücreleri 5 dakika 200 x g&#39;de, temiz, steril 15 ml konik bir tüp ve santrifüj içine 2ml&#39;si çıkarın. 50 ul ortam kaplama pelletini </li><li> Hemositometre ile hücreleri saymak. Yük mikroakışkan odasının bir bölmesine 15-20 ul hücre süspansiyonu (~ 40.000 hücre). Bölmenininkübatör 10 dakika hücreler lamel takmak için izin için. 10 dakika sonra, bölmenin her iki bölümü doldurmak için daha fazla kaplama ortamı eklenir. </li><li> Diseksiyon ikinci gününde, tamamen bir hücre gövdesi ve akson bölmesinin her iki (2 mM glutamax,% 2 B27 ile Neurobasal) bakım medya ile kaplama ortamı değiştirin. Hipokampal nöronların aksonları, 3. gün Mikroyivlerin çapraz ve günün 5-7 arasındaki akson bölüme ulaşmak başlar. Bu süre boyunca, Bakım ortamının her 24 ~ 48 saat ile kültür ortamının yarısı değiştirin. </li></ol> QD-BDNF 4. Akson Taşıma <ol><li> , QD-BDNF aksonal taşıma Canlı görüntülemeye mikroakışkan odasının hücre gövdesi ve akson hem bölmelerden BDNF tüketmek için önce iyice ile 2 saat boyunca, BDNF içermeyen serumsuz Neuro temelli ortam her 30 dakikada bir, her iki bölümü ile yıkayın. </li><li> BDNF tükenmesi sırasında, QD-BDNF eşleniklerini hazırlamak. Mono-biyotinile BD 50 nM karıştırınNF Neurobasal ortam içinde, 50 nM QD655 streptavidin eşlenikleri ile, dimer ve 60 dakika boyunca buz üzerinde inkübe edilir. </li><li> Akson bölmesinde Ortamı çıkarın ve 37 ° C&#39;de 4 saat boyunca 0.25 nM&#39;lik nihai bir konsantrasyona sahip 300 ul QD-BDNF ekleyin. Hücre gövdesi bölmesine QD-BDNF difüzyon en aza indirmek için, her zaman akson bölmesine göre hücre gövdesi bölmesinde ortamın daha yüksek bir düzeyde tutmak için çok önemlidir. Canlı görüntüleme önce kuluçkadan sonra bağlanmamış QD-BDNF yıkayın. </li><li> Bir 100X yağ objektif lens ile donatılmış ters bir mikroskop kullanılarak QD-BDNF taşıma canlı görüntüleme yürütmek. Kapsam ve sabit bir sıcaklıkta (37 ° C), CO2 (% 5), kendisine bağlı kazanı ısıtın. QD655 sinyalini görselleştirmek için Texas kırmızı uyarma / emisyon küpler bir dizi kullanın. </li><li> Edinme ve CCD kamera kullanarak 2 dakika bir toplam 1 kare / sn hızında orta akson içindeki time-lapse görüntüleri yakalayabilir. Aksonlar ile kullanımı microgrooves That hiçbir QD ve dolayısıyla infiltrasyon için bir kontrol olarak sinyal yok. </li><li> Herhangi bir görüntü analizi yazılımı veya NIH ImageJ kullanarak BDNF taşıma analiz edin. </li></ol>

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Çıkar çatışması ilan etti.

Bu çalışmada, biyolojik olarak aktif bir üretmek için yeni tekniğin gelişimleri, BDNF aksonal taşınması takip etmek için kullanılabilir BDNF (mBtBDNF) monobiotinylated. Kuantum dot streptavidin konjüge proteini ve bir mikroakışkan odacığı kullanılarak, yöntem, bir gerçek zamanlı olarak ve uzamsal ve zamansal çözünürlüğe kadar tek molekül hassasiyet ile primer nöronlarda aksonal taşınması BDNF algılamasını sağlar. Burada kullanılan aletler sağlık ve hastalık nöronlar BDNF / TrkB ...

Nöronlar yüksek olan uzun ve genellikle son derece özenli süreçleri kurmak ve nöral devrelerin yapısını ve işlevini korumak için temel olan hücreler polarize. Akson ve sinapsların yük taşıyan hayati bir rol oynar. Hücre soma sentezlenen proteinler ve organelleri nöronal fonksiyonu desteklemek için presinaptik terminale ulaşmak için aksonlar aracılığıyla taşınması gerekir. Buna karşılık, sinyalleri uzak aksonlar transdüse gerekir alınan ve soma aktarıldı. Bu işlemler, nöronal canlı kalmasını, farklılaşmasını ve bakım için çok önemlidir. Bazı nöronlarda aksonal taşınmasının bu mesafeler üzerinden 1.000 &#39;den fazla kez hücre gövdesi çapının yürütülmesi gereken olarak, olasılık bile hali hazırda küçük eksikliklerin belirgin nöronal devre fonksiyonunu etkileyebilir düşünülmüştür. , Beyin türevli nörotropik faktör (BDNF), büyüme faktörlerinin nörotrofin ailesinin bir üyesi, ma mevcutturhipokampus, serebral korteks ve bazal ön dahil ny beyin bölgeleri,. BDNF, bilişsel devrelerinde katılma nöronların hayatta kalmasını, farklılaşmasını ve fonksiyon destekleyerek idrak ve hafıza oluşumunda önemli bir rol oynar. BDNF, bu mitojen tarafından aktive edilen protein kinaz / hücre dışı sinyal ile düzenlenen protein kinaz (MAPK / ERK), fosfatidilinositol-3-kinaz dahil olmak üzere, TrkB aracılı sinyalleme yolunu aktive akson terminal, kendi reseptörü, tirosin kinaz TrkB bağlanan (PI3K) ve fosfolipaz C-gama (PLCγ). Bu sinyal yolları katılan proteinler daha sonra retrograd nöronal soma taşınır endosome 1-6 sinyalizasyon BDNF / TrkB oluşturmak için endositik veziküler yapıların üzerine paketlenir. Mikroakışkan kültür oda normal şartlar altında yanı sıra yaralanma ve hastalık 7,8 ayarı aksonal biyoloji eğitimi için çok yararlı bir platform. Izole aksonlarlahücre gövdeleri, cihaz aksonlar 8-10, özellikle taşıma anlaşılmasına bir sağladı. Bu çalışmada kullanılan ve 450 um microgroove bariyerli PDMS göre mikro-akışkan platformlar ticari (Malzemeler tabloya bakınız) satın alınmıştır. BDNF, taşıma incelemek için monobiotinylated BDNF (mBtBDNF) üretimi için yeni bir teknoloji geliştirilmiştir. (Da AviTag olarak da bilinir), biyotin alıcı peptidin, AP yararlandı. Bu, özellikle Escherichia coli enziminin biyotin ligazı, Bira ile biyotine bağlanabilir bir lisin kalıntısı ihtiva eden bir 15 amino asit dizisidir. Bu PCR (Şekil 1 A), fare pre-proBDNF cDNA&#39;nın C terminaline AviTag kaynaşık. Bu yapı, memeli ekspresyon vektörü pcDNA3.1 myc-his vektörü içine klonlandı. Ayrıca, pcDNA3.1 myc-his vektörü içine bakteri, BirA DNA klonlanmıştır. Iki plasmid geçici olarak her iki proteini ifade etmek için HEK293FT hücrelerine eş-transfekte edilmiştir. BirA biotun ligasyonu katalizeözel olarak bir lizin 1 de BDNF C-terminalindeki AviTag içinde ikamet: 1 oranında, BDNF monomer monobiotinylated üretmek. Biyotinile edilmiş, ~ 18 kDa bir moleküler kütleye sahip BDNF olgun geri kazanılmış ve Ni-reçinesi (Şekil 1C) kullanılarak ortamdan saflaştırılmıştır. (Şekil 1D) imüno-benekleme ile değiştirilmemiş BDNF saptamak mümkün ile karar kılındığı üzere BDNF biyotinilasyon, tamamlandı. Streptavidin konjüge kuantum noktaları, QD 655, QD-BDNF yapmak mBtBDNF etiketlemek için kullanıldı. MBtBDNF fosforile TrkB (Şekil 1E) ve rekombinant insan aktive BDNF (rhBDNF) ölçüsünde nörit gelişimini (Şekil 1F) teşvik etmek için mümkün olduğu gibi AviTag varlığı BDNF aktivitesine müdahale etmedi. QD-BDNF QD-BDNF biyoaktif (Şekil 1G) olduğunu belirten, hipokampal aksonlardaki trkB ile birlikte yerleştiğini göstermiştir immünolekeleme. BDNF taşıma incelemek için, QD-BDNF distal akson bölmesine eklenmiştirsıçan E18 hipokampal nöronlar (Şekil 2A) içeren mikrosıvı kültürler. Akson içindeki QD-BDNF retrograd taşıma kırmızı floresan etiketi (Desteklenmesi videolar S1, S2) gerçek-zamanlı canlı görüntüleme tarafından yakalandı. Oluşturulan kymograph analiz ederek, QD-BDNF yaklaşık 1.06 mm / sn (Şekil 3A) bir hızda hareket eden bir retrograd taşınacak gözlenmiştir. GFP veya MCherry etiketli BDNF BDNF aksonal hareketini izlemek için kullanılır olmuştur. Büyük dezavantajı, tek bir molekülün çalışmaları için yeterince parlak değil olmasıdır. Ayrıca, her iki anterograd ve retrograd BDNF hareketlerinin bulunması zor retrograd olarak taşınan, BDNF bir nörotrofinin / reseptör kompleksi olup olmadığını değerlendirmek için yapar. Bu videoda, biz birincil nöronları kullanarak mikroakışkan odaları QD-BDNF canlı ticaretini incelemek için kullanılan teknikleri göstermek. Ultrabrightness ve kuantum noktaları mak mükemmel fotostabilitees mümkün BDNF ulaşım uzun vadeli izleme gerçekleştirmek için. Bu teknikler, MS, HD ve diğer nörodejeneratif hastalıklar aksonal çalışmalar fonksiyonunun arttırılması için kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF nöronal hayatta kalmasını, farklılaşmasını ve fonksiyon bir çok alanda önemli bir rol oynar. Akson yapısal ve fonksiyonel açıkları giderek Alzheimer hastalığı (AH) ve Huntington hastalığı (HD) olmak üzere nörodejeneratif hastalıklar, erken bir özelliği olarak görülmektedir. Henüz tam olarak açıklanamamıştır aksonal yaralanma kaynaklı olduğu mekanizma (ler) idir. Bu biyolojik olarak aktif bir üretmek için yeni bir teknik gelişme rapor BDNF aksonal taşınması takip etmek için kullanılabilir BDNF (mBtBDNF) monobiotinylated. Kuantum nokta-etiketli BDNF (QD-BDNF) mBtBDNF kuantum nokta 655 konjuge tarafından üretildi. Mikroakışkan cihaz nöron hücre gövdeleri aksonlar izole etmek için kullanılmıştır. Aksonal bölmesine QD-BDNF eklenmesi akson BDNF ulaşım canlı görüntüleme izin verdi. Biz, QD-BDNF yaklaşık 1.06 mm / sn bir hızda hareket azından çok az duraklar, retro-esas olarak sadece hareket ettiğini gösterdi. Bu sistem beni araştırmak için kullanılabilirAH veya bozulmuş HD aksonal fonksiyonu gibi başka dejeneratif bozukluklar chanisms.

Üretim ve biyolojik olarak aktif mono-biyotinlenmiş BDNF saflaştırılması Bir AviTag dizisi (GGGLNDIFEAQKIEWHE) ile birleştirilir BDNF sentezleme vektörü, daha önce yayınlanan bir protokolün 10 ile tespit edildi. Tam uzunluklu füzyon proteininin moleküler kütlesi yaklaşık 32 kDa (olduğu kestirilmiştir <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ) 18 kDa (Şekil 1A) tahmin edilen mo...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF, bir nörotrofik faktör, bir aksonal taşınması birçok nöron popülasyonlarının hayatta kalma ve fonksiyonu için kritik öneme sahiptir. Bazı dejeneratif bozukluklar aksonal yapı ve fonksiyon bozulması ile işaretlenmiştir. Biz birincil nöronları kullanarak mikroakışkan odaları QD-BDNF canlı ticaretini incelemek için kullanılan teknikler gösterdi.

Quantum Akson Ulaştırma Gerçek zamanlı Görüntüleme İlköğretim Nöronlar BDNF Dot-etiketli

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Research

JoVE Journal

Neuroscience

15.0K Görüntüleme.  University of California, San Diego.  BDNF, bir nörotrofik faktör, bir aksonal taşınması birçok nöron popülasyonlarının hayatta kalma ve fonksiyonu için kritik öneme sahiptir. Bazı dejeneratif bozukluklar aksonal yapı ve fonksiyon bozulması ile işaretlenmiştir. Biz birincil nöronları kullanarak mikroakışkan odaları QD-BDNF canlı ticaretini incelemek için kullanılan teknikler gösterdi. 

Video: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Nöronlar Aksonal Geliştirme Kontrol Mekanik Manipülasyon

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Nörobilim

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Nöromodülasyon ve Mitokondrial Ulaştırma: Uzun Süreleri içinde Hipokampal Nöronlar Canlı Görüntüleme

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Gerçek zamanlı yönetim fMRI psikoterapi eğitimi için bir iletişim kuralı

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Motor Nöron benzeri NSC-34 Hücrelerinde Gerçek Zamanlı Nükleositoplazmik Taşınımı Ölçüm Testi

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Embriyonik Motor Nöronlarının Aksonal Projeksiyon Paterninin Görselleştirilmesi<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Mikroakışkan Odacıksistemi Kullanılarak Motor Nöron Kültürlerinde Organellerin Aksonal Taşınması

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Hayvanlarda Gerçek Zamanlı fMRI Beyin Haritalaması

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Aksonal Taşımacılığın In Vivo Görüntülemesi için Araç Kitinin Genişletilmesi

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

İn Vivo Bozulmamış Dorsal Kök Ganglialarında Primer Duyusal Nöronların Ağlarında Nöronal Toplulukların Kalsiyum Görüntülenmesi

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...