Yazarlar kısmen çekimler sırasında ona yardım için Sheffield ve Bayan Jaimee Mayne Üniversitesi bu araştırmaları misafirini ağırlayan seyahat finansman desteği için NanoVentures Avustralya ve Prof. John Haycock kabul etmek istiyorum.

1. Au NR&#39;ler Hazırlık Not: Au NR&#39;ler tarifleri 16 bir dizi ile sentezlenebilir, ya ticari satıcılardan satın alınabilir. <ol><li> UV-Vis spektroskopisi yoluyla, 0.5-2 nm çözünürlüğe sahip 300 nm 1.000 ila emilim değerleri kaydederek Au NR çözeltisinin ilk optik yoğunluk (OD) ölçün. Çözeltinin hacmi mevcut küvet ile kullanılmak üzere değişir. </li><li> Uygun bir teknik 17 ile ilk NP molar konsantrasyonu (örneğin UV-Vis spektroskopisi, tek parçacık indüktif eşleşmiş plazma kütle spektrometresi, transmisyon elektron mikroskobu) değerlendirmek veya satıcı tarafından sağlanan konsantrasyon değerleri kullanın. </li><li> En iyi tekrarlanabilirlik için, 1. = OD ulaşmak test edilen örneklerin tüm OD sabit tutmak için ilk Au NR örnek seyreltilmesi ile 5 ml stok çözelti hazırlayın. Seyreltme çözücü bileşimi için ticari satıcının protokolünü izleyin. Emin değilsenizDeiyonize su kullanın. </li><li> Iki kez 15 dakika süreyle, Au NR solüsyonun 1 ml&#39;lik 7,800 x g&#39;de santrifüjleyin çözeltiden herhangi bir kimyasal fazlasının ayrılması için. Santrifüj çevrimi kuvvet ve zaman (örneğin 20 dk 5.600 × g) 18 değişebilir. </li><li> Süpernatantlar çıkarın ve deiyonize su içinde NRs yeniden askıya. Yeniden partikülleri çözelti içinde agrega oluşturacak edebileceğiniz gibi, en iyi sonuçlara sahip günlük onları hazırlamak. Alternatif olarak, 1 hafta daha uzun süre buzdolabında saklayın. Donma yok. </li><li> Hücre kültürü koşulları altında kullanmadan önce, 5 dakika boyunca, Au NR çözeltisi sonikasyon ve sonra, (mW m ∙ en az 400 daha -2 254 nm&#39;de UV radyasyon şiddeti), 30 dakika boyunca UV ışığı ile sterilize edilir. </li></ol> 2. NG108-15 Nöronal Hücre Hattı Kültür ve Farklılaşma <ol><li> Hücre kültür ortamı için, steril Dulbecco tadil edilmiş Eagle ortamı (DMEM) cenin dana serumu (w / v) (FCS) ihtiva eden% 10,% 1 (ağırlık / hacim), L-glutamin,% 1, 500 ml hazırlamak(Ağırlık / hacim) penisilin / streptomisin ve% 0.5 (ağırlık / hacim) amfoterisin B Not: Takviyeler, -20 ° C&#39;de saklandı, bölünmüş ve gereken gün ortama ilave edilebilir. Hücre kültür ortamı, 1 aylık bir süre için steril bir durumda frigorifik edilebilir. </li><li> Hücre farklılaşması ortamı için% 1 ihtiva eden steril DMEM 50 ml hazırlamak L-glutamin,% 1 (ağ / h), penisilin / streptomisin ve% 0.5 (ağırlık / hacim) amfoterisin B (a / h) </li><li> (37 ° C&#39;de% 5 CO2), nemli bir atmosfere sahip bir kuluçka makinesi içinde polistiren T75 şişeleri içinde hücre kültür ortamı, 10 ml NG108-15 nöronal hücreler büyür. Normalde, 3-4 gün içinde hazır olmak için her şişede 1.5-2 × 10 5 hücre tohum. Her iki günde hücre kültürü ortamı değiştirin. Not: deneyler için geçit 21 daha eski hücreleri kullanmayın, genetik sürüklenir veya varyasyonunu önlemek için. </li><li> Zaman% 70-80 konfluent kültüründe, sıcak taze hücre kültür ortamı ile orta değiştirmek. Mekanik hafifçe Knoc hücreleri ayırmakKral birleşen Şişenin alt. Tripsin kullanmayın. </li><li> Santrifüj 600 x g&#39;de ve 5 dakika boyunca hücre süspansiyonu yeniden askıya sıcak hücre farklılaştırma ortamının 2 ml hücre pelletini. </li><li> Hücre farklılaştırma ortamının 200 ul doku kültürü polistiren 96 oyuklu bir plaka içerisinde tohum 2 x 10 4 hücre / cm2. % 5 CO 2/37 ° C&#39;de 1 gün boyunca deney inkübe edin. </li><li> 2 gün Au NR çözeltisi 10 0 1 x -4,2 9 10 x 3.2 arasında parçacık / ml ilave edilir ve ek 24 saat için inkübe edilir. Kontrol deneyleri için parçacıkları katmayın. Not: Alternatif bir kontrol olarak, bir iyi farklılaşmış zirve absorpsiyon dalga boyuna sahip Au NPler, karşılaştırma amacıyla 14 için kullanılabilir. Bir daha tutarlı hücre davranışı için, sabit hücre yoğunluğu tutmak ve hücre ekimi öncesinde kuyu yüzeyi değişiklik yok. </li></ol> 3. akson akıbet Geliştirme <ol><li> ÇiftBir tek modlu fiber optik lazer (sayısal açıklık = 0.13) ve (YP konnektörleri uygun ve yaygın mevcuttur) fiber konnektörü ile sona erdirebilir. Standart bir elektrik metre ile çıkış lazer gücünü ölçün. En etkili sonuçlar elde etmek için, NR&#39;lerin plazmon rezonans zirvesine lazer dalga boyu tepe maç. </li><li> Günde 3 Aşağıdaki NR kuluçka üzerinde, iyi FC konnektörünü düzeltmek. Farklı lazer güçler için, sürekli bir dalga 1 dakika için oda sıcaklığında örnekleri ve kontrol ışın tedavisi. Kültür% 5 CO 2/37 ° C&#39;de 3 gün daha devam izin verin. 3 bağımsız ölçümün en az bir lazer ışınlama tekrarlayın. Not: Farklı ışınlama süresi ve darbe frekansları, uygulamaya bağlı olarak, seçilebilmektedir. </li><li> Işın çapı ve lazer ışınım açısından lazer karakterize (W ∙ cm -2). <ol><li> Standart tek modlu fiber için, NA = n ∙ sin θ kullanınN Kullanılan ortamın kırılma endeksi ve θ elyaf çıkan ışık konisinin yarım açı olduğu (Şekil 1A bakınız). R ışın yarıçapı trigonometri, r = tan θ ∙ d ve d lif ve numune arasındaki mesafedir. FC bağlayıcı bu nedenle, bir d mesafesinde örnek = 2.70 ± 0.20 mm aydınlatıcı, iyi çapı ile eşleşir. Işık, r = tan (sin-1 (NA / n)) veren d ∙ (n = 1.33), su içinde lif çıkar. </li><li> İkinci denklemi kullanarak, lazer ışını yarıçapı, ilgili ışın alanı ve hedef ortalama lazer ışınlama (ışın alanına bölünmesiyle lazer güç) hesaplamak (Şekil 1B örnek grafiğe bakınız). Bu değerler ışıklı alana ortalama parlama temsil eder. </li><li> Hata yayılma genel teorisi ile ölçümler için hataları değerlendirin. Not: mesafe between FC konektörü ve örnek uygun yazılım (örn ImageJ) kullanarak görüntünün deneysel düzenleme ve post-processing fotoğrafları ile ölçülebilir. </li></ol></li><li> 5. günde, deneylerden elde edilen hücre farklılaşması orta kaldırmak ve% 3.7 10 dakika süre ile (h / h) formaldehid çözeltisi ile örnekleri saptamak, daha sonra% 0.1 20 dakika boyunca (h / h) Triton X-100 ile hücrelerin geçirgen. </li><li> (W / v), 60 dakika süre ile numuneler, büyükbaş hayvan serum albümini (BSA) bağlayıcı siteleri girmemiş protein bloke etmek için% 3 ekleyin. 4 ° C&#39;de anti-βIII-tübülin gece boyunca (PBS / ml 5 ug BSA% 1 ile takviye edilmiş) için numuneler etiketlenir. </li><li> PBS içinde% 1 BSA içinde 0,4-2 mg / ml &#39;lik bir konsantrasyon ile (örneğin, TRITC-konjuge edilmiş anti-fare IgG antikoru) uygun bir ikincil antikor ile karanlıkta 90 dakika süreyle inkübe hücreleri. 10 dakika boyunca DAPI (0.1 ug / ml de-iyonize edilmiş su), hücre, çekirdeklerini etiketleyin. Not: Antikor çekimi ve konsantrasyon MIGht şirket protokolüne göre değişir. Iki kez boyama safhasından sonra 5 dakika boyunca PBS ile yıkayın örnekler. <img alt="Şekil 1," src="/files/ftp_upload/52566/52566fig1.jpg" /></li><li> En az bir 20 × objektif kullanarak Epifloresans veya konfokal mikroskobu ile görüntü örnekleri. Mikroskop sekonder antikor buna göre filtreleri seçin. Bir DAPI filtre seçin (λ EX = 358 nm; λ EM 488 nm =) hücre çekirdekleri görselleştirmek için. </li><li> Değerlendirerek fotoğrafları analiz: i) maksimum nörit uzunluğu (kayıt hücre gövdesi başlangıcına akson ucundan uzunluğu), ii) nöronal hücre başına nöritlerin sayısı () hücre başına nöritlerin tüm Özetle iii) nöritleri olan hücrelerin yüzdesi (pozitif lekeli çekirdeğe sahip olan hücrelerin toplam sayısı) 19 ile βIII-tubulin sentezleyen hücrelerin toplam sayısına bölün. </li></ol> 4. Hücre içi Ca 2+ Görüntüleme Lazer kaynaklı <ol&gt; <li> Pluronic F-127 stok çözeltisi (w / v), susuz dimetil sülfoksit (DMSO), 10 ml çözünen 2 g çözülmesiyle bir% 20 hazırlayın. Çözünürlüğünü arttırmak için yaklaşık 20 dakika boyunca, 40 ° C&#39;de, Pluronic F-127 çözeltisi ısıtın. Oda sıcaklığında saklanan önceden hazırlayın. </li><li> ND, inkübasyonu takiben 3. günde, bir dengelenmiş tuz solüsyonu hazırlamak (BSS, 135 mM NaCI, 4.5 mM KCI, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glükoz ve 10 mM HEPES, pH 7.4) ve 5 ile desteklenmiş DMSO içinde Fluo-4:00 ve% 0.1 uM (a / h), Pluronic F-127 çözeltisi eklenmiştir. Not: BSS çözümü önceden hazırlanmış ve 1 aylık bir süre için buzdolabında olabilir. Deney gününde takviyesi (Fluo-4:00 ve Pluronic ™ F-127) ilave edin. </li><li> Hücre farklılaşması orta çıkarın ve desteklenen BSS çözümü ile değiştirin. Bir ters konfokal mikroskop ile en iyi sonuçları elde etmek için, iyi bir mikro-slayt hücreleri kuluçkaya yatmaktadır. </li><li> Fluo-4:00 ile yük NG108-15 hücreleri% 5 CO 2/37 ° C&#39;de karanlıkta 30 dakika karıştırıldı. Inkübasyon süresi sonrasında, herhangi bir hücre dışı yüksüz floroforu kaldırmak için BSS ile iki kez numune yıkayın. </li><li> Çift bir tek modlu fiber optik, lazer ve standart teknikler kullanılarak ucu bölmektedir. (Uç mikroskopi incelenmesi üzerine lif eksenine dik ve düz olmalıdır, yani), yüksek kalitede bir yüzey sağlamak için, bir optik mikroskop altında elde edilen uç dikkate alınmalıdır. <ol><li> Standart bir elektrik metre ile çıkış lazer gücünü ölçün. Bir fiber optik tutucu içine ışık teslimat lif yerleştirin ve bir micropositioner takınız. Brown ve ark bakın. 20 fiber optik hazırlama ve konumlandırma hakkında daha fazla bilgi için. Dikkat: (örn lazer ışınına doğrudan bakmayın taşıma sırasında lazer güvenlik gözlük, diğer laboratuvar kullanıcılara kaçak ışık maruziyeti önlemek) lazer gücünün ölçümü sırasında lazer güvenliği için genel kurallara uyun &lt;./ Li&gt; </ol></li><li> Optik modülasyonu izlemek için taşınabilir lazer bir osiloskop bağlayın. Değişken frekans (0.5-2 Hz) ve darbe uzunlukları (20-100 ms) olan bir ikili sinyali kullan. Paviolo ark. 13 gösterilen kurulum sonrasında mikroskop için transistör-transistör mantığı (TTL) olarak modülasyon sinyali kullanarak lazer girişini bağlayın. <ol><li> Bir ters konfokal mikroskop altında hücrelerin yerleştirin ve uzak iletim aydınlatma modunda hedef hücrenin ışık teslimat fiber 250 ± 50 mikron yerleştirin. Bu mesafede, hedefe ışın yarıçapını hesaplamak için 3.3.1 sunulan denklemleri kullanın. </li></ol></li><li> Içselleştirilmiş Fluo-4:00 boya heyecanlandırmak için argon-iyon lazeri (488 nm) kullanın (λ EX = 494 nm; λ EM 516 nm =) ve endocytosed NR&#39;lerin uyarılması için senkronize LD. Zaman serisi tarama toplayarak en az 40 x objektif kullanılarak, örneklerin ve kontrollerin oda sıcaklığında görüntüleme gerçekleştirmek256 256 x piksel / en az 4 Hz frekans ile gidiş-dönüş modunda çerçeve çözünürlüğünde. Herhangi bir argon-iyon lazer girişimi (bazal gürültü) tanımlamak için herhangi bir LD uyarma olmadan kayıt yapın. </li><li> Uyarlamalı ağırlıklı cezalandırılmış en-küçük kareler algoritmaları kullanarak 21 verilerden arka plan çıkarın. Zamanın bir fonksiyonu olarak bağlı Ca2 + varyasyonları çizilir. Bir seviyede temel gürültü (3σ) üç kez standart sapması eşikleme görüntü sonrası işleme yazılımı kullanarak floresan yoğunluğu geçici zirveleri analiz. </li></ol>

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The authors have no competing interests to disclose.

Bu sunumda belirtilen protokoller kültüre, farklılaştırmak ve optik dışsal emici kullanarak nöronal hücreleri uyarmak nasıl açıklar. NR özellikleri (örneğin boyutlar, şekil, plazmon rezonans dalga boyu ve yüzey kimyası) ve (vb dalga boyu, darbe uzunluğu, tekrarlama oranı, gibi) lazer stimülasyon parametreleri farklı deneysel ihtiyaçlarına uygun değiştirilebilir. Hücre davranışı üzerindeki etkisi standart biyolojik tahliller ve malzemeler kullanılarak izlenebilir. Genel y...

Son zamanlarda yapılan çalışmalar, su (dalga boyu&gt; 1400 nm) kızılötesi ışık emme ile bağlantılı geçici ısıtma sinir dokusu 1 kardiyomiyositlerde 2 hücre içi kalsiyum geçişlerdeki aksiyon potansiyelleri uyarılması için kullanılabileceğini göstermişlerdir. kızılötesi ışığın kullanımı nedeniyle potansiyel alansal çözünürlük, nöral protez uygulamaları için büyük ilgisini çekmiştir, genetik doğrudan doku ile temas, uyarım eserler minimize, ve ihtiyaç çıkarılması eksikliği (uyarılması öncesi hücreleri değiştirmek ) Optogenetik gerekli olduğu şekilde 1. Tüm bu avantajlar rağmen, son zamanlarda geliştirilen termal modeller hedef doku / hücre birden fazla uyaran siteleri ve / veya yüksek tekrarlama oranları 3,4 kullanılan kümülatif ısıtma etkileri, etkilenebilir önerdi. Bu sorunlara cevap olarak, araştırmacılar dışsal Akustik kullanma potansiyeli tanıdısinir uyarımı için mektir dokuda daha lokalize ısıtma etkiler üretmek için. Huang ve ark., Uzaktan bir radyo frekansı manyetik alanı 5, HEK 293 hücrelerinde sıcaklığa duyarlı TRPV1 kanalları etkinleştirmek için süperparamanyetik ferrit nanopartiküller kullanarak bu prensip gösterdi. Bu teknik, (manyetik alan doku ile nispeten zayıf etkileşim) daha derin bir penetrasyon için izin vermesine rağmen, tepkiler sadece oldukça biyonik cihazlar 5 ihtiyaç milisaniye süreleri daha saniye süre boyunca kaydedildi. In vitro siyah mikro-parçacıklar ile sıçan kortikal nöronların Benzer şekilde, Farah ark. Gösterdi elektriksel stimülasyon. Onlar potansiyel hızlı tekrarlama oranları 6 için izin μJ aralığında us ve enerjilerin yüzlerce sipariş üzerine darbe süreleri kullanılarak uyarılması hücre düzeyinde hassasiyet gösterdi. dış emicilerin kullanılması da uyarılması için tatbik edilmiştirin vitro morfolojik değişimler. Ciofani ark. Ultrason 7 heyecan piezoelektrik bor nitrür nanotüpler kullanılarak nöronal hücre akıbet bir ~% 40 artış gösterdi. Benzer şekilde, PC12 hücrelerinde endocytosed demir oksit nanopartiküller nedeniyle demir oksit 8 hücre yapışma moleküllerinin aktivasyonuna, doza bağımlı bir şekilde nörit farklılaşmasını arttırmak için rapor edilmiştir. Son zamanlarda, sinir uyarımı yardımcı olmak için dışsal emiciler ilgi de altın nanopartiküllerin kullanımı (Au NPS) odaklanmıştır. Au NPler etkili bir plasmonik tepe lazer ışığı absorbe ettiği ve ısı 9 şeklinde çevreleyen ortam içine dağıtmak yeteneğine sahiptir. 10 - partikül şekilleri tüm arasında, altın nanoçubuklar optik soğurma (Au NR&#39;ler) elverişli (NUR, 750-1,400 nm arasında dalga boyu yakın kızılötesi) Biyolojik dokuların terapötik pencere eşleşir. Ayrıca, cont içindesinir stimülasyon ext, Au NR&#39;lerin kullanımı nispeten olumlu biyouyumluluk ve yüzey fonksiyonlandırmalar seçenekleri 11 geniş bir yelpazede sunmaktadır. Son zamanlarda yapılan çalışmalar farklılaşması üzerinde bir uyarıcı etkisi NG108-15 nöronal hücrelerin 12 Au NR&#39;lerin sürekli lazer maruz sonra indüklenebilir göstermiştir. Benzer şekilde, hücre içi kalsiyum geçici değişken frekansı ve darbe ile modüle edilmiş lazer ışını 13 uzunlukları sonra Au NR&#39;lerin ile kültürlenmiştir nöronal hücrelerde kaydedildi. Hücre membran depolarizasyon aynı zamanda, spiral gangliyon nöronları 14 primer kültürlerinde Au NR&#39;lerin NIR lazer aydınlatma sonra kaydedildi. ışınlanmış Au NR&#39;lerin ile in vivo uygulamada ilk sadece son zamanlarda kanıtlanmıştır. Eom ve arkadaşları onların plasmonik zirvesinde Au NRS maruz kalan ve bileşik sinir aksiyon potansiyellerinin (CNaPS) genliği bir altı kat artış ve sıçan siyatik sinir stimülasyon eşiği üç kat azalma kaydedildi. trzenginleştirilebilir yanıtı NR plasmonik tepe 15 uyarılması kaynaklanan lokal ısıtma etkilerine bağlandı. Bu yazıda, Au NR&#39;lerin ile kültüre NG108-15 nöronal hücrelerin lazer stimülasyon etkilerini araştırmak için protokoller belirtilir. Bu yöntemler, standart biyolojik teknikler ve malzemeler kullanılarak in vitro hücre popülasyonlarının saçmak için basit, ama güçlü, bir yol sağlar. Protokol güvenli bir şekilde çalışmasını ve tekrarlanabilir dengelenmesini sağlayan fiber lazer diyot (LD) dayanmaktadır. Au NR örnek hazırlama ve lazer ışınlama yöntemi daha spesifik sentez ve kültür protokolleri sırasıyla bilinen şartıyla, farklı parçacık şekil ve nöronal hücre kültürlerine uzatılabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Au NR</td>
			<td>Sigma Aldrich</td>
			<td>716812</td>
			<td></td>
		</tr>
		<tr>
			<td>NG108-15</td>
			<td>Sigma Aldrich</td>
			<td>8811230</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6546</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life Technologies</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;III-tubulin</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated anti-mouse IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>T5393</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P6867</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Varian Medical Systems Inc.</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Eppendorf</td>
			<td>Mini Spin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic bath</td>
			<td>Unisonics Australia</td>
			<td>FPX 10D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Kendro</td>
			<td>Hera Cell 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Hettich</td>
			<td>Rotofix 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diode</td>
			<td>Optotech</td>
			<td>780 nm single mode fibre - coupled LD</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>780 HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Coherent</td>
			<td>Laser Check</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>http://rsb.info.nih.gov/ij/index.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Axon Instruments</td>
			<td>ImageX-press 5000A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-slide well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy Ltd.</td>
			<td>LSM 510 meta-confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS210</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Burada tarif edilen Protokolleri 1, 2 ve 3 kullanılarak, farklılaşması üzerinde bir uyarıcı etkisi Au NP ile kültüre NG108-15 nöronal hücrelerde gözlenmiştir laser (Au NR&#39;ler, poli (sitiren) Au NRS ve silis kaplı Au NR&#39;ler -kaplı) 1.25 ve 7.5 W arasındaki poz · cm -2. RhodamineB etiketli Au NR&#39;lerin Konfokal görüntüler partiküller inkübasyonun 12 1. gününden itibaren içsel olduğunu göstermiştir. lokalizasyonları alım tercih mekanizması hücre gövdesi memb...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Altın Nöronal Hücrelerde optik Uyarım Nanorod destekli

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K Görüntüleme.  Swinburne University of Technology. This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Video: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

Varsayılan Modu Ağ inceleyin Transkraniyal Manyetik Uyarım ve fMRI birleştiren

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Nörobilim

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Fragile X Sendromu ve Otizm Bireylerde Plastisite, Mechasnism keşfedin Theta-Burst Protokolü kullanma

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS Aygıt: FDA yürütülmesi Depresyon Tedavisinde Protokolü Onaylandı

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Nöronlar bir Genetiği kodlanmış doğal olmayan Amino Asit kullanarak Nöronal Protein Optik Kontrol

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Mikro Elektrod Dizilerinde Nöronal Hücre Kültürlerinin Uyarılmasına Ağ Yanıtına Zamana Bağlı Artış

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Optik Koherens tomografi: Fare retina Ganglion hücreleri Vivo içinde görüntüleme

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Kronik Transkraniyal elektriksel stimülasyon ve Sıçanlarda Intracortical kayıt

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Fare Embriyonik Kök Hücre in vitro nöronal farklılaşma

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Kan Türevi Nöronal Hücrelerin GM'den Arındırılmış Üretimi

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...