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><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-&amp;beta;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>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>&amp;mu;-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

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Nöronlar Foto-Stimülasyon için uygun fiyatlı LED Diziler

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Nörobilim

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Kronik Yetişkin doğmuş Koku Nöronlar Seçici Viral İletimi In vivo Optogenetic Uyarım

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Beyin Dokusu Kronik Optogenetic uyarılması için Fiber-optik İmplantasyon

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Bireysel piramidal nöronlar High Fidelity optogenetic Kontrolü için Bir Yöntem<em&gt; In vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

Gelişmiş nörokimyasal algılama için altın Nanopmakale modifiye karbon fiber Mikroelektrotlar

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

Kimya

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Altın nanorods İçeren Hücresel Araçların Hazırlama ve foto akustik Analizi

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Biyomühendislik

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Konjuge Polimerler tarafından Hücreler Elektrik Etkinliği Yaşam Optik Kontrol

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Kemirgen Santral Sinir Sistemi vivo optogenetic Stimülasyon içinde

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

İşitsel Sinir optogenetic uyarılması

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Altın Nöronal Hücrelerde optik Uyarım Nanorod destekli

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