Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1. Hazırlık <ol><li> 37 ° C&#39;de ve% 5 CO2 ile ayarlanmış bir doku kültürü inkübatörü içinde, gerçek zamanlı hücre analiz cihazının istasyonu yerleştirin. , Steril koşullar altında, bir doku kültürü kaputu tüm hücre kültürü işleme ve farmakolojik tedaviler yürütmek. </li><li> Not: kültür standart koşullar ile karşılaştırıldığında farklı bir sıcaklık ayarı gerektiren Nöronal hücre çizgileri, buna göre ayarlanmalıdır. Zayıf, yapışan hücre hatları için, E-Levha 96 kuyu altındaki altın mikroelektrotla hücre etkileşimi kolaylaştırmak için kaplama maddeleri kullanılır. </li><li> (Uygun bir çözücü içinde nöro-koruyucu maddeler ya da ikinci haberci yollar inhibitörleri - dimetilsülfoksit ya da steril su) 1,000X hazır hücre kültürü tedavisi için farmakolojik bileşimlerin çözelti hazırlayın ve -20 ° C&#39;de saklayın. Aşağıdaki tedavilerinde araç olarak çözücü kullanın. </li><li> DMEM / F1 Kültürü insan nöroblastoma SK-N-SH hücreleri2 ortamının, yaklaşık% 75 bir yoğunluğa 175 cm2 yüzey ile hücre kültürü şişeleri içinde% 10 fetal sığır serumu (FBS) (çoğalma ortamı) ile takviye edilmiştir. Hücre çoğalması ve sitotoksisite tepki hızı açısından deney arasında tutarlılık tespit etmek için (yaklaşık 10 geçitlerin) aralığında bir hücre kanalı sayısı tutun. Alt geçit numaraları tercih edilir. </li><li> PBS ile yapışık SK-N-SH hücre tabakasını yıkayın ve 37 ° C&#39;de 5 dakika boyunca% 0.05 tripsin-EDTA ile tripsinize edin çözeltisi. Santrifüj, 5 dakika boyunca 170 x g &#39;de, hücreler tripsin kaldırın. 10 ml çoğalma ortamı hücreleri tekrar süspansiyon. Scepter hücre sayacı ile hücrelerin sayısı ve çoğalma ortamı ile hücrelerin sulandırarak 300,000 hücre / ml hücre sayısını ayarlayın. </li></ol> 2. Kaplama ve SK-N-SH hücrelerinin çoğalma <ol><li> E-96 Levha her çukuruna 100 ul çoğalma ortamı ilave edin ve oda sıcaklığında dengelenmeye doku kültürü kaputu 30 dakika boyunca bırakın. E-Pla takın37 ° C&#39;de CO2 inkübatöründe gerçek zamanlı hücre analiz istasyonunda 96 te. </li><li> Gerçek zamanlı hücre analiz yazılımını başlatın. Düzeni sayfa kuyular deneye dahil seçin ve düzenleme kutularını hücre tipi, hücre sayısı, isimleri ve hücre tedavisinde kullanılan kimyasal bileşiklerin konsantrasyonları hakkında bilgi girin. NOT: Bu protokolün amaçları için en az 4 replika her tedavi için tavsiye edilir. </li><li> Program yazılım sayfada deneyde, Hücre İndeksi ve her adım için yapılan taramalar arasındaki aralığı ölçüm silme sayısını seçerek dahil &quot;Adımlar&quot; belirler. Adım 1 arka plan ölçümü için önceden belirlenmiş olduğundan, hücre indeksi 96 saat boyunca her 15 dakikada ölçmek ve set Adım 2 &quot;bir adım ekleyin&quot; seçeneğini seçin. NOT: tedaviler için, hangi Hızlı kinetik ile etkiler, beklenen daha kısa aralıklarla süpürür ayarlayın. </li><li> Otomatik olarak önceden belirlenmiş Adım başlatmak için Başlat &#39;1ve ortamın arka empedansı ölçmek. Hücreler, oyuklara ilave edilir sonra bu değeri otomatik olarak her bir veri noktasında yazılım tarafından çıkartılır. </li><li> Çoğalma ortamı içinde 300,000 hücre / ml &#39;lik, daha önce 1.5 adım hazırlandı) hücre süspansiyonu kullanmaktadır. / Oyuk hücre toksisitesi / nöro çalışmalar ve iyi hücre çoğalması çalışmaları için 15,000 hücre / için 30,000 hücre. E-Plate çıkarın ve hücre toksisitesi / nöro çalışmaları için göz başına 100 ul hücre süspansiyonu ekleyin ve hücre çoğalma çalışmaları için oyuk başına 50 ul hücre süspansiyonu, 50 ul çoğalması orta ekleyin. Gözenek başına plakalanan hücre sayısı deneysel olarak her bir hücre hattı için optimize edilmesi gerekmektedir. </li><li> Yavaşça hücrelerin bile kaplanması için E-Plate 96 döndürün. Hücreler kuyuların alt eşit yerleşmek izin vermek için oda sıcaklığında doku kültürü kaputu 30 dakika için E-Plate 96 bırakın. </li><li> Gerçek zamanlı hücre analiz istasyonu E-Plate 96 takın ve zamanlama p Adım 2 başlayacakyaş. Plot sayfa ve yazılımın Hücre İndeks sayfasına ham Hücre Endeksi verilerine hücre eğrileri görüntüleyerek deney boyunca Hücre Endeksi değerleri izleyin. </li></ol> 3. Serum Yoksunluk <ol><li> 24 saat hücreleri kaplama sonra, deney duraklatmak ve gerçek zamanlı hücre analiz istasyonuna E-Plakasını 96 kaldırın. DMEM / F12, serum-içermeyen ortam ile 1,000X stoktan ikinci haberci inhibitörleri seyreltin - 200 x son konsantrasyon. Ilgili yolları inhibe etmek için, ikinci haberci yollarının 1 ul önleyicileri 30 dakika geçmeden önce sitotoksisite nörotoksisite / nöro katılımları için test edilecek olan hücrelerin tedavi edin. Istasyonuna E-Plate 96 İade ve deneysel Hücre Endeksi ölçümleri devam. </li><li> 30 dakika sonra tekrar denemenizi durdurabilirsiniz. Dikkatle yapışık hücre tabakasını bozmak için özen bir pipet (veya çok-kanallı pipet) ile e-Plate tam olarak 96 kuyu çoğalması orta kaldırmakKuyunun köşesine pipet kenarını yönlendirerek. 200 ul / serum içermeyen DMEM / F12 ortamında (serum yoksunluğu ortamı) oyuk ilave edin. Hücrelerin mekanik ajitasyon en aza indirmek için, hücre kültür ortamı hızlı bir değişiminin sağlanması. </li><li> Seyreltik bileşikleri nihai konsantrasyon x 200 olacağı şekilde, serum içermeyen DMEM / F12 ortamı ile 1,000X stoktan bunların nöroprotektif etkileri için test edilecek. 1 ul ekle bileşikleri bunların nöroprotektif etkileri, deneysel planına uygun olarak, tek tek oyuklara ikinci haberci yollarının 1 ul inhibitörleri için test edilecek. </li><li> Çoğalma çalışmaları için hücreler ortam içinde değişmez. Oyuk başına 1 ul ile tedavi etmek ve çoğalma ortamı içinde kültüre devam çoğalma ortamı içinde nihai konsantrasyon x 200 1,000X stoktan, test edilecek bileşikler seyreltin. </li><li> Önceden ayarlanmış olarak 96 saat boyunca Hücre Endeksi ölçümleri ile her 15 dk devam deney sürdürün. </li></ol> 4. Veri AANALİZ <ol><li> Nörotoksisite için / nöroproteksiyon veri Plot sayfada &quot;Saat normalleştirmek&quot; &quot;normalize Hücre Göstergesi&quot; seçerek deneyler arasındaki farklılıkları azaltmak ve farmakolojik tedavi veya orta değişiminden önce son kez noktaya Hücre Endeksi normalleştirmek. </li><li> Ilgi deney koşulları için Plot sayfasında kuyuları vurgulayın ve normalize Hücre Endeksi eğrileri çizmek için &quot;Ekle&quot; butonuna tıklayınız. Ya da zamanın bir fonksiyonu olarak normalize Celi Endeksi eğrileri gibi ikinci habercileri inhibisyon nörotoksik / nöro-koruyucu etkinin kinetiğinin dikkate alınmalıdır. </li><li> Çoğalması üzerinde bir etkisi nöro-koruyucu / nörotoksik etkisi olarak yer alıp almadığını değerlendirmek için, zamanın bir fonksiyonu olarak çoğalma ortamı içinde, test edilen ilaç tedavileri için Hücre Endeksi eğriler dikkat edin. </li><li> Sonuçların istatistiksel olarak değerlendirilmesi başlatmak için bir Excel dosyası içine Hücre Endeksi yazılım sayfadaki tüm Cep Endeksi zaman noktaları için deneysel bilgi İhracat <br/&gt; Not: İstatistiksel analizde bir ilk adım p-değeri ile, Welch&#39;in t-testi kullanmak modifiye MATLAB programları bir dizi semilogarithmically çizilen gibi, (ek kod dosyaları olarak verilmiştir) zaman ölçeğinde karşı ters ekseni kullanmaktadır. Bu programlar, bütün gerçek zamanlı bir analiz hücre deney süresi boyunca-p-değerleri tespit edilmesini sağlar ve bu nedenle daha detaylı bir istatistiksel analiz için zaman noktalarının seçimi oldukça kolaylaşmıştır. Programları hakkında ayrıntılı bilgi için ek malzeme bakın. </li><li> Belirli zaman noktalarında farklı tedavi koşullarında Hücre Endeksi değerlerindeki farklılıkların istatistiksel analizi için, dışa aktarılan Excel dosyasından ilgili zaman noktalarında seçin Hücre Endeks değerleri. Istatistiksel yazılım kullanarak bir post-hoc testi varyansın tek-yönlü veya iki yönlü analizi (ANOVA) ile seçilen zaman noktaları için Hücre Endeksi değerleri analiz edin. </li></ol>

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Geçerli video-makale için yayın masrafları &quot;ACEA Biyobilimlerdeki&quot; tarafından desteklenmiştir.

Geçerli protokol sürekli olarak, etiket içermeyen koşullar altında, nöronal hücre hatlarında bileşiklerin nöro-koruyucu / nörotoksik etkilerini değerlendirmek için ve etkisi dahil ikinci haberici yoluna daha iyi anlamak için, gerçek zamanlı bir analiz hücre yararını sunar. Sitotoksisite ve hücre çoğalması üzerindeki ilaçların etkilerini incelemek için gerçek zamanlı hücre analiz cihazının programı genel kabul görmüş olsa da, sadece birkaç çalışmalarda ...

Nöronal hücre ölümü, birçok beyin-1 ile ilişkili bozuklukların patofizyolojisinde önemli bir rol oynar. In vitro toksisite deneyleri güvenilir ve yüksek verimlilik kullanılabilirliği nörotoksisitenin mekanizmaları daha iyi anlamanıza ve ilaç geliştirme 2 terapötik aday olarak nöro molekülleri seçmenize yardımcı için çok önemlidir. Bununla birlikte, en yaygın olarak kinetik çözülmesine izin veren tek bir zaman noktasında nörotoksisite / değerlendirmek için nöro-nitro nörotoksisite assays.They olarak kullanılan bir çok sınırlamalar mevcuttur; çoğu zaman sinyal yollarına müdahale ve aynı hücre popülasyonunda ek çalışmalar sınırlamak ve genellikle bir iş gücünü gerektirir ve pek çok durumda mekanik bir bilgi sağlar yok olabilir veya bir etiket sonda kullanma. Bu çalışmada gerçek zamanlı ve altında, nöronal hücre hattında nöro-toksisiteyi ve nöro-belirlemek için, gerçek zamanlı bir empedans tabanlı hücre analiz yararlılığını göstermek etiketsizkoşulları etkilenmesinde rol ikinci haberci yollara analizi yoluyla aşağı mekanizmaları içgörü sağlamak ve. Önceki çalışmalar, standart teknikler ile karşılaştırıldığında, 3,4,5,6 sitotoksisite gibi hücre hatları, hücre çoğalması üzerindeki etkisini belirlemek için, gerçek zamanlı hücre analiz geçerliliğini teyit etmiştir. Örneğin, bir iyi bir korelasyon bazal koşullar altında çoğalması ve HeLa hücrelerinde 3, iki farklı toksik paradigmaların ardından çeşitli zaman noktalarında standart hücre canlılığı, WST-1 deneyinin okumalar ve hücre endeksi değerleri arasında gözlenmiştir. Celi Endeksi ölçümleri ile ve standart olarak kullanılan sülforodamin B (SRB) 4 deney sırasında mikrotübül dengeleyici paklitaksel ile tahrik A549 ve MDA-MB-231 hücreleri çoğalması ve sitotoksisitesinde çok benzer sonuçlar göstermiştir. Ölümsüzleştirilmiş hipokampal nöronlarının nöronal hücre hattı HT-22 Hücre indeksi ölçümlerinin yetenekleri t doğrulanmıştıro yaygın olarak kullanılan 3-(4,5-dimetiltiazol-2-yl) karşı, hücre proliferasyonunu, glutamat sitotoksisitesinin ve sitoproteksiyonunu 2,5-difeniltetrazolyum bromür (MTT) deneyi 5 algılar. Aynı çalışmada MTT deneyi sonuçları ve Hücre Endeksi ölçümleri de pan-kaspaz inhibitörü Qvd 5 ile büyüme faktörleri yoksunluk ve sitotoksisite kurtarılması sonrasında nöronal progenitör hücrelerin çoğalması, sitotoksitesi ölçme pozitif ilişkilidir. Vandetanib (vasküler endotel büyüme faktörü reseptör ve epidermal büyüme faktörü reseptörü inhibitörü) ile NIH 3T3 hücrelerinde olduğu sitotoksisitesi, hücre endeksi değerleri ya da nötr kırmızı alım deneyi 6 ölçülen benzer sonuçlar göstermiştir. Son zamanlarda, serotonin 2A nöro-koruyucu etkisini değerlendirmek için gerçek zaman hücre analiz sistemi kullanılmaktadır (5-HT2A) alıcı agonisti (±) -2,5-dimetoksi-4-iodoamphetamine hidroklorür, nöronal bir hücre hattında (DOI) ( SK-N-SH hücreleri) ve Seco katılımı açısından tarandıgözlenen nöro-7 kimyasal inhibisyon etkinin izlenmesi sayesinde nci mesaj yolakları. İlginç bir şekilde, 5-HT2A reseptör yaygın ve farklı bir ikinci haberci yolları 8 hem de aktive edebilmektedir (Doi ve lisurid, sırasıyla gibi) ve halüsinojen nonhallucinogenic hem agonistler hem de vardır. Sunulan tekniğin avantajları, gün içerisinde hücre sağkalım üzerine gerçek zamanlı bilgi toplamak için nörokoruma çoğalması etkilerin olası katkısını değerlendirmek için, ve optimal zamanı seçmek için, ikinci-haberci yollar dahil belirginleştiren için izin vardır Aynı hücre popülasyonu üzerinde ek uç nokta çalışmaları için. Mevcut protokolde iş akışının şematik bir diyagramı Şekil 1&#39;de sunulmuştur.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

Birçok beyin ile ilgili bozukluklar kendi patofizyolojisinde rol nöronal hücre ölümünü var. Uyuşturucu ve nöro / nörotoksisitenin moleküler mekanizmaları anlamak ve potansiyel ilaç gelişimini kolaylaştırabilir yardımcı olacağını çıkıyor aşağı yollar nöroprotektif veya nörotoksik etkilerini incelemek için in vitro modellerinde geliştirilmiş. Ancak, birçok mevcut in vitro toksisite deneyleri büyük sınırlamaları var - çoğu değil zaman ders ve etkisi kinetiğini gözlemlemek için izin, tek bir zaman noktasında nörotoksisiteyi ve nöro değerlendirmek. Ayrıca, gerçek zamanlı olarak nöro katılan aşağı sinyal yollar hakkında bilgi toplamak için fırsat büyük önem olurdu. Mevcut protokolde, etiket içermeyen ve gerçek zamanlı iklimlendirme altında, nöronal hücre hattında serotonin 2A (5-HT2A) reseptör agonistlerinin nöro-koruyucu etkisini araştırmak üzere bir reel-zamanlı empedans tabanlı hücre analiz kullanılmasını tarif ederempedans ölçümleri kullanarak iyonları. Ayrıca, ikinci haberci yollarının inhibitörleri nöro-koruyucu etkisinin dahil alt molekülleri tanımlamak için kullanılabileceğini göstermektedir. Ayrıca hücre proliferasyonu üzerinde bir etkiye sahip bir gözlenen nöro-koruyucu etkisine katkıda olup olmadığını belirlemek için bu tekniğin programı açıklanmaktadır. Sistem, kuyuların alt yüzeyi üzerinde altın mikro-elektrot diziler alternatif hücre sensör olarak işlev gören ihtiva e-Plakalar olarak adlandırılır özel mikroelektronik plakalar kullanır. Empedans okuma yapışık hücreler, hücre canlılığı, morfolojisi ve yapışma sayısına göre modifiye edilir. Hücre Endeksi olarak adlandırılan bir boyutsuz parametre elektrik empedans ölçümleri elde edilir ve hücre durumunu temsil etmek için kullanılır. Genel olarak, gerçek zamanlı empedans tabanlı hücre analiz fazla katkı, gerçek zamanlı, nöro-korumanın ve nörotoksisite etiketsiz değerlendirmesi, haberci ve ikinci yollar katılımının değerlendirilmesine izin verirtedavi ilaçlarını seçilmesi için in vitro nöro-koruyucu potansiyel bileşiklerin ayrıntılı ve yüksek verim değerlendirmesi.

<html> , Serum yoksunluğu, gerçek-zaman hücre analiz sürekli izlenebilir Celi endeksi değerlerine azalmaya yol açar Nörotoksik uyarıcı hücrelere sunulan teknik ve spesifik nörotoksik uyaran ve testlerde kullanılan hücre türüne bağımlı olan dinamikleri ile gerçek zamanlı olarak takip edilebilir Celi endeksi değerleri bir azalmaya yol açmaktadır. Şekil 2 &#39;de artış göstermektedir SK-N-SH hücreleri bir plato (yeşil hat) ve mahrumi...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Bir Nöronal Hücre Line Neurotoxicity ve nöro ilgilendim Moleküler Pathways Delineate bir aracı olarak Real-Time Empedans tabanlı Cell Analyzer

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

real-time-impedance-based-cell-analyzer-as-tool-to-delineate

Research

JoVE Journal

Neuroscience

11.3K Görüntüleme.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Video: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Çoklu Ölçeklerde Sayısallaştırılmış Nöronal Morfoloji Otomatik Sholl Analizi

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Nörobilim

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Nöronal atalarıdır ve Nöronlar Verimli türetilmesi

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Eşzamanlı Elektroensefalografi, Kemirgen Serebral Korteks Laktat Konsantrasyonu ve Nöronal Faaliyet Optogenetic Manipülasyon Gerçek zamanlı ölçüm

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

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

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

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

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

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

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

İskemik Hücre Ölümü Pathways Eğitim için seks Katmanlı Nöronal Kültürleri

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Nöronal Ateşleme Oranlarının Manipülasyon tarafından Astrositik reseptörleri Plastisite Inducing

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Kombine Metodolojileri kullanan Akson Dallanman ve Nöronal Morfogenez sırasında Plazma Membran Teslimat Rolü tanımlayın

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparçacık Neurotoksite Belirleyen Bir Vekil Biosensor olarak mikroglia

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

Rahimde Elektroporasyon Nöronal alt popülasyonlar heyecanlanma ve Tek hücreli Bağlantı Eğitim Yaklaşımları

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...