Bu çalışma Radyasyon Onkolojisi, Alabama-Birmingham Üniversitesi Kapsamlı Kanser Merkezi&#39;nde, Dövüş Çocuk Kanser Vakfı ve Gabrielle&#39;in Angel Vakfı (ESY için.) Bölümü IMPACT Ödülü ile desteklenmiştir.

1. Hücre Kültürü <ol><li> Kültür nöronal hücrelerin ve onları gerektiği gibi davranın. </li><li> 15 ml&#39;lik tüp içine hasat hücreleri: aspiratı ortam, fosfat ile durulama (PBS, kalsiyum ve magnezyum serbest) tamponlu tuzlu su, 15 ml tüp içinde toplamak ve uygun bir serum ihtiva eden ortam ile nötralize tripsin, tripsin ekleyin. </li><li> 5 dakika boyunca 1000 xg Spin. </li><li> Medya aspire. </li><li> PBS içinde hücreler yeniden süspanse edin. </li><li> 5 dakika boyunca 1000 xg Spin. </li><li> Taze PBS içinde medya ve tekrar süspansiyon hücreleri aspire. </li><li> Bir hemasitometre veya tercih ettiğiniz hücre sayıcı kullanarak hücreleri saymak. </li><li> Hücre numuneleri deney başlamadan hemen önce hazırlanmalıdır. </li><li> Tüm numuneler, morötesi ışık DNA hasarı önlemek için ya da koyu sarı ışık altında ele alınmalıdır. </li></ol> 2. Comet Assay 1. Slayt Hazırlama <ol><li> % 1 agaroz (1X Tris Base, Borik asit, EDTA, TB ve 1 g / 100 ml eritinE) 3 dakika süreyle mikrodalga tüm granül kayboluncaya kadar. </li><li> Dip erimiş agaroz içine slaytlar ve bir tarafı silin bir Kimwipe ile temizleyin. Şeffaf bir film kuruması için agaroz izin verin. Bu muhafaza edilebilir ve önceden kızak içinde yapılabilir. </li></ol> 2. Nötr Comet Assay <ol><li> Kullanımdan önce en az 1 saat boyunca 4 ° C sıcaklıkta erimesi Chill çözeltisi (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris baz,% 1 sodyum lauril sarkosinat, ve% 1 Triton X-100, pH 10). </li><li> 2.2.2. Tüm granül kayboluncaya kadar bir mikrodalga% 1 düşük erime noktası agaroz (1X Tris Bankası&#39;ndaki 1 g / 100ml, Borik asit, EDTA) 3 dakika eritin. Agaroz kuyruklu kuyruk yapay indüksiyon önlemek için ° C&#39;de bir su banyosu içinde 37 kadar soğutuldu gerekmektedir. İdeal olarak, agaroz kullanımdan önce yarım saat için soğutulması gerekmektedir. </li><li> 2.2.3. , Ml başına 100,000 hücre olup, böylece hücre süspansiyonu ile seyreltilir. Düşük erime noktası agaroz (37 ° C) ile hücre süspansiyonu Kombine1:10 (v / v), girdap kısaca, oranı ve hemen Comet slayt üzerine 50 ul pipetle. Örnek alan üzerinde eşit hücre süspansiyonu yaymak için pipet ucu tarafını kullanın. Her tedavi grubu en az üç kopya halinde olmalıdır. </li><li> Bir daire slayt periferinde görünene kadar Sıra 30 dakika boyunca buzdolabında düz kayar. </li><li> Prechill 4 karanlıkta 30 dakika için bu çözelti içinde lizis çözelti daldırın ve slaytlar ° C&#39;de (ya da bir soğutma cihazı olarak). </li><li> Kapalı dökün veya lizis çözüm aspirat ve 1X nötr elektroforez tamponu (Tris baz, Borik asit, EDTA, 1X TBE) ekleyin. Buzdolabında yarım saat bu tampona slaytlar bırakın. </li><li> Elektroforez odasında önceden soğutulmuş 1X Nötr Elektroforez Tamponu (TBE), elektroforez slayt tepsisine yerleştirin slayt ekleyin. Onlar elektrotlar eşit uzaklıkta olacak şekilde slaytlar hizalayın. </li><li> 1X nötr elektroforez slaytlar üzerinde 0,2 inç kadar tampon dökün. Aşırı tampon arası olacakelektroforezi ile Fere. </li><li> Cm başına 1 V (elektrot elektrot ölçülür) güç kaynağı gerilimini ayarlayın ve karanlıkta 4 ° C (veya soğuk oda) 30 dakika boyunca çalıştırın. </li></ol> 3. Alkali Comet Assay <ol><li> Kullanımdan önce en az 1 saat boyunca 4 ° C sıcaklıkta erimesi Chill çözeltisi (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris baz,% 1 sodyum lauril sarkosinat, ve% 1 Triton X-100, pH 10). </li><li> Tüm granül kayboluncaya kadar 3 dakika boyunca mikrodalga içinde% 1 düşük erime noktası agaroz (1X Tris Bankası&#39;ndaki 1 g / 100 ml, Borik asit, EDTA) eritin. Sonra en az 30 dakika süreyle 37 ° C su banyosunda soğutulur. </li><li> 1:10 (v / v), girdap kısaca, bir oranında erimiş düşük bir erime noktasına sahip agaroz 1 x 105 / ml arasındadır (37 ° C) hücreleri ile birleştirin ve hemen Comet slayt üzerine 50 ul pipetle. Örnek alan üzerinde eşit hücre süspansiyonu yaymak için pipet ucu tarafını kullanın. Her tedavi grubu en az üç kopya halinde olmalıdır. </li><li> Sıra kaydırdıes düz 30 dakika buzdolabında bir daire slayt periferinde görünene kadar. </li><li> Lizis çözelti içinde önceden soğutulmuş ve 4 ° C&#39;de bırakmak karanlıkta gece boyunca, 1 saat için Immerse kayar. </li><li> Lizis çözüm slaytları çıkarın, slaytlar boşaltın ve alkali gevşemek adım öncesinde kalan deterjan ve tuzları kaldırmak için 5 dakika süreyle soğuk nötralizasyon tamponu ile bir kez yıkayın. </li><li> Bir jel elektroforez odasında Sıra slaytlar slaytları yukarıda 0.5 cm geçmeyecek şekilde önceden soğutulmuş taze yapılmış elektroforez tamponu (300 mM NaOH, 1 mM EDTA,&gt; 13 pH) ile doldurulur. Onlar elektrotlar eşit uzaklıkta olacak şekilde slaytlar hizalayın. </li><li> Slaytlar DNA ve alkali sorumlu hasar ifade çözülmesi için izin vermek için karanlıkta 30 dakika süreyle alkalin tampon bekletin. </li><li> Cm başına 1 V (elektrot elektrot ölçülen) güç kaynağı gerilimi ayarlayın ve 4 az 30 dakika çalıştırın ° C (veya soğuk oda). </li></ol> 4. Tespit ve Staining Hücreler <ol><li> Aşırı Elektroforez Tampon boşaltın </li><li> Daldırın oda sıcaklığında 5 dakika boyunca önceden soğutulmuş damıtılmış su içinde kayar. </li><li> Oda sıcaklığında 5 dakika boyunca önceden soğutulmuş% 70 etanol içinde Immerse kayar. </li><li> Gecede Kuru örnekleri. Parlak ışık slaytlar maruz bırakmayın. Numuneler bu aşamada puanlama için oda sıcaklığında ay boyunca muhafaza edilebilir. </li><li> Buzdolabında 20 dakika SYBR Green (PBS içinde seyreltilmesi 1X) daldırarak slaytlar Leke. </li><li> Slaytları çıkarın ve onları karanlıkta tamamen kurumasını bekleyin. Tamamen kuru agaroz şeffaf hale gelecektir. </li></ol> 4. Görüntü Toplama ve Analizi <ol><li> Yeşil filtre (Zeiss AxioVision) ve Comet Assay yazılım (Algısal Instruments) kullanılarak analiz ayarlanır floresan mikroskop kullanılarak görüntü elde edin. </li><li> &quot;Kuyruklu kafa&quot; (çekirdeği) üzerine tıklayın ve yazılımı içinde ortalama kuyruk momenti ve DNA miktarı dahil parametreleri hesaplarçekirdek. </li><li> Tedavi başına en az 200 hücre analiz edin. </li><li> Microsoft Excel&#39;e veri verme. </li><li> Her tedavi grubu ve uygun arsa verileri için kuyruk momenti ortalama hesaplayın. </li></ol> 5. Temsilcisi Sonuçlar Nöron hücreleri üzerinde kuyruklu tahlil analizinin bir örneği Şekil 2 ve Şekil 3 &#39;de gösterilmiştir. Bu durumda, nöronal hücrelerin ışınlama DNA hasarına neden olmaktadır. Hücreler elektrik alanına maruz gibi, DNA daha sonra Comet Test yazılımı kullanılarak analiz edilmiştir boyut farklılıklara bağlı olarak farklı hızlarda geçirir. Daha fazla DNA hasarı, uzak DNA çekirdek dışında geçirir. Bu sonradan yüksek kuyruk anda yazılım ve sonuçları tarafından alınır çekirdeğinde floresan yoğunluğu azalır. Tablo 1 comet assay analizi ve Şekil 4 bir temsilci tablo gösteriyor bir temsilcisi gösterirniteliksel grafiği comet assay ile ölçülen radyasyon sonrasında nöronal hücrelerde DNA tahribatına karşılaştırarak. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Şekil 1"/> Şekil 1. Kuyruklu testin Akış şeması. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Şekil 2,"/> Şekil 2. Nöron hücrelerinin Örnek görüntü (A) olmaksızın ve (B) kuyrukluyıldız kuyruk ile. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Şekil 3"/> Comet Assay yazılımı kullanarak Şekil 3. Comet assay analizi. (A) görüntü Temsilcisi ekran görüntüleri Carl Zeiss floresan mikroskop kullanılarak kazanılmış ve Comet kullanılarak analizTest yazılımı. (B) nukleus ya da &quot;kuyruklu kafa&quot; (yeşil daire) tıkladığınızda bir floresan harita ve grafik (sarı daire içinde) ve (C) veri tablosu (kırmızı daire) oluşturur. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Şekil 4,"/> Şekil 4. Nötr comet assay kullanarak ışınlanmış ve ışınlanmamış nöronal hücrelerde DNA tahribatına çözümlenmesiyle elde edilen ortalama kuyruk momenti komplo ile elde Temsilcisi grafiği. Beklendiği gibi ışınlanmış nöronal hücrelerde yüksek ortalama kuyruk momenti tarafından gösterildiği gibi, 3 Gy radyasyon (x-ray), DNA hasarı indükler. , ** P &lt;0.01 - ortalama kuyruk momenti (Standart hata + /) olduğu gösterilir. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tablo 1"/> Tablo 1. </strong&gt; ışınlanmış ve ışınlanmamış nöronal hücreler üzerinde kuyruklu testi analizi ile elde Örnek tablo. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> .

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Çıkar çatışması ilan etti.

Comet assay bireysel hücrelerin analiz eşsiz kapasitesine sahiptir. Bu sitotoksik ajanlara diferansiyel tepki göstermek hücrelerinin alt popülasyonlarının belirlenmesi avantajlıdır. Birkaç pratik sınırlamaları dikkate alınmalıdır. Ayrı ayrı değerlendirilebilir hücrelerin sayısı her bir bağlı olarak değişebilir. Bir popülasyon içindeki DNA hasarı varyans varsa örneklem büyüklüğü artırılmalıdır. Nekrotik ya apoptotik hücrelerin baskın varlığı hata katacak yana Yaşayabilir tek h...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> 1.5 ml&#39;lik tüplere </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris baz, borik asit, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml tüp </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agaroz </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Alüminyum folyo </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Deney şişesi </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Santrifüj </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Comet Assay yazılımı </td><td> Comet Assay IV Görüntü Analiz Sistemi </td><td></td><td></td></tr><tr><td> Silindir </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Elektroforez odası </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> Etanol </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Floresan mikroskop </td><td> Zeiss Axio Vizyon </td><td></td><td> Yeşil filtre ile herhangi bir floresan mikroskop yeterli olacaktır </td></tr><tr><td> Hemasitometre </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Düşük erime noktası agaroz </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Mikrodalga </td><td> Sears </td><td></td><td></td></tr><tr><td> Fosfat ücretsiz tuzlu, kalsiyum içermeyen, magnezyum tamponlu </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Güç kaynağı </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Buzdolabı </td><td> Sears </td><td></td><td></td></tr><tr><td> Cetvel </td><td> Staples </td><td></td><td></td></tr><tr><td> Slayt </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Sodyum klorür </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Sodyum hidroksit </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Sodyum lauril sarkosinat </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Tepsi </td><td> FisOnu Bilimsel </td><td> 15242B </td><td></td></tr><tr><td> Tris Baz </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Tripsin </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Girdap </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Su banyosu </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Ilaçların bir dizi DNA onarım yolakları hedef ve hücre DNA hasarı oluşturarak öldürmeye teşvik. Böylece, doğrudan DNA hasarı ölçmek için süreçlerin kapsamlı değerlendirilmiştir. Geleneksel yöntemler zaman alıcı, pahalı, kaynak yoğun ve kopyalayan hücrelerin gerektirir. Buna karşılık, kuyruklu testi, tek hücre jel elektroforezi testi, DNA hasarı ve onarımı daha hızlı, non-invaziv, ucuz, direkt ve duyarlı bir ölçüsüdür. DNA hasarı yanı sıra DNA tamir bütün formları bu güçlü tekniği kullanarak tek hücre düzeyinde görüntülenebilir. Comet assay yatan prensip DNA hasarı bu organizasyon bozan ise intakt DNA çok sipariş olmasıdır. Hasarlı DNA&#39;nın agaroz matriks içine girer ve bir elektrik alanına maruz kaldığında, negatif yüklü DNA pozitif yüklüdür katoda doğru göç eder. Büyük hasarsız DNA ipliklerini çok çekirdek göç etmek mümkün değildir. DNA hasaruzak bozulmamış DNA daha seyahatlerinizde küçük DNA parçaları oluşturur. Comet Assay, bir görüntü analiz yazılımı, tedbirler ve çekirdeğin dışına göç etmiş DNA ile DNA&#39;dan genel floresan yoğunluğu karşılaştırır. Göç DNA&#39;dan Floresan sinyal DNA hasarı ile doğru orantılıdır. Uzun parlak DNA kuyruk artmış DNA hasarı anlamına gelir. Ölçülen parametreler bazıları kuyruk DNA&#39;sının miktarı ve kuyruk, kuyruk uzunluğundaki DNA dağıtımı ve DNA yüzdesi her ikisi de bir ölçüsüdür kuyruk an vardır. Bu testte DNA hasarının çözünürlüğü tamir yerini almıştır anlamına beri yanı DNA onarım ölçmek için sağlar. Duyarlılık sınırı diploid memeli hücre 1,2 başına yaklaşık 50 iplikli kırılır. Örneğin etoposid gibi herhangi bir DNA hasarına neden olan ajan ile muamele Hücreler, bir pozitif kontrol olarak da kullanılabilir. Böylece comet assay DNA hasarı ölçmek için hızlı ve etkili bir yöntemdir.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Comet assay alkali labil siteleri ve hipokampal nöronlar da dahil olmak üzere tüm hücrelerde DNA üzerinde DNA-DNA/DNA-protein çapraz bağlantılar dahil tek ve çift zincir kırıkları, tespit etmenin en etkin yoludur. Yöntem DNA hasar miktarının farklılıklara bağlı olarak, bir elektrik alanı içinde DNA diferansiyel göç yararlanır.

Comet Assay kullanma Hipokampal Nöronlar DNA Hasarı assaying

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.1K Görüntüleme.  University of Alabama-Birmingham. Comet assay alkali labil siteleri ve hipokampal nöronlar da dahil olmak üzere tüm hücrelerde DNA üzerinde DNA-DNA/DNA-protein çapraz bağlantılar dahil tek ve çift zincir kırıkları, tespit etmenin en etkin yoludur. Yöntem DNA hasar miktarının farklılıklara bağlı olarak, bir elektrik alanı içinde DNA diferansiyel göç yararlanır.

Video: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Transgenik kullanarak Assaying β-amiloid Toksisite C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Nörobilim

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

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

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

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

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Primer hipokampal nöronlar kalsiyum fosfat transfeksiyonu

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Yapısal Aydınlatma Mikroskobu kullanılarak Rat İlköğretim hipokampalinden Dentritik Spine görüntüleme

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Subselüler Ca Floresans ve Biyoparlaklık Görüntüleme<sup&gt; 2+</sup&gt; Yaşlı Hipokampal Nöronlar

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Çizgili Deneyi Disosiye Hipokampal Nöronlar kullanarak Protein Substrat çekici veya itici Aktivite Eğitim için

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Yaşlı Bir Farede Hipokampal Dendritik Karmaşıklığın Golgi-Cox Yöntemi ile Değerlendirilmesi

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Sinaptik vezikül endositoz kültürlü Hipokampal nöronlar içinde ölçme

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Yetişkin hipokampal sinirsel precursor hücrelerinin devre spesifik Yönetmeliği

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...