Name: assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method
Uploaded: 2017-06-22T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

Bu çalışma, NIH P20 GM109005 (ARA) kapsamında bir pilot yardım ve Translational Neuroscience IDeA Programı ödül P30 GM110702 tarafından desteklenmiştir.

Denemeler, UAMS &#39;da Kurumsal Hayvan Bakımı ve Kullanım Komitesi tarafından onaylanan etik standartlara uygun olarak yürütülmüştür. 1. Hayvanlar ve 5-Fu Enjeksiyon Paradigması <ol><li> 6 aylık erkek C57Bl6 / J vahşi tipli fareler satın alarak bunları 1 yaşına gelene kadar sabit 12 saat aydınlık / karanlık döngü içinde yerleştirin. </li><li> 5-Fu&#39;yı% 0.9 steril tuzlu suda inceltin. Fare başına gerekli doz olarak 60 mg / kg kullanın. </li><li> 5-Fu&...

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Daha modern tekniklerle karşılaştırıldığında, Golgi-Cox yöntemi, omurga morfolojisini incelemek için tercih edilen yöntem olan çeşitli avantajlara sahiptir: 1) Boyama esasen herhangi bir doku için kullanılabilir 2) Basit bir ışık mikroskopu kurulumu, 3) Golgi-Cox görüntüleme konfokal görüntülemeden daha hızlıdır ve 4) Golgi lekeli bölümler birkaç ay ila yıllarca floresanla işaretlenmiş örneklerden daha uzun süre yaşayabilir. Bu avantajlarla bile, Golgi-Cox yönteminin hala bazı sın?...

<html> Dendritler, presinaptik girdi 1&#39;i alan ve işleyen nöronların en büyük kısmıdır. Dendritik süreçleri, proksimal dalların distal dallardan daha büyük bir çapa sahip olduğu karmaşık bir geometriye sahiptir. Dendritler geliştikçe, dendritik arborizasyon olarak adlandırılan bir süreçte diğer nöronlarla birkaç bağlantı oluştururlar. Bu dallanma miktarı ve paterni, bir dendrite&#39;nin 2&#39;yi yeterince işleyebildiği sinaptik girdilerin miktarını belirler. Dendritik koordinasyon, aktiviteye bağlı plastisite ve nöronal devrelerin düzgün gelişimi için gerekli bir süreçtir. Uzatma, geri çekme, dallanma ve sinaptojenez intrensek genetik programlar ve ekstrensek faktörlerden etkilenen karmaşık süreçlerdir. Hipokampus içerisindeki nöronal dendritlerin morfolojik ve dallanmış varyasyonları biliş ve hafıza oluşumuyla ilişkilendirilirF &quot;&gt; 3 , 4. Dendritik karmaşıklıktaki değişiklikler patofizyolojik ve davranışsal değişikliklerle ilişkilidir 5. Anormallikler Kırılgan X Sendromu ve Down Sendromu 6 dahil olmak üzere çeşitli hastalık durumlarıyla ilişkilidir. Dendritik omurgalar, merkezi sinir sistemi içinde heyecan verici girdiler alan dendritik arbelerin uzmanlaşmış subselüler bölmeleridir. Dendritik dikenlerin üç morfolojik sınıfı vardır, her sınıfa boyutları ve şekilleri temel alınarak adı verilmiştir: 1) diğer dikenlere göre daha fazla glutamat reseptörü ile kompleks postsinaptik yoğunluklara sahip olan mantar dikenleri; 2) kökten yoksun sağlam dikenler; Ve 3) uzunlamasına dar bir gövdeden ve küresel bir baştan oluşan ince dikenler. Dendritik omurga hacmi, onları kısmen tanımlamak için kullanılır; genellikle küçük ince dikenler (0.01 μm3 </sup&gt;) Mantar omurgalarına kıyasla (0.8 μm 3 ) 9 , 10 . Omurgalar olgunlaşma ile dengelenir. Örneğin, ince dikenler birkaç gün sonra geri çekilir veya mantar omurgalarına dönüşür. Alternatif olarak, mantar dikenleri nispeten kararlıdır ve uzun süre hayatta kalabilir. Nöronal bağlantıların kuvvetinin dikenlerin sayısına ve / veya hacmine 11 , 12 , 13 dayalı olduğu düşünülmektedir. Klasik Golgi boyama yöntemi ve daha modern varyasyonları, dendritik omurga morfolojisi ve yoğunluğunu incelemek için kullanışlıdır. Golgi boyamasının benzersiz bir özelliği, rasgele toplam nöronların% 5&#39;ini lekelediğidir, bu da bireysel nöronların izini sağlar 14,15. Golgi metasının tam mekanizması olsa daOd lekeleri bireysel nöronlar hala bilinmemektedir, yöntemin prensibi, gümüş kromat (Ag 2 CrO 4 ) 16 , 17&#39;nin kristalleşmesine dayanmaktadır. Golgi yönteminin üç ana türü vardır: hızlı Golgi, Golgi-Cox ve Golgi-Kopsch 18 , 19 . Her üç yöntem de birkaç gün ila aylarca krom tuzlarında ilk inkübasyon evresi ile başlar ancak aralarında bazı önemli farklar vardır. Hızlı Golgi, ilk aşamada osmiyum tetroksit kullanırken, Golgi-Kopsch paraformaldehid içerir. Hem hızlı Golgi hem de Golgi-Kopsch boyama sonrasında, yaklaşık% 7 oranında,% 1-2 gümüş nitrat solüsyonunda inkübe edilir. Golgi-Cox yöntemi gümüş nitrat yerine mercurik klorür ve potasyum dikromat kullanır ve 2-4 hafta süreyle emprenyasyon süresine sahiptir. Dokular daha sonra kesitlendirilir ve seyreltilmiş amonyakÇözeltiyi, ardından tuzları gidermek için bir fotoğrafik düzeltici uygulayın. Üç tipten Golgi-Cox yönteminin, kısmen arka plana müdahale etmeden dendritik aplikleri boyamakta en iyi olduğu düşünülmektedir, çünkü kısmen kristal yapılar doku yüzeyinde oluşmaz (hızlı Golgi yönteminde olduğu gibi) 17 , 20 , 21 . Mevcut yöntem, ticari bir Golgi boyama kiti ile sağlanan protokolün hafif bir varyasyonudur ve nispeten düşük bir dozda 5-Fu&#39;nın dendritik morfolojik özellikleri ve omurganın yoğunluğunu nasıl etkilediğini değerlendirmek üzere tasarlanmıştır. Elde edilen veriler, kemoterapötik tedavinin nöronal devreleri nasıl etkilediği konusunda daha fazla bilgi sağlayabilir.

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assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method

Dendritik dikenler, eksitatör sinapslar içeren nöronal dendritik millerin çıkıntılarıdır. Hipokampus içerisindeki nöronal dendritlerin morfolojik ve dallanmış varyasyonları biliş ve hafıza oluşumuyla ilişkilendirilir. Golgi boyama için çeşitli yaklaşımlar vardır, bunların hepsi dendritik alandların morfolojik özelliklerini belirlemek ve belirgin bir arka plan oluşturmak için kullanışlıdır. Mevcut Golgi-Cox yöntemi (ticari bir Golgi boyama kiti ile sağlanan protokolün ufak bir varyasyonu), kemoterapötik ilaç olan 5-flurouracil&#39;in (5-Fu) nispeten düşük dozunun dendritik morfolojiyi nasıl etkilediğini değerlendirmek üzere tasarlanmıştır , Omurganın sayısı ve hipokampüs içindeki işlenmesi karmaşıklığı. 5-Fu, dendritik karmaşıklığı önemli derecede modüle etti ve bölgeye özel bir şekilde, hipokampustaki omurga yoğunluğunu azalttı. Sunulan veriler, Golgi boyama yönteminin efOlgun nöronları, CA1, CA3 ve hipokampus diş açısı (DG) lekelemiştir. Bu protokol, diğer araştırmacıların beyne dokuları yüksek kaliteli sonuçlar ve minimum sorun giderme ile güvenilir şekilde boyayabilmesi için her adımın ayrıntılarını bildirir.

5-Fu işleminin, Golgi ile boyanmış beyin bölümlerinin hipokampusundaki dendritik taşlama ve kompleksite üzerindeki etkileri, ticari olarak mevcut bir görüntüleme yazılımı kullanılarak nicelleştirildi ve izlendi. İzlemeden sonra, dendritik kabarcıklanma, omurga yoğunluğu ve omurga morfolojisi Sholl analizi ve dendritik karmaşıklık indeksi (DCI) kullanılarak analiz edildi. Sholl analizi, dendritik çember morfolojisini belirlemek için kullanılan nicel bir analitik ...

Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

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

Burada ayrıntılı bir Golgi-Cox protokolünü sunuyoruz. Bu güvenilir doku lekesi metodu, minimal sorun giderme ile hipokampustaki ve tüm beyindeki sitolojik mimarinin yüksek kaliteli bir değerlendirmesini sağlar.

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 ...

The overall goal of this methodology is to properly Golgi stain brain tissue in order to examine and quantify dendritic morphology and complexity. This method can help answer key questions in neuroscience. Since Golgi staining only stains a small subset of cells, this technique could be used to trace the structure of neurons.The main advantage of this technique is that brain sections can be easily stained without risk to them drying out or falling off during any of the washing steps. Demonstrating the procedure will be Fred Kiffer, Tyler Alexander and Julie Anderson, graduate students in Dr.Allen's laboratory. Begin by immersing freshly harvested, unfixed rodent half brains in five milliliters or mercuric chloride-based solution.As mercuric chloride solution is light sensitive, cover the tube with foil. Store the samples in the dark at room temperature. The following day, slowly decant the solution into a temporary container before properly disposing of it in a biohazard waste container.Add five milliliters of fresh mercuric chloride solution and ensure that the brains are immersed. Return the samples to the dark at room temperature for 13 more days. After 14 days, prepare post-impregnation buffer by adding 30 grams of powered post-impregnation buffer to 90 milliliters of distilled or deionized water, DH2O.Fill each well of the six-well plate with 6.5 milliliters of the post-impregnation buffer, one well per brain. Then, remove the mercuric chloride solution from the brain tissue as before. Rinse the tissue with DH2O, then transfer the tissue to the six-well plates with post-impregnation buffer.Cover the plates with foil and store at room temperature in the dark. The next day, aspirate and replace the post-impregnation buffer. Re-cover the plate and store in a four degree Celsius fridge.Prepare two or three 12-well plates per brain by first labeling the plates on their lids with numbers in ascending order from left to right and up to down. Add the identification number of the brain sectioned. Pipette two milliliters of 1X PBS into each well of each plate, then set up the stage and turn on the vibratome.Then, put tape on the tissue block of the specimen holder and add a little cyanoacrylate glue on top. Remove a brain from the six-well plate and place it on a plastic or glass dish. Use a stainless steel double edged blade to cut the cerebellum caudally, making sure that the portion of the cerebellum remaining on the brain is flat.Then, place the flat surface of the remaining cerebellum on the glue. After the glue is dry, place the specimen holder with the tissue into the specimen bath. Place the specimen bath on the vibratome and cover the tissue sample in PBS.Then, attach the blade to the blade holder of the vibratome and slowly lower the blade into the specimen bath until it is fully submerged and slightly below the top of the tissue. Next, set the vibratome speed to seven, the amplitude to six and the cutting angle to 12 degrees. Start the vibration and cut 200-micron sections.Use a large paint brush to move the tissue sections from the specimen bath into each designated well of the 12-well plates. Continue to cut the tissue until the desired number of tissue sections is reached. After sectioning, use a transfer pipette to aspirate the PBS from the wells of the 12-well plate, then add two milliliters of PBS-T and wash for 30 minutes with agitation.Close to the end of the wash period, dilute three parts ammonium hydroxide solution with five parts DH2O. Then, after removing the PBS-T, add the diluted ammonium hydroxide solution. Protect from light using foil and stain the sections for 19 to 21 minutes.Then, after safely removing the ammonium hydroxide solution, add post-staining buffer. Protect with foil and stain for another 19 to 21 minutes. Finally, rinse the sections in PBS-T three times for five to 10 minutes each time.Mount the sections on 1%gelatin coated slides with the large paint brush. Allow them to dry for 20 to 30 minutes at room temperature, then place them in a Coplin Jar and leave overnight. Remove the slides from the Coplin Jar and place them in a plastic rack in the fume hood.Place in a staining dish containing 100%ethanol for five minutes. Dehydrate the sections through two further five-minute ethanol washes. Next, place the slides in a glass rack and use a metal handle to lower the rack into a glass staining dish containing 99%xylene.Wash the slides in two changes of xylene for five minutes each. After the time for the second wash has elapsed, remove the slides from the xylene one at a time and quickly cover the tissue with approximately 0.25 milliliters of mounting media. Next, take a slide cover and lay it over the media.Push any trapped air bubbles underneath the slide covers to the edge with a blunt object, such as the end of paint brush. Place the slides in an area out of direct sunlight and allow them to dry for two to three days. After drying, proceed to image acquisition using a microscope and analysis with the appropriate software.This bright field image shows a Golgi-stained neuron that meets the criteria for imaging and further analysis. All three spine types can easily be seen. Staining is consistent across the entire length of the dendrite and the dendrite is isolated from other neurons.This figure shows that Golgi stained neurons within the Dentate Gyrus showed decreased length at the fourth and sixth order following 5-fluorouracil treatment compared to the saline-treated group. Golgi staining can be used to detect significant differences in spine density. In the CA1 apical pyramidal dendrites, 5-fluorouracil administration to live mice significantly decreased spine density.In the basal dendrites, there were no significant changes in the overall density of spines. Sholl Analysis of CA1 apical pyramidal dendrites revealed that 5-fluorouracil treatment significantly decreased dendritic arborization at distances between 40 to 160 microns away from the soma. In the basal dendrites, the 5-fluorouracil treatment decreased the dendritic arborization at 30 to 110 microns away from the soma.Once mastered, this technique, accounting for both the Golgi staining process and mounting, cleaning and covering can be done in three hours per brain if performed properly. After its development, this technique paved the way for researchers in the field of neurobiology to explore neuroanatomical connections in a murine model. While attempting this procedure, it's important to remember to keep the sections in ammonium hydroxide solution for only 19 to 21 minutes as to not overstain the neurons.After watching this video, you should have a good understanding of how to properly stain, section and rinse brain tissue. Don't forget that working with the mercuric chloride-based solution, ammonium hydroxide solution and xylene can be extremely hazardous, so take precautions and wear lab gloves and work in a fume hood when performing this procedure.

Research

JoVE Journal

Neuroscience

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Denge kullanarak Fare Motor Denge ve Koordinasyon Değerlendirme

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Nörobilim

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Kültür Hipokampal Nöronlar Dendritik Spine Floresan Takip edilen Proteinler Photobleaching sonra Floresan Kurtarma (sıkı bağlamak)

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

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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 ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

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Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

Golgi-Cox Boyama ile dendritik morfoloji ve sinaptik bağlantı üzerinde olumlu bir Erken Deneyimi, Tactile uyarılması Etkileri, görselleştirme

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

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

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 ...

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

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. ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Farelerde Hipokampal Bölgesi Dentate Gyrus dendritik arborization Değerlendirilmesi

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Fareler Soğuk Hassasiyeti ve Uyum belirlenmesi için Basit ve ucuz Yöntem

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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

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

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 ...