Name: an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices
Uploaded: 2018-04-11T15:00:00.000+00:00
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Bu eser nöro-Bio Ltd. tarafından finanse edildi Dr. Giovanni Ferrati ve doktor Sergio Rotondo (nöro-BIO) teşekkür etmek onların yorum ve tavsiyeler el yazması için istiyoruz.

Tüm hayvan çalışmaları onaylı protokolleri altında gerçekleştirilmiş.
Not: Bu bölümde ana aşamadan dizisi deneysel işlem sırasında gerçekleştirilen ve önerilen zaman aralığı (şekil 1) sağlanır. Ayrıca, protokol, adım adım açıklamasını kritik eylemler doku homojenizasyon kuluçka dönemi (Şekil 2) sonra beyin kaldırılması arasında gösterilen açıklayıcı bir panel tarafından desteklenmiştir. Yukarıda açıklanan22ile ilgili malzeme ve cihazları ve sonraki aşama WB analiz için oluşturmak için yönergeleri ayrıntılardır.
1. cerrahi aletler, Vibratome ayarları ve tedavi hazırlık
Not: deneysel bir işlem başlatmadan önce aşağıdaki adımları yürütün:
<ol><li>İki farklı yapay serebrospinal sıvı (aCSFs), bir beyin Dilimleme için kullanılan hazırlamak ve diğer kuluçka adım için daha önce29açıklandığı gibi. Kısaca, "Dilimleme" aCSF için iki aCSFs (içinde mmol) çalışma konsantrasyonları şunlardır: 120 NaCl, 5 KCl, 20 NaHCO3, 2.4 CaCl2, 2 MgSO4, 1.2 KH2PO4ve 10 glikoz; 6,7 HEPES tuz ve 3.3 HEPES asit; pH: 7.1. "Kayıt" aCSF süre: 124 NaCl, 3.7 KCl, 26 NaHCO3, 2 CaCl2, 1.3 MgSO4, 1.3 KH2PO4ve 10 glikoz; pH: 7.1.</li>
	<li>Mix iyi çözümleri ve buza koy ve dilimleme aCSF için bu soğuk beyin ve sonraki kesit kaldırma sırasında oksijen. Oda sıcaklığında (RT) ve tedarik oksijen kayıt aCSF tutun.</li>
	<li>Diseksiyon başlık altında aletleri işten çıkarma ve beyin kaldırma, Yani, giyotin, cerrahi disseksiyon seti (makas, pens, bıçaklar) ve spatula için yerleştirin.</li>
	<li>Vibratome parametreleri dilim kalınlığı (Bu hazırlık 300 µm) ve 7 frekans ve 6 hızda seçerek ayarlayın. Vibratome odası uygun, boşluk ve buz ile surround.</li>
	<li>"Dilimleme" (aCSF) beyin kesit sırasında oksijen carbogen (% 95'i O2, % 5 CO2) Vanayı aç. 2 mL/dk civarında en düşük akış hızı seçin.</li>
	<li>Test etmek için koşulları hazırlamak için iki 15 mL tüp al. Ekle bir tüp 10 mL "kayıt" aCSF yalnız (kontrol grubu) ve diğer, "kayıt" aCSF 10 mL 2 µM (tedavi grubu) bir konsantrasyon T30 ile zenginleştirilmiş. Girdap hem tüpler ve kullanımları kurulum hazırlama aşaması (Bölüm 3) kadar buz üzerinde tutun.</li>
</ol>2. anestezi, beyin çıkarma ve dilimleme
Not: Bu çalışmada, 9 vahşi türü P14 erkek Wistar rats kullanılmıştır. Onlar hızlı bir şekilde (10 dk içinde) önlemek veya en az doku dejenerasyonu süreçleri yavaşlatmak için gerçekleştirilmesi gereken bu yana beyin ayıklama ve sonraki Dilimleme protokolün kritik bir parçası oluşturur.
<ol><li>% 100 w/w isoflurane 1,5 mL bir pamuk katmanda indüksiyon odasına ekleyin, hayvan içine yerleştirin ve kapağını kapatın. Hayvan derinden pedal refleks, yokluğunda teyit anestezi kadar bekleyin ve Giyotin kullanarak hayvan başını kesmek.</li>
	<li>Buz gibi baş hayvan içeren bir kap içinde aCSF beyin kaldırma adımı sırasında Dilimleme tutmak (şekil 2A -C).</li>
	<li>Cilt üzerinde kafatası, cerrahi bıçak deşmek sonra kafatasının orta hat posterior bölümünden takip makas kullanarak kesmek ve anteriorly kemik OBs (şekil 2A) yukarıda erişene dek devam edin.</li>
	<li>Beyin (şekil 2B) erişim sağlamak için bir çift forseps kullanarak kafatası yan yana iki tarafına çek. Bir spatula ventrally beyin için eklemek, yavaşça dışarı ve buz gibi "Dilimleme" aCSF için yaklaşık 5 dk (şekil 2C) içinde nemli tutmak Kepçe.</li>
	<li>Beyin filtre kağıdı üzerine atın ve bir bıçak (şekil 2B) kullanarak beyincik kesti. Dikey olarak vibratome disk beyin tutkal ve parça odası ekleyin, buz gibi "Dilimleme" aCSF ile doldurun ve oksijen (şekil 2E) sağlar.</li>
	<li>Kesme aralığını ayarlamak ve beyin (şekil 2F) kesit ile devam edin. Medial septum (MS), Broca (DBB) ve gözlemliyorum innominata (SI), çapraz grup gibi ilgi bölge içeren bölümler yukarıda açıklanan22toplamak. Yukarıdan-aşağıya üç dilim rostral (R), orta (I) ve (Şekil 2 g) BF Kaudal (C) bölümünü içerir.</li>
	<li>Orta çizgi her bölüm küçük makasla bölmek tek tek kontrol ve tedavi durum aynı anatomik uçağı olarak kullanılan iki aynasal hemislices (Resim 2 H), alma.</li>
	<li>Hemisections 5-10 dakika vibratome odasında tutmak, o zaman onlara bir cam pipet ile kayıt aCSF içeren bir kabarcıklanma pota transfer. Yeniden elde etmek için yaklaşık 30 dakika RT devam et.</li>
	<li>Bu arada, üç cam şişeleri ile kayıt aCSF yalnız 3 (Adım 1.6 kontrol grubu için önceden hazırlanmış) mL ve T30 (1.6 adımda önceden hazırlanmış) ile tedavi grubu için zenginleştirilmiş aCSF 3 mL ile üç şişeleri doldurun.</li>
</ol>3. Kurulum hazırlama
<ol><li>Hemisections kontrol grubu (yeşil çerçeve, şekil 2J) (rostral, ara ve Kaudal BF bölge dahil) üç ardışık hemislices için karşılık gelen cam şişe (şekil 2I) aktarmak ve onların tedavi grubu (kırmızı çerçeve, şekil 2J) için meslektaşları eşleştirme.</li>
	<li>Koşullar arasında herhangi bir kontaminasyonu önlemek için iki farklı fırçalar, bir denetim hemislices ve başka için tedavi meslektaşları ile beyin dokusu aktarın.</li>
	<li>Her cam şişe oxygenating iğne (21 G) (şekil 2J) sunma karşılık gelen kapak ile kapatın. Cihazları ana tüp carbogen kaynak (Şekil 2 K) bağlayın. Deneme sırasında beyin dokusu ve mekanik zarar herhangi bir hareketi önlemek için hemislices 2 mL/dak bir minimum akış hızı ile kuluçka süresi boyunca için oksijen sağlamak. 5 h kuluçka başlatın. 5 dakika içinde 3.1-3.3 adımları yürütün. Not: Onay sık (her 15-20 dk) oksijen tüpleri sağlanan ve akışını aşağıdan doku kıpırdamıyor.</li>
</ol>4. örnek homojenizasyon
<ol><li>Kuluçka sonra oksijen akışını durdurmak ve tüpleri tüm kapakları kaldırın. 250 µL üzerine buz (Şekil 2 L) boruları Bakımı lizis tamponunun içeren 1,5 mL tüpler içine uygun fırçalar kullanarak hemislices aktarın. Arabellek, PBS 1 mix lizis yapmak x fosfataz ve proteaz inhibitörleri kokteyller ile hem 1: 100 seyreltilmiş ve doku örnekleri arasında önlemek amacıyla farklı dibekler ile homojenize. 10-15 dk içinde bu eylemleri tamamlayın.</li>
	<li>1000 x g 4 ° C'de 5 min için de lysate beyin santrifüj kapasitesi Süpernatant yeni tüpler içine aktarmak ve örnekleri-80 ° C'de tutmak</li>
</ol>5. okuma Protein konsantrasyonu ve Western Blot analizi
<ol><li>Her örnek protein konsantrasyonu belirlemek ve daha sonra yukarıda açıklanan22olarak Western blot analizi için aliquots hazırlamak.</li>
	<li>Protein ifadesi üzerinde tedavinin etkilerini değerlendirmek için istatistiksel çözümleme gerçekleştirme.</li>
</ol>

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Yazarlar rakip mali çıkarlarının bildirin. Susan A. Greenfield nöro-Bio Limited, özel sektöre ait bir şirket ve şirket tutar hisseleri kurucusu ve başkanıdır. Emanuele Brai ve Antonella Cogoni nöro-Bio Ltd. çalışanları olan

Bu iletişim kuralı, köklü ex vivo beyin tekniğine dayalı asıl yönünü zaman uyumlu olarak belirli bir uygulama sonra onların yanıt izleme aynı anatomik uçak elde edilen iki aynasal hemislices test etmek için izin verir Koşul (kontrol veya tedavi); Bu nedenle deneysel bir paradigma olarak sıkı mümkün olduğunca kontrol sunuyor. Bir saat, doz ve nöronal bozukluğu için ilgili siteye özgü şekilde farklı fizyolojinin nörodejeneratif olaylar22sırasında görüldüğ?...

Kronik bir patoloji entorhinal korteks (EC), BF, Hipokampus (HC) ve olfaktör ampul gibi farklı beyin bölgeleri etkileyen kademeli nörodejeneratif bozukluğu (OB)1,2,3ile karakterize reklamdır, 4,5. Reklam gelişimin geç yaklaşık % 70'ini tüm durumlarda6muhasebe demans en sık görülen bu hastalık yapma ilerici bir bilişsel gerileme yol. Reklam neden ilk etap anlamak için yoğun girişimlerine rağmen bir şey yok şu anda tanımlanmış bir deneysel gösterge onları elucidating. Reklam pathobiology, ne de etkili7,8 kanıtlamıştır bir ilaç hedef açıklayan tam bir profil sağlamaz beri Ayrıca, en popüler teori - "amiloid hipotezi" - giderek sorgulanmaktadır ,9.
Ateş sırasında meydana gelen ilk mekanizmaları nöronal küme reklam3,10,11 ' öncelikle duyarlı ilişkili artan ilgi alma alternatif bir teori önerir , 12 , 13 , 14. bu türdeş olmayan hücresel Merkez AK, HC ve OB15,16gibi birden çok bölgeye BF, orta ve beyin sapı, projeler içinde kapsıyordu. Nöronal morfoloji ve nörotransmitter sentez çeşitliliği rağmen bu çekirdek hücrelerin enzimatik olmayan işlev17,18de içerebilir ağrısı ifade içinde ortak bir özelliği paylaşır. Molekül trofik veya toksik olayları ile ilgili olarak Ca2 + doz, kullanılabilirlik ve nöronal yaş17,18 geçirmek için nöronlar kalsiyum (Ca2 +) akışına aracılık sinyal bir roman olarak klasik olmayan bu rolü , 19.
Ateş sırasında gözlenen hücresel kaybı bu nedenle ağrısı C-terminus i ciddi bir 30mer peptid (T30) atfedilebilecek olduğu bu enzimatik olmayan işlev17,18,20' ye, ilişkili olabilir 20. hücre kültürü ve optik görüntüleme18,21 hazırlıklar, önceki sonuçları doğrultusunda biz, ex vivo sıçan beyin dilimleri T30 indüklenen BF yapıları içeren dayalı yeni bir yaklaşım ile gösterdi bir reklam gibi profil22. Özellikle, birçok anatomik devre koruma için bir zaman penceresi saat de olsa için arasında değişen bir sağlam doku özelliklerini korur beri bu yeni yöntem daha fizyolojik bir senaryo hücre kültürü daha sunuyor. Biz ateş, erken aşamalarında yer alan olayları keşfetmek için bu iletişim kurallarını T30 başvurusu üzerine akut yanıt izleme uygulanır.
Beyin kullanma edebiyatının büyük beden rağmen moleküler yolları araştırmak için dilimler nöronal hasar örtük veya neurogenesis23,24, bu protokolü ilk kez daha acil sağlar ve okumak duyarlı karşılaştırıldığında organotypic dilimleri yaygın kullanımı için. Beyin bölümleri organotypic için olduğu gibi Ancak, bu akut dilim yordamı da değerlendirilmesi nöroprotektif veya Nörotoksik molekülleri, belirli bir süreç içinde birincil moleküler değişimleri keşfi gibi çeşitli amaçlar için kabul edilebilir, immunohistokimyasal analizleri ve merkezi sinir sistemi için farmakolojik deneyleri patolojiler ilgili.

<table><tbody><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich, Germany</td><td>S7653</td><td>Reagent for aCSF preparation</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich, Germany</td><td>P9333</td><td>Reagent for aCSF preparation</td></tr><tr><td>Sodium bicarbonate (NaHCO3)</td><td>Sigma-Aldrich, Germany</td><td>S5761</td><td>Reagent for aCSF preparation</td></tr><tr><td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td><td>Sigma-Aldrich, Germany</td><td>63138</td><td>Reagent for aCSF preparation</td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma-Aldrich, Germany</td><td>P5655</td><td>Reagent for aCSF preparation</td></tr><tr><td>Hepes salt</td><td>Sigma-Aldrich, Germany</td><td>H7006</td><td>Reagent for aCSF preparation</td></tr><tr><td>Hepes acid</td><td>Sigma-Aldrich, Germany</td><td>H3375</td><td>Reagent for aCSF preparation</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Germany</td><td>G7528</td><td>Reagent for aCSF preparation</td></tr><tr><td>Calcium chloride dehydrate</td><td>Sigma-Aldrich, Germany</td><td>223506</td><td>Reagent for aCSF preparation</td></tr><tr><td>T30 peptide</td><td>Genosphere Biotechnologies, France</td><td></td><td>AChE-derived peptide tested</td></tr><tr><td>Surgical dissecting kit</td><td>World Precision Instruments, USA</td><td>Item #: MOUSEKIT</td><td>Brain removal step</td></tr><tr><td>Surgical blades</td><td>Swann-Morton, UK</td><td>BS 2982</td><td>Brain removal step</td></tr><tr><td>Filter paper</td><td>Fisher Scientific, USA</td><td>11566873</td><td>Brain preparation for slicing</td></tr><tr><td>Glue</td><td></td><td></td><td>Brain preparation for slicing</td></tr><tr><td>Vibratome</td><td>Leica, Germany</td><td>VT1000 S</td><td>Slicing</td></tr><tr><td>Brushes</td><td></td><td></td><td>Tissue handling</td></tr><tr><td>Oxygen canister</td><td></td><td></td><td>Sectioning and incubation phase</td></tr><tr><td>1x Phosphate buffer saline (PBS)</td><td>Fisher Scientific, USA</td><td>BP2438-4</td><td>Homogenization step</td></tr><tr><td>Phosphatase inhibitors</td><td>Fisher Scientific, USA</td><td>1284-1650</td><td>Homogenization step</td></tr><tr><td>Protease inhibitors</td><td>Roche complete PIC, USA</td><td>4693116001</td><td>Homogenization step</td></tr><tr><td>Pestles</td><td>Starlab, UK</td><td>I1415-5390</td><td>Homogenization step</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Pierce 660 nm Protein Assay</td><td>Thermo Scientific, USA</td><td>22660</td><td>Protein concentration</td></tr></tbody></table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

Hangi birincil yansıtan temel ateş işler güvenilir hayvan modelleri geliştirmek için çalışan çok sayıda çalışma rağmen çok az yaygın olarak kabul etmiştir. Burada, biz daha yakın vivo içindesunan iyi bilinen ex vivo beyin dilim tekniği, uyarlanmış yeni bir yordam teklif- vitro hücre dejenerasyonu, uyarının harekete geçirilmesine karşılık olarak erken olayları araştıran için hazırlıklar, daha senaryo gibi Alzheimer hastalığı içinde (Ah) görülmektedir. Bu varyasyon seçili beyin bölgesi ve yerel işlevselliğini fizyolojik ortamında bir anatomik cytoarchitecture korunması etkinleştirme basit ve kolayca tekrarlanabilir adımlar oluşur. Farklı anatomik alanlarda birden fazla deneyler söz konusu tedavileri ile bir site-, doz- ve saat-bağımlı bir şekilde gerçekleştirmek için fırsat sağlayan aynı beyin elde edilebilir. Bu metodoloji ile ilgili sonuçları etkileyebilecek potansiyel sınırlamalar doku, Yani, anatomik bütünlüğünü sırasında dilimleme ve kuluçka adımları ve bölüm kalınlık bakımından korunması için ile ilgili hangi biyokimyasal ve immunohistokimyasal analiz etkileyebilir. Bu yaklaşım moleküler mekanizmaları fizyolojik ve patolojik koşulları, uyuşturucu tarama veya doz-yanıt deneyleri dahil keşfetmek gibi farklı amaçlar için istihdam edilebilir. Son olarak, bu iletişim kuralını da hayvan davranış çalışmalarda istihdam sayısını azaltabilir. Rapor burada uygulama son zamanlarda açıklanan ve ex vivo sıçan beyin dilimler içeren bir reklam öncelikle etkilenen beyin bölgelerinden biri olan Bazal ön (BF), tarihinde ilk kez test. Asetilkolinesteraz (AChE) C-terminus elde edilen zehirli bir peptid yönetim tetikleme, BF, antero-posterior ekseni boyunca farklı bir ifade bir reklam gibi profil sor kanıtlanmıştır özellikle, Reklamda, alpha7 nikotinik reseptör (α7-nAChR), gibi değişmiş proteinlerin Tau (p-Tau) ve amiloid beta (Aβ) fosforile.

Burada sunulan Protokolü toksik bir peptid, T30, yönetim α7-nAChR, p ifadesi bir site bağlı şekilde modüle gösterir-Tau ve Aβ BF içeren bölümler (şekil 3A) içinde. Nikotinik reseptör denetim muadili karşılaştırıldığında rostral tedavi hemislice önemli bir artış gösterir (dilim 1, p = 0.0310) (ara dilim arasında iki koşul (herhangi bir değişiklik ortaya koymuyor ikenşekil 3B), dilim 2, ...

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Biz daha fazla ateş altında yatan erken etkinlik kazandırabileceğini kurulan eski-vivo beyin tekniğine dayalı bir yöntemini in vivo ve in vitro deneyler avantajlarını birleştiren mevcut. Ayrıca, tedavi ve tedavi edilmezse grubun aynı anatomik düzlemde doğrudan karşılaştırma için eşsiz bir fırsat temsil eder.

Ateş Ex Vivo sıçan beyin dilimleri kullanarak birincil olayları incelemek için alternatif bir yaklaşım

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

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Neuroscience

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

6.9K Görüntüleme.  Culham Science Centre. Biz daha fazla ateş altında yatan erken etkinlik kazandırabileceğini kurulan eski-vivo beyin tekniğine dayalı bir yöntemini in vivo ve in vitro deneyler avantajlarını birleştiren mevcut. Ayrıca, tedavi ve tedavi edilmezse grubun aynı anatomik düzlemde doğrudan karşılaştırma için eşsiz bir fırsat temsil eder.

Video: An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

POSTNATAL nöron Çalışmaları için organotipik Dilim Kültürler

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Gelişim Biyolojisi

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Yaşlanma ve Amiloid Patoloji sırasında sinaptik değişiklikler Çalışması Sıçanlar ve Transgenik Farelerde akut Hipokampal Dilimleri hazırlanması

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Nörobilim

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Kemirgen Beyin Dilimleri Nöronlar Vibrodissociation Sinaptik İletim ve Görüntü Presinaptik Terminalleri Eğitim

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Tıp

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Beyin Dilim Biyotinilasyonu: An<em&gt; Ex Vivo</emYetişkin Nöronlar Bölge özgü Plazma Membran Protein Ticaretiyle Tedbir&gt; Yaklaşım

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Içinde Neurovascular Yaptırma Analizi Entorhino-hipokampal Organotipik Dilim Kültürler

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

En iyi duruma getirilmiş bir Nkullanarak akut beyin dilimleri hazırlanması-metil-D-glucamine koruyucu kurtarma yöntemi

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Spontan Hipokampal Ağ Aktivitesini Araştırmak için Akut Fare Beyin Dilimlemeyi

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Source: Carvalho, M., et al., <a href="https://app.jove.com/v/61330">A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures &#8212; resembling in vivo epilepsy &#8212; and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.

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