Bu çalışmada IPSEN Vakfı tarafından finanse edildi. IMV, Moleküler Biyoloji Amerikan Kanser Derneği Profesörü ve Örnek Yaşam Bilimleri Irwin ve Joan Jacobs Başkanı tutar. Bu çalışma NIH, Leducq Vakfı, Meriaux Vakfı, Ellison Tıp Vakfı, Ipsen / Biomeasure, Sanofi Aventis, Prostat Kanseri Vakfı, HN ve Frances C. Berger Vakfı hibe kısmen desteklenmiştir. PAS Nörobilim ve Uyuşturucu (R01 DA019022) Ulusal Enstitüsü McKnight Endowment Fund tarafından finanse edilmektedir. Yazarlar Mark Schmitt teşekkür etmek istiyorum. Grafik sanat, Murat T. Simon tarafından dizayn edilmiştir. Sponsorluk nazik Invitrogen tarafından sağlanmıştır.

1. Organotipik Serebellar Kültürler: <ol><li> Organotipik serebellar kültürler başarıyla oluşturmak için P0-P3 veya P8-P12 arasındaki fareler kullanın. Deneme için bu gelişimsel aşamada, bizim çalışmamız için daha uygun olması nedeniyle, P8-P12 serisi fareler kullanıldı. </li><li> Diseksiyon ve kültür ortamı hazırlayın: <ol><li> Beyin diseksiyonu ve dilim hazırlama 1mm kalsiyum klorür içeren düşük sodyumlu Yapay Beyin Omurilik Sıvısı (ACSF), 10mM D-Glukoz, 4mm potasyum klorür, 5mM magnezyum klorür, 26mm sodyum bikarbonat, 246mM sakaroz ve fenol kırmızısı çözeltisi (1:1000) yürütülmektedir pH 7.3. Çözüm sterilize etmek için, 4 .22 mikron filtre ve mağaza üzerinden filtre ° C </li><li> Serebellar dilimleri% 75 Minimum Essential Orta Kartalı (MEM),% 25 ısı ile inaktive edilmiş at serumu, 25mm Hepes, 1mm glutamin, 5 mg / ml glikoz, penisilin (100 U / ml) ve streptomisin (100 U / ml) kültür. Iki hafta en fazla 4 0.22 mikron filtre ve mağaza ° C ile orta Filtresi. </li></ol></li><li> Kırmızıdan turuncuya kayacak önceden Carbogen (% 95 oksijen ve% 5 karbon dioksit) Not kabarmış buz ACSF orta beyin teşrih, Carbogen doyma çözeltinin pH değişecek. Diseksiyonu beyin yapısına zarar vermemek için dikkatli olmak mümkün zaman minimum tutar yapılmalıdır. </li><li> Vibratome kullanarak 400 mikron sagital bölümlerde beyin dilimleyin. <ol><li> Beyincik mümkün olduğunca az kesmeye çalışırken bir yarımkürede bir jilet ile sagital kesme olun; bu dilimleme için düz bir yüzey sağlayacaktır. Korteks destek için dilimleme kolaylaştırmak için kullanılır ama zaman önemli bir konudur çünkü bir jiletle kesip korteksin rostral yarısında daha uygun. </li><li> (Superglue), metal vibratome plaka üzerinde beyin konumunu düzeltmek için siyanoakrilat kullanın. Tutkal eklemeden önce buz üzerinde plaka soğuk ve ince bir yapışkan tabaka oluşturacak şekilde tüp ucunu kullanarak iyi yaymak için çok önemlidir. Superglue fazlalığı varsa ACSF ve örnek kaybolur temas geldiğinde, plaka terketmez. </li><li> Sonra beyin, beyin kapak ve levha, soğuk tutmak için aşağıda buz koymak için yeterli ACSF medya eklemek vibratome plaka takılı. </li><li> 400 mikron dilim kesin ve bir plastik Pasteur pipeti (genişletmek ve dilimleri zarar görmesini önlemek için ucu kesilmiş) 6-iyi hücre kültürü plaka buz üzerinde ACSF içeren bir transfer. </li><li> Diseksiyon mikroskobu altında iki insülin şırıngaları (28 gauge iğne ile küçük çaplı şırınga) yardımıyla beynin geri kalanı beyincik ayırın. Bazı dilim superglue kenarlarına bağlı olacaktır: Bu dilim canlılığı etkileyebilir olarak kaldırmak için denemek için bir an. Serebellar yapıya zarar vermeden dikkatli bir şekilde bunu yapmak için çok önemlidir. </li></ol></li><li> 0.4 mikron gözenekleri ile 30mm kültür plaka ekler içeren 6-kuyucuğu serebellar dilim aktarın. 1 ml kültür ortamı başına iyi, 37 en az 2 saat süreyle inkübasyon tarafından önceden klimalı ° C ve% 5 CO 2 (hücre kültürü inkübatörde). Ekler altında hapsolmuş hava kabarcıkları ve daha sonra tamamen genişletilmiş emin olun ekler ve sıvı onları kapsayan üst serebellar dilimleri yerleştirin (insert ortalama 1-3) emin olun medyanın üstüne yerleştirin. Gerekirse medya aşan aspire edin. 37 inkübe ° C ve% 5 CO 2 kültür ortamı her 2 ya da 3 gün değişiyor. Kullanılan orta dilim kaynaklı hücre sağkalım için önemli olan trofik faktörler içerir, çünkü bir seferde orta (örneğin ½ hacmi) yalnızca bir bölümünü değiştirmek için en iyisidir. </li></ol> 2. Sülfüroz Tedavi ve Serebellar Dilimleri boyanması: <ol><li> Apoptotik meydan deneme için, öncelikle kültür, in vitro 3 (DIV3) günü medya değişen 5 gün dilimleri . DIV5 kültürleri hücre ölümü temiz bir arka plan ortam değişiklikleri beri sahip olmak için doğrudan medyaya ekleyerek Fas agonist antikoru Jo2 0.5 mikrogram / ml ile tedavi edilir kültürlerde bazı ölüm neden olabilir. Kuyuların yarısı kontrol grubu olarak tedavi edilmezse. </li><li> 24 saat emme ekler altındaki medya kaldırdıktan sonra. Dilimleri düzeltmek için, altındaki buz gibi% 4 PFA 1 ml ve 1 ml eklemek yukarıda ekleyin. Oda sıcaklığında 10 dakika inkübe edin. </li><li> 10 dakika PFA kaldırın ve kısa bir süre soğuk PBS ile yıkama sonra. </li><li> Sonraki PBS çıkartın ve oda sıcaklığında 5 dakika inkübe PBS ve% 20 buz metanol eklemek üzerinde 1 ml altında ve 1 ml ekleyin. </li><li> PBS ile tekrar yıkayın ve altına 1 ml ve üzerinde 1 ml Triton X-100 PBS içinde% 0,5 permeabilization ekleme ekleyin. 4 en az 12 saat inkübe ° C. </li><li> Permeabilization sonra, en az PBS içinde% 20 BSA ile bloke4 saat oda sıcaklığında ya da geceleme 4 ° C Bu aşamada, dilim, en az 3 gün, 4 ° C engelleme çözüm tutulabilir </li><li> Dilimleri dikkatle serebellar dilim bağlı eklemek membran parça kesme ve PBS içeren 24 kuyucuğu transfer leke için. </li><li> Immün TUNEL reaktif ile ilk bir saat ve Calbidin antikor ile bir gecede, sırayla yapılır. <ol><li> PBS aspire edin ve her dilim tüm yüzeyini kaplayan emin TUNEL reaktif 250 mcL ekleyin. Karanlık bir saat için 37 ° C&#39;de inkübe edin. </li><li> TUNEL karışımı kaldırmak ve bir saat 10 dakika PBS ile üç defa yıkamak sonra. </li><li> Son yıkamadan sonra, PBS kaldırmak ve 1 / 1000 PBS içinde Calbindin antikor seyreltme 250 mcL ekleyin ve 4 gece boyunca inkübe ° C de karanlıkta. </li><li> Birincil antikor çözüm çıkarın ve 10 dakika PBS ile üç kez yıkayın. </li><li> Ikincil antikor ekle 250 mcL karanlıkta oda sıcaklığında en az üç saat süreyle inkübe PBS ve 1 / 500 sulandırılmış. TUNEL kiti kullanılan TMR kırmızı etiketli bu nedenle Calbindin için sekonder antikoru Alexa-488 ile konjuge olur. </li><li> PBS ile 10 dakika boyunca üç kez yıkayın ve dilim DAPI içeren bir montaj medya kullanarak cam bir mikroskop lamı üzerine monte. </li><li> Sırasıyla 488 nm ve 561 nm eksitasyon dalga boyu Calbindin ve TUNEL kullanarak konfokal mikroskop ile resim dilimleri. </li></ol></li></ol> 3. Temsilcisi Sonuçlar: Bu protokolün temel zorluk, bizim son okuma hücre ölümü olacaktır, özellikle de sağlıklı serebellar dilim kültürlerini oluşturmak için muktedir. Sağlıklı bir kültür, bir hücre gövdesi katmanı saydam olarak görünür ve mikroskop altında beyincik (Şekil 1) yapraklanmış yapısını görmenizi sağlar. Kültür Birkaç gün sonra dilimleri ince ve non-nöronal hücrelerin başlar dilim marjları uzak göç görülebilir. Kültür birkaç gün sonra dilimleri kapsayan koyu renkli yuvarlak hücreler (büyük olasılıkla makrofajlar), sonunda 10-14 gün sonra, in vitro (Şekil 2) 5 sayısı azalacak. Dilim canlılığı için nihai kanıtı Purkinje hücreleri gibi Calbindin gibi farklı hücresel belirteçleri, leke. Şekil 3 Purkinje hücre tabakasının çeşitli TUNEL pozitif çekirdeklerin bir temsilcisi konfokal mikroskop görüntü gösterir. Bazı dilim apoptotik uyaran aldı bile, onlar sadece 24 saat açık olduğunu göz önünde bulundurmak önemlidir. Bu TUNEL boyama ile ölmekte olan hücrelerin DNA parçalanması gözlemlemek için yeterli zaman izin verilir, ancak belirli bir Purkinje hücre tabakası hala mevcut. <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="Şekil 1" /> Şekil 1. DIV2 Sağlıklı serebellar dilim Bu görüntü, saydam hücre gövdesi tabaka ve sağlıklı bir dilim yapraklanmış serebellar yapı gösterir . <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="Şekil 2" /> Şekil 2. DIV 7 Sağlıklı serebellar dilim Bu resimde biz hala serebellum ve karanlık bir hücreye organları görünümünü yapraklanmış yapısı izleyebilirsiniz. <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="Şekil 3" /> Şekil 3. Fas tedavi serebellar dilim Temsilcisi görüntü konfokal Bu görüntü Calbindin (yeşil) ve TUNEL boyama (kırmızı) için pozitif apoptotik hücreleri ile boyandı Purkinje hücreleri gösterir. Purkinje hücreleri tek bir satır görünmüyor ama Folium gibi bir yapı oluşturan kümelenmiş.

<ol>
	<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+ependymal+cells+in+tissue+cultures.">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures.">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocol+for+organotypic+hippocampal+slice+cultures.">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Beat, H., G&#228;hwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells. , 461-498 (1998).</li></ol>

IACUC tarafından belirlenen politikalara uygun olarak yapılan tüm hayvan çalışmaları yapıldı. Salk Enstitüsü&#39;nden AAALAC akredite bir tesistir.

Bu yöntem, nöronal Organotipik kültürlerin birçok olası uygulamalar biri açıklar ve yabani tip ve mutant serebellar dilim apoptotik uyaranların etkisini araştırmak için izin verdi. Bu deneyin en önemli parçası, sürekli olarak sağlıklı serebellar dilim kültürü oluşturmak mümkün olmaktır. Önemli faktörler diseksiyon ve dilimleme tekniği ve mümkün olan en kısa zamanda protokol gerçekleştirme yeteneği, ama aynı zamanda dikkatli dilim zarar görmemesi için. Bir diğer önemli faktör, kültür ortamının hazırlanması ve kompozisyon, bu kültürlerin çok hassastır, bu yüzden biz, medyanın çeşitli yemek tarifleri ve markalar denemek zorunda kaldık. , Serum veya medya herhangi bir diğer bileşenler bile farklı partiler dilim canlılığı etkileyebilir. Biz Invitrogen Lot # 480.116 At Serumu ile en iyi sonuçları var. Serebellar dilim sonra bir serebellar gelişim, elektrofizyoloji, hücre yaşamını tek başına veya apoptotik uyaranlara eksitotoksisitesi veya oksijensizlik yanıt olarak eğitim görebilirsiniz. Yabani tip ve mutant serebellar dilimleri kullanarak karşılaştırmalı çalışmalar serebellar fonksiyon ve gelişiminin pek çok açıdan çok anlayışlı edilmiştir.

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<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11090</td>
<td>&nbsp;</td>
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<td>Horse Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
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<td>Hepes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
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<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/ Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Superglue</td>
<td>&nbsp;</td>
<td>Krazy Glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well / 24 well cell culture plates</td>
<td>&nbsp;</td>
<td>Corning Incorporated</td>
<td>3506/ 3527</td>
<td>&nbsp;</td>
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<td>30mm culture plate inserts (0.4-&mu;m pore)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>554254</td>
<td>&nbsp;</td>
<tr>
<td>In situ cell death detection kit, TMR red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>12 156 792 910</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-Calbindin D-28k antibody</td>
<td>&nbsp;</td>
<td>Swant</td>
<td>CB-38a</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 488-goat anti-rabbit IgG secondary antibody</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular probes)</td>
<td>A-11008</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector labs</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Zeiss Stemi SV 11</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica TCS SP2 AOBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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organotypic cerebellar cultures: apoptotic challenges and detection

Nöronal doku Organotipik kültürleri, ilk kez 1947 1,2 Hogue tarafından tanıtıldı ve nörobilim alanında önemli bir atılım teşkil etmiştir . O zamandan beri, bu teknik daha da geliştirilmiş ve henüz Organotipik kültürleri hazırlamak için birçok farklı yolu vardır. Stoppini ve arkadaşları tarafından açıklanan bir uyarlanmış yöntemi burada sundu. Dilimleri hazırlanması ve Gogolla ark. Boyama işlemi 3,4 için. Bu tekniğin eşsiz bir özelliği, tüm hücresel organizasyonu ve nöron ağları bozulduğu olduğu ayrışmış kültürleri üzerinde büyük bir avantaj sağlayarak, kendi özgün yapısı hipokampus ve serebellum gibi beynin farklı yerlerinde çalışmaya olanak veren. Serebellumun durumda Purkinje hücrelerinin çalışma, ayrışmış temel kültür olarak elde etmek için son derece zor izin verdiğinden bile daha avantajlıdır. Bu yöntem, in vitro olarak serebellum bazı gelişimsel özellikleri yanı sıra çalışmada, yabani tip ve mutant fareler hem de elektrofizyolojik ve farmakolojik deneyler için kullanılabilir . Apoptotik hücre ölümü ölçmek için TUNEL boyama yöntemi, burada açıklanan, gelişmekte olan beyincik Fas ligand olarak apoptotik uyaranlara etkisini araştırmak için dizayn edilmiştir. TUNEL boyama gibi Calbindin Purkinje hücreleri için hücre tipi özgül belirteçler ile birlikte ise, bir hücre popülasyonu belirli bir şekilde hücre ölümünü değerlendirmek mümkündür. Calbindin boyama da serebellar kültürlerin kalitesini değerlendirmek amacıyla hizmet vermektedir.

Watch this Scientific Journal Video about Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection at JoVE.com

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Bu yöntem, Organotipik serebellar kültürlerin üretimi ve farklı serebellar hücre tiplerinin canlılığı belli apoptotik uyaranların etkisi açıklanmıştır.

Organotipik Serebellar Kültürler: Apoptotik Zorluklar ve Tayin

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

Research

JoVE Journal

Neuroscience

18.1K Görüntüleme.  The Salk Institute for Biological Studies. Bu yöntem, Organotipik serebellar kültürlerin üretimi ve farklı serebellar hücre tiplerinin canlılığı belli apoptotik uyaranların etkisi açıklanmıştır.

Video: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotipik Hipokampal Dilim Kültürler

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Nörobilim

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotipik Kültürleri nöronal Çığ Çok elektrot Dizi Kayıtlar

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Embriyonik ventral orta beyin Organotipik Dilim Kültürler: dopaminerjik Nöronal Gelişim Eğitim Bir Sistem In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotipik Dilim Kültürleri Purkinje Hücre Dendritik Morfoloji Analizi

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotipik Dilim Kültürler Oligodendrosit Dinamikler ve miyelinasyonun incelemek için

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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 ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Beyindeki granü hücre kültürlerinden mikroglia zayıflatılması, L-lösin metil ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Çok elektrot Dizileri Yetiştirilen Organotipik Omurilik Co-kültürler Fonksiyonel Rejenerasyon incelenmesi

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

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

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

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

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

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

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Eski Utero Elektroporasyon ve embriyonik fare beyni canlı görüntüleme, GABAergic Interneurons geçiş için Organotypic dilim kültürleri

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...