Name: organotypic slice cultures for studies of postnatal neurogenesis
Uploaded: 2015-03-04T13:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

NOT: Tüm hayvan prosedürleri Toronto Üniversitesi Karşılaştırmalı Tıp Anabilim hayvan sağlığı ve refahı kurallarına uyduğu. Hipokampal Dilimleri hazırlanması 1. <ol><li> 125 ° C&#39;de kuru otoklav kullanarak aşağıdaki aletleri sterilize: Neşter kolu (# 3) (2), Standart desen forseps, büyük (1), Küçük disektör makas (yan açılı) (1), Mikro kaşık (kaşık ve düz spatula uçları) (1), yuvarlak Mikro-spatula (ve konik uçları yuvarlatılmış) (2), İnce fırça (1), Yangın parlatılmış Pasteur pipeti (2), tül kareler, 2 x 2 inç (5). </li><li> Ne zaman steril, steril bir kap içine koyun ve aletleri kullanıma kadar kapalı tutun. Hemen diseksiyon önce% 70&#39;lik bir etanol çözeltisi içinde aletleri bırakın. </li><li> 35 ° C a kuvöz plaka de / kültür ortamı, 1 ml ilave edilmesi ve depolanması ile diseksiyon işlemine başlamadan önce, bir kültür olarak yerleştirilmiş bir 6-yuvalı kültür plakası hazırlayınnd% 5 CO 2. </li></ol> 2. Steril Laminer Akış kaputu Diseksiyon Araçlar düzenleyin <ol><li> % 70 etanol ile laminer akış kaputu Sprey ve alkolden sterilize diseksiyon aletleri çıkarın. Steril Petri kabı üzerinde duran alkol ve disseke beyin dokusu arasındaki teması önlemek için ise araçlar kurumasını bekleyin. </li><li> 2 büyük steril Petri kapları içinde steril, buz gibi soğuk kesme çözeltisi Deposit 5-7 mi. Bir tabak soğuk ve kafa (kirli), soğutma ve scooped beyin (temiz) durulama için diğerini temizler. Beyin dışarı Anatomi için Petri kabı kapaklarının birinde steril filtre kağıdı yerleştirin. </li><li> Hipokampus diseksiyon küçük Petri kabı kapağının birinde küçük, sterilize filtre kağıdı yerleştirin. 2 küçük steril Petri kapları içinde sterilize buz soğukluğunda kesme çözeltisi Deposit 3-5 mi. Bir çanak, bir hipokampal bölümlere ayrılması sırasında bölümleri yapacak, scooped hipokampus yapacakdiseksiyon mikroskop altında. </li><li> Kesme aşamasına steril filtre kağıdı parçası ölçme ve steril bir traş makinesi bıçak ağzı tarafından, doku kesicisi hazırlayın. Sterilize diseksiyon çözümü ile filtre kağıdı ıslatın. </li><li> Alkol ile temiz bir biyo-çanta Sprey ve laminer akış temiz tezgah yerleştirin. </li></ol> 3. Hipokampal Diseksiyon <ol><li> Laminer akımlı temiz tezgah dışında% 70 etanol ile P7 Sprague Dawley sıçan yavru Sprey ve hızlı bir şekilde laminer akış tezgah içinde büyük steril cerrahi makas kullanarak hayvan başını kesmek. Baş Petri yemekleri biri buz gibi soğuk diseksiyon çözüm içine bırakalım. </li><li> Petri kabındaki, kan durulayın ve hızlı bir şekilde aşağı sterilize filtre kağıdına kafa, ventral tarafı aktarmak. </li><li> Sagital düzlemde dorsal yüzeyi boyunca kesilmiş neşter kullanarak altta yatan kafatası ortaya çıkarmak. Yumuşak ve t sıçanlarda kolayca nüfuz altta yatan kemik, deri yoluyla kesin değilOnun yaşı. Kenara bu &quot;kirli&quot; neşter ayarlayın ve beyin dokusu üzerinde kullanmayın. </li><li> Küçük disektör (yan açılı) makas ve forseps kullanarak, bregmaya kafatası sagital sütür boyunca koronal sütür sagital sütür ile dik kesişen kafatası üzerinde anatomik nokta kafatasını açık kesti. Kafatasının orta hattan yukarı ve uzağa kafatası kanatları çekmek için forseps kullanın. </li><li> Yavaşça kafatasının dışında beyin kaldırmak için, beyin sapı altındaki beynin alt, mikro kaşık yerleştirin. Beynin bazal yüzeyinde optik sinirler ve koku ampul maruz beyin kaldırın. Tam kafatası beyin ayırmak için küçük makas ile bu yapıları kesin. Çıkarın ve buz soğukluğunda bir kesme çözeltisini ihtiva eden başka bir büyük bir Petri tabağına sağlam beyin aktarın. </li><li> Mikro kaşık kullanarak, steril filtre kağıdı içeren küçük bir Petri-çanak kapak beyin aktarın. Steril bir Pasteur pipeti ile dis birkaç damla ile beyin durulayınçözüm secting doku nemli tutmak için. </li><li> &quot;Temiz&quot; neşter bıçak kullanarak arayla iki hemisfer kesti. Mikro kaşıkla büyük Petri kabı sol arka yarımkürede aktarın ve sonraki kullanım için buz gibi soğuk diseksiyon çözeltisi içinde aşağı yarımkürede pia tarafı yerleştirin. </li><li> Sağ hemisfer medial yüzünü görüntüleyin ve kenar forniksinin, hipokampusun medial kenarı boyunca beyaz cevher önemli bir bant tespit. Steril bir neşter kullanarak, forniksten aracılığıyla bir sagital kesim yapmak, ancak neşter ucu sadece 0,5 cm forniksinin kesmek için yeterli olacaktır, çünkü dikkat çekmek. </li><li> 2 mikro-spatula kullanarak, beyin sapı üzerinde sağ-spatula yerleştirerek ve sol spatula ile örten korteks kaldırarak sağ hemisfer ilk hipokampus çıkarın. Yavaşça hipokampus lateral ventrikül ve medial yüzeyi ortaya çıkarmak için korteks kaldırın. Beyaz eğri çizgi, Fimbria, şimdi görünür olmalıdır. </li><li> Curva ile spatula eğrilik hizalayınfimbrial Ture ve yavaşça fimbrial altında spatula basın. Sol spatula kaydırın ve rostral-kaudal ekseni boyunca binmek ve daha sonra hipokampus çıkarmak için dorsal yönde spatula kaldırın. </li><li> Buz gibi soğuk diseksiyon çözeltisi ile bir 2. küçük Petri kabı hipokampus aktarın. Sol hipokampus kaldırmak için sol yarımkürede aynı işlemi tekrarlayın. </li><li> Bir mikro spatula kullanarak, dikkatle doku kıyıcı aşamasına hippocampi aktarın. Kıyıcı bıçak eksenine birbirine dik bunları bitişik ve paralel düzenlemek. Doku konumuna Hipokampuslardan ve üst kesme solüsyonu ve bir kaç damla eklemek için bir fırça kullanılır. </li><li> Bireysel dilimleri kaldırmak için duraklamadan 400 mikron dilimleri doku kesin (genellikle bıçak uygun olmaz). Bütün hipokamp kesilmiş sonra, yavaşça diseksiyon çözeltisi ile 2. küçük bir Petri tabağına bölümleri aktarmak için fırça kullanılır. </li><li> Reklamın altındamikroskop issecting, dikkatle amicro-spatula ve fırça kullanarak kayan dilimleri ayırmak. </li><li> Bir laminar akış başlığı içinde kuluçka ve yerine kültür girişi ile önceden hazırlanmış bir kültür tabağı çıkarın. </li><li> Bir yangın cilalı Pasteur pipet kullanarak, kültür insert membran apikal yüzeye pipet ve transfer dilimler halinde 4-5 dilim çizin. Sonraki, bir fırça ile konumlandırma ayarlayın ve bireysel bölümlere ve kültür insert sınırında arasında boşluk bırakın. </li><li> Steril bir Pasteur pipeti kullanılarak, zarın tepe yüzey aşırı kesme uzaklaştırın. NOT: pipet içine doku bölümleri çizim çözüm kaçınınız çıkarırken. Alternatif olarak, yavaş yavaş çözüm kaldırmak için sterilize pipet uçları ile düzenli pipet (P200 veya P1000) kullanın. </li><li> Serum içeren kültür ortamı ile, 35 ° C&#39;de tekrar inkübatör içine hipokampal dilimler ve% 5 CO2 ile birlikte kültür plakası yerleştirin. NOT: Deney aramaları durumundaBirden hayvanlardan kültürleri üretmek için, iyice diseksiyonların arasındaki laminer akış temiz tezgah temizleyin. Alkol çözüm araçları dönün ve tüm Petri-yemekleri, filtre kağıtları ve ikinci diseksiyon öncesinde steril Jilet değiştirin. </li></ol> 4. Beslenme ve Organotipik Dilimleri bakımı <ol><li> Steril bir laminer akımlı temiz tezgah Yem kültürleri. </li><li> Kültürlü bölümlerde 2 gün sonrası diseksiyon ilk beslenmesini yapın. Steril cam pipet kullanarak aspire eski kültür ortamı. </li><li> Çukurlara, taze, steril, serum ihtiva eden ortamda 1 ml eklemek için 5 ml steril serolojik pipet kullanın. </li><li> Yavaşça kültür yuvasını yerine ve membran yüzeyinde altında oluşmuş olabilecek herhangi bir hava kabarcıklarını çıkarmak için özen gösterin. </li><li> İlk besleme sonra, her gün orta değiştirin. </li></ol> Timidin Analoglarının 5. Enkübe Doku Dilimleri Yenidoğan Nöronlar Etiket için <ol><li> Incelemek için,olgunlaşma ve hipokampus dentat granül hücrelerin entegrasyonu Bromodoksiüridin (BrdU) veya Chlorodeoxyuridine (CldU) gibi bir timidin analoğu olan Organotipik dilimleri inkübe edin. Burada, çünkü tuzlu su içinde daha yüksek çözünürlüğe sahip CldU kullanır. </li><li> 3DIV sonra, 10 ug / ml&#39;lik bir son konsantrasyon için, kültür ortamı içinde 1 ml tuzlu su içinde 10 mg / ml CldU stok çözeltisi 1 ul ekle. BrdU kullanılırsa, molekül ağırlığı farklılıkları konsantrasyonu düzeltin. Kültür çukurlarına CldU ile ortamı ilave edin ve 35 ° C&#39;de 2 saat boyunca ortam CldU-içeren doku inkübe edin. </li><li> 35 ° C&#39;de inkübasyon 2 saat sonra, CldU içeren orta kaldırmak ve (aşağıda belirtilmiştir), normal beslenme programı devam etmek için düzenli olarak besleme ortamı ile değiştirin. </li></ol> 6. Doku Fiksasyon ve Depolama <ol><li> Tedavileri uygulayarak ve kültür deneyleri (Şekil 2B) başlamadan önce doku örnekleri sabitlemek için bir zaman çizelgesi oluşturulması. </li> &lt;önceden tespit edilmiş bir gün sonraki diseksiyon Li&gt;, bir laboratuar bir davlumbaz aşağıdaki hazırlamak: 10-50 ml&#39;lik beher cam, fosfat tamponlu tuzlu su (PBS) içinde% 4 paraformaldehit (PFA) ihtiva etmektedir; atılan kültür ortamı için boş bir beher; küçük forseps; tek kullanımlık uçları olan ve 1.000 ml&#39;lik pipet. </li><li> Davlumbaz için kuluçka odası ve transfer kültür plakası (ler) sökün. Forseps ile bireysel kuyu plaka ekler eğin. Sonraki, bir bertaraf beher kültür ortamı ve transferi kaldırmak için bir pipet kullanın. Tüm kültür ortamı çıkarılır emin olmak için bir açıyla kültürü plakası yatırın. </li><li> Bir seferde bir kültür plaka için ortamı çıkarmadan bitirin. Orta kaldırıldığında, her bir kültür için% 4 PFA 1 ml ekleyin ve iyice parafilm plaka sızdırmaz. 24 saat süre ile 4 ° C&#39;de bir buzdolabı için gerekli ve aktarma levhaları kadar kültür plakaları için tekrarlayın. </li><li> 24 saat sonra, bir çeker ocak içinde, aşağıdaki hazırlama: 10-50 PBS içinde% 0.1 sodyum azid ihtiva eden mi beher; Boşatılan PFA (toksik maddelerin imha ederken güvenlik önlemlerine uyun) için beher; küçük forseps; tek kullanımlık uçları olan ve 1.000 ml pipet. </li><li> PFA eklemek için 6.4 özetlenen prosedürü uygulayın, ancak bunun yerine sodyumazid ile PBS 1 ml ekleyin. Bir kez tamamen parafilm kenarları sarılması ve 4 ° C&#39;de buzdolabında muhafaza gelecekteki kullanım için, sızdırmaz kuyucuğu aktarılır. </li></ol> 7. İmmünohistokimya için Doku Kesit <ol><li> Bir vibratome kullanarak kesit doku gerçekleştirin. adımlar aşağıdaki serisi organotipik kültürlerden kullanılabilir doku kesitlerinin verimi üst düzeye çıkarmanıza yardımcı. Not: hemen hipokampal diseksiyon ve dilimlerin kaplama ardından doku yaklaşık 400 um&#39;lik bir kalınlığa sahiptir. Bununla birlikte, inkübasyon odasında 2-3 hafta sonra doku dilimleri 150-300 um&#39;lik bir son kısmı kalınlığı ile sonuçlanarak, düzleştirmek başlayacaktır. </li><li> Bölüm kültürlü dokuya aşağıdaki öğeleri hazırlayın: # 11 neşter bLade ve idare, doku monte buz soğuk PBS, bir mikro-diseksiyonu forseps ve temiz bir vibratome kesme aşamasını içeren bir cam Petri. </li><li> Plastik uç gövdesinden ayrılmış, böylece bir neşter kullanarak dikkatli bir şekilde dairesel uç zarın çevresi boyunca kesmek için kullanılır. Kültürlü dilim ve neşter arasındaki geniş boşluk bırakın. </li><li> PBS içeren bir petri tabağına konulacak ve müstakil uç membran aktarın. PBS ile durulandıktan sonra vibratome montaj aşamasına membran aktarmak için forseps kullanır. </li><li> Sonraki, kültürlü dilimleri çevreleyen membran aşırı ortadan kaldırmak ve temiz kenarları oluşturmak için fazla malzeme kesip neşter kullanın. Membran sağlayacak bu adım, düz ve kolayca kesme yüzeyine yapışabilir. </li><li> Vibratome kesme sahnede yapışkan 1-2 damla koyun ve hatta 22 G iğne kullanılarak katmanda yayıldı. Vibratome kesme bıçağının uzun kenar paralel bir dikdörtgen şekil yapıştırıcıyı yayıldı. Bu adımı gerçekleştirinhızlı bir şekilde, kurutma yapıştırıcıyı önler. </li><li> Kesme aşamasına hipokampal dilimleri içeren kesilmiş membran aktarmak ve yavaşça tutkal membran konumlandırmak ve hiçbir hava kabarcığı olmadığından emin olmak için forseps kullanın. </li><li> Süper yapıştırıcı kurudukça geri içeren PBS Petri kabı yapıştırılmış membran ile vibratome aşamasına aktarılır. Vibratome bıçak ve doku bölümleri depolamak için bir 48 oyuklu plaka ihtiva eden sodyum azid hazırlayın. </li><li> Depolama ve daha sonra immünohistokimyasal boyanması için bir 48 oyuklu plaka içeren sodyum azide Organotipik dilim doku ve transferin 30 um bölümler oluşturmak için vibratome kullanın. </li><li> Immünohistokimyasal boyama 26,29 için protokolleri mevcut önceden başvurun. </li></ol>

<ol>
	<li>Buchs, P. A., Stoppini, L., 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=Structural+modifications+associated+with+synaptic+development+in+area+CA1+of+rat+hippocampal+organotypic+cultures.">Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.</a> Brain research. Developmental Brain Research. 71 (1), 81-91 (1993).</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> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Opitz-Araya, X., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures.">Organotypic hippocampal slice cultures.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Muller, D., Buchs, P. A., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+synaptic+development+in+hippocampal+organotypic+cultures.">Time course of synaptic development in hippocampal organotypic cultures.</a> Developmental Brain Research. 71 (1), 93-100 (1993).</li><li>Rio, J. A., Heimrich, B., Soriano, E., Schwegler, H., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+glial+fibrillary+acidic+protein-immunoreactive+glial+cells+in+organotypic+slice+cultures+of+rat+hippocampus.">Proliferation and differentiation of glial fibrillary acidic protein-immunoreactive glial cells in organotypic slice cultures of rat hippocampus.</a> Neuroscience. 43 (2-3), 335-347 (1991).</li><li>Ziemka-Nalecz, M., Stanaszek, L., Zalewska, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-glucose+deprivation+promotes+gliogenesis+and+microglia+activation+in+organotypic+hippocampal+slice+culture:+involvement+of+metalloproteinases.">Oxygen-glucose deprivation promotes gliogenesis and microglia activation in organotypic hippocampal slice culture: involvement of metalloproteinases.</a> Acta Neurobiologiae Experimentalis. 73 (1), 130-142 (2013).</li><li>Subramanian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+Lhx2+is+necessary+and+sufficient+to+suppress+astrogliogenesis+and+promote+neurogenesis+in+the+developing+hippocampus.">Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), E265-E274 (2011).</li><li>Strassburger, M., Braun, H., Reymann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+treatment+with+the+p38+mitogen-activated+protein+kinase+inhibitor+SB239063+is+neuroprotective,+decreases+the+number+of+activated+microglia+and+facilitates+neurogenesis+in+oxygen-glucose-deprived+hippocampal+slice+cultures.">Anti-inflammatory treatment with the p38 mitogen-activated protein kinase inhibitor SB239063 is neuroprotective, decreases the number of activated microglia and facilitates neurogenesis in oxygen-glucose-deprived hippocampal slice cultures.</a> European Journal Of Pharmacology. 592 (1-3), 55-61 (2008).</li><li>Sadgrove, M. P., Chad, J. E., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kainic+acid+induces+rapid+cell+death+followed+by+transiently+reduced+cell+proliferation+in+the+immature+granule+cell+layer+of+rat+organotypic+hippocampal+slice+cultures.">Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures.</a> Brain Research. 1035 (2), 111-119 (2005).</li><li>Wise-Faberowski, L., Robinson, P. N., Rich, S., Warner, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+deprivation+in+an+organotypic+hippocampal+slice+model+of+the+developing+rat+brain:+the+effects+on+N-methyl-D-aspartate+subunit+composition.">Oxygen and glucose deprivation in an organotypic hippocampal slice model of the developing rat brain: the effects on N-methyl-D-aspartate subunit composition.</a> Anesthesia and Analgesia. 109 (1), 205-210 (2009).</li><li>Cho, S., Wood, A., Brain Bowlby, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slices+as+models+for+neurodegenerative+disease+and+screening+platforms+to+identify+novel+therapeutics.">slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics.</a> Current Neuropharmacology. 5 (1), 19-33 (2007).</li><li>Noraberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures+for+studies+of+brain+damage,+neuroprotection+and+neurorepair.+Current+Drug+Targets.">Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Current Drug Targets.</a> CNS And Neurological Disorders. 4 (4), 435-452 (2005).</li><li>Berdichevsky, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K-Akt+signaling+activates+mTOR-mediated+epileptogenesis+in+organotypic+hippocampal+culture+model+of+post-traumatic+epilepsy.">PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 33 (21), 9056-9067 (2013).</li><li>Koyama, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+excitation+after+febrile+seizures+induces+ectopic+granule+cells+and+adult+epilepsy.">GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.</a> Nature Medicine. 18 (8), 1271 (2012).</li><li>Staley, K. J., White, A., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+spikes:+harbingers+or+causes+of+epilepsy.">Interictal spikes: harbingers or causes of epilepsy.</a> Neuroscience Letters. 497 (3), 247-250 (2011).</li><li>Lee, H., Lee, D., Park, C. H., Ho, W. K., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+mediates+the+network+activity-dependent+facilitation+of+axonal+outgrowth+from+the+newborn+granule+cells+in+the+early+postnatal+rat+hippocampus.">GABA mediates the network activity-dependent facilitation of axonal outgrowth from the newborn granule cells in the early postnatal rat hippocampus.</a> The European Journal Of Neuroscience. 36 (6), 2743-2752 (2012).</li><li>Raineteau, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+labeling+of+newborn+granule+cells+to+visualize+their+integration+into+established+circuits+in+hippocampal+slice+cultures.">Conditional labeling of newborn granule cells to visualize their integration into established circuits in hippocampal slice cultures.</a> Molecular and Cellular Neurosciences. 32 (4), 344-355 (2006).</li><li>Raineteau, O., Rietschin, L., Gradwohl, G., Guillemot, F., Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

CldU (veya BrdU) uygulamasını takiben, farmakolojik ajanların uygulama çizelgesi belirli bir gelişme pencereleri sırasında doğan KEGM hedef olarak seçilebilir. Örneğin, bir varsayımsal madde, GABA depolarize yerde bir gelişme aşamasında olgunlaşmamış nöronların yaşı denk önerilmiştir ikinci hafta sonra CldU enjeksiyon sırasında uygulanabilir. Bu protokolü kullanarak gelecekteki çalışmalar farmakolojik ajan ve &quot;terzi&quot; özel ilgi deneysel soruya yaklaşım maruz kalma penceresi adap...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

<table><tbody><tr><td>5-chloro-2&#039;-deoxyuridine (CldU)</td><td>MP Biomedicals</td><td>105478</td><td>Hazardous, Carcinogenic</td></tr><tr><td>Cell culture inserts, 30 mm diameter, 0.4 &amp;micro;m pore size</td><td>Thermo scientific&amp;nbsp;</td><td>140660</td><td>Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td></tr><tr><td>Conical Centrifuge tubes (sterile)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Dissector scissors (angled to side)</td><td>Fine Science Tools&amp;nbsp;</td><td>14082-09</td><td></td></tr><tr><td>Minimum essential medium (MEM)</td><td>Gibco</td><td>11095; liquid</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Eclipse Ni-U fluorescent microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Glue for tissue</td><td>Krazy Glue</td><td>KG585</td><td>Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS) (500 ml)</td><td>Gibco</td><td>14025-092</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Horse Serum Heat Inactivated (500 ml)</td><td>Gibco</td><td>16050-122</td><td>Make 50 ml aliquots and store at -20 &amp;deg;C</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clarke</td><td>TW 31KYPBX</td><td></td></tr><tr><td>Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td><td></td><td></td><td></td></tr><tr><td>Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)</td><td>Fisher Brand</td><td>FB0875712 and FB0875713A</td><td></td></tr><tr><td>Scalpel blades #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Serological Pipettes</td><td>Sorfa Medical Plastic Co.</td><td>P8050</td><td></td></tr><tr><td>Standard Pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Sterile vacuum filter</td><td>Thermo-Scientific</td><td>565-0020</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr><tr><td>Syringe driven filter unit</td><td>Millipore-Millex</td><td>SLGP033RS</td><td></td></tr><tr><td>Tissue chopper with moveable stage</td><td>Stoelting&amp;nbsp;</td><td>51425</td><td></td></tr><tr><td>Fine tip paintbrush</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures for studies of postnatal neurogenesis

Burada organotipik dilim kültür tekniği kullanılarak kemirgen beyin hipokampal doğum sonrası nöron incelemek için bir teknik tarif. Gelişmekte olan hipokamp kıvrımlarının farmakolojik maddelerin doğrudan uygulanmasını sağlayan, bu yöntem, hipokampus karakteristik topografik morfolojisini muhafaza eder. Buna ek olarak, dilim kültürlerinin kadar, 4 hafta boyunca muhafaza edilebilir ve böylece, tek bir yeni doğmuş granül nöronların olgunlaşma sürecini çalışma sağlar. Karmaşık değişkenler hariç ederken Dilim kültürleri gibi hipokampus derin anatomik konumu yanı sıra, kan beyin bariyerinin ilgili belirsizlikler gibi hipokampal dilim verimli farmakolojik manipülasyon için izin verir. Bu nedenlerden dolayı, doğum sonrası nöron araştırma için özel Organotipik dilim kültürlerinin optimize etmek için çalışmıştır.

1) dilimleri, in vitro olarak 10-21 gün sonra (DIV), sonra hipokampal dilimler karakteristik morfolojik özelliklerini korumak ve 2) yenidoğan KEGM sayısal edilebilir: Organotipik kültürler yetişkin nöron araştırma için uygun olacağını belirlemek iki ana kriterlerine uygun olması gerekmektedir erişkin nöron araştırma kullanılan standart immünohistokimyasal teknikler kullanılarak. İlk kriter ilgili olarak, Şekil 1A ve 1B korunmuş hipokampal morfolojisi vu...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Burada organotipik dilim kültür tekniği kullanılarak hipokampal doğum sonrası nöron incelemek için bir teknik tarif. Bu yöntem, yetişkin nöron in vitro müdahale edilebilmesini sağlar ve kültürlenmiş hipokampus farmakolojik maddelerin doğrudan uygulama için izin verir.

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 ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

Research

JoVE Journal

Developmental Biology

12.2K Görüntüleme.  University of Toronto.  Burada organotipik dilim kültür tekniği kullanılarak hipokampal doğum sonrası nöron incelemek için bir teknik tarif. Bu yöntem, yetişkin nöron in vitro müdahale edilebilmesini sağlar ve kültürlenmiş hipokampus farmakolojik maddelerin doğrudan uygulama için izin verir. 

Video: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18 Rat Brain Organotipik Dilim Kültür

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Biyoloji

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

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Doğum Sonrası Beyin Nöronal Göç Yüksek Çözünürlüklü Zaman Aşımı Görüntüleme için bir Organotipik Dilim Testi

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Nöronal Morphogenesis Çalışması Yöntemleri:<em&gt; Ex vivo</emEmbriyonik Mürin Serebral Korteks olarak&gt; RNAi Elektroporasyon

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

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. ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Yetişkin İnsan Beyninden Kısa Süreli Serbest Kayan Dilim Kültürleri

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Olomotor sinir gelişimi için zaman aşımı görüntüleme için embriyonik GFP-Ifade fareler dan ex vivo okülomotor dilim kültürü

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotipik Serebellar Dilim Kültürlerde Purkinje Hücre Sağkalım

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

POSTNATAL nöron Çalışmaları için organotipik Dilim Kültürler

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