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>

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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.

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			<td height="46" rowspan="2" style="height:47px;width:265px;">Name of Reagent/ Equipment</td>
			<td rowspan="2" style="width:131px;">Company</td>
			<td rowspan="2" style="width:135px;">Catalog Number</td>
			<td rowspan="2" style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">5-chloro-2&#39;-deoxyuridine (CldU)</td>
			<td style="width:131px;">MP Biomedicals</td>
			<td align="right" style="width:135px;">105478</td>
			<td style="width:239px;">Hazardous, Carcinogenic</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Cell culture inserts, 30mm diameter, 0.4&#181;m pore size</td>
			<td style="width:131px;">Thermo scientific&#160;</td>
			<td align="right" style="width:135px;">140660</td>
			<td style="width:239px;">Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Conical Centrifuge tubes (sterile)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">14-432-22</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Dissector scissors (angled to side)</td>
			<td style="width:131px;">Fine Science Tools&#160;</td>
			<td style="width:135px;">14082-09</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Minimum essential medium (MEM)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">11095; liquid</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Eclipse Ni-U fluorescent microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Glue for tissue</td>
			<td style="width:131px;">Krazy Glue</td>
			<td style="width:135px;">KG585</td>
			<td style="width:239px;">Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Hank&#8217;s Balanced Salt Solution (HBSS) (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">14025-092</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Horse Serum Heat Inactivated (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">16050-122</td>
			<td style="width:239px;">Make 50 mL aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Kimwipes</td>
			<td style="width:131px;">Kimberly-Clarke</td>
			<td style="width:135px;">TW 31KYPBX</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:265px;">Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Petri Dish (100mm x 15mm) and (60mm x 15mm)</td>
			<td style="width:131px;">Fisher Brand</td>
			<td style="width:135px;">FB0875712 and FB0875713A</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel blades #11</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10011-00</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel handle #3</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10003-12</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Serological Pipettes</td>
			<td style="width:131px;">Sorfa Medical Plastic Co.</td>
			<td style="width:135px;">P8050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Standard Pattern forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Sterile vacuum filter</td>
			<td style="width:131px;">Thermo-Scientific</td>
			<td style="width:135px;">565-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Surgical Scissors</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">14054-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Syringe driven filter unit</td>
			<td style="width:131px;">Millipore-Millex</td>
			<td style="width:135px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Tissue chopper with moveable stage</td>
			<td style="width:131px;">Stoelting&#160;</td>
			<td align="right" style="width:135px;">51425</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Fine tip paintbrush</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></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

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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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Fare Hipokampal Doku Ex Utero Elektroporasyon ve Organotipik Dilim Kültür

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Gelişim Biyolojisi

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Güçsüz İnsan dermis kullanma Genetiği Değiştirilmiş Organotipik Cilt Kültürlerin Üretimi

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Fare Kulak ve Kuyruk Dokular Yaratma İlköğretim Fibroblast Kültürleri

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Görüntüleme Tek hücreli Çözünürlük embriyonik ve Postnatal Kalpleri Temizlendi

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Doğum sonrası kardiyomiyositlerde Hücre Döngüsü Varyasyonları görselleştirme ve Çekirdeklenme Belirlenmesi

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Diseksiyon ve fare hipofiz bezi geliştirme koronal dilim hazırlık

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Vestibüler ve işitme duyu organları kültürleri üç boyutlu Organotypic

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Fare pankreatik endokrin hücreleri tek hücre Transcriptomic analizleri

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

Nörojenez P19 embriyonal karsinom hücrelerini kullanarak

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Murin Nörogenezi Sırasında Çentik Sinyal Dinamiğinin Gerçek Zamanlı Biyolüminesans Görüntülemesi

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...