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.

NOTA: Tutte le procedure sugli animali conformi alle linee guida per la salute e il benessere degli animali del Dipartimento di Medicina comparata presso l&#39;Università di Toronto. 1. Preparazione di fettine di ippocampo <ol><li> Sterilizzare i seguenti strumenti che utilizzano l&#39;autoclave secco a 125 ° C: manico bisturi (# 3) (2), pinze modello standard, di grandi dimensioni (1), Piccolo dissettore forbice (angolato a lato) (1), Micro cucchiaio (cucchiaio e piatto estremità spatola) (1), Micro-spatole (arrotondati e arrotondati estremità rastremate) (2), pennello fine (1), Fuoco lucidato pipetta Pasteur (2), le piazze garza, 2 x 2 pollici (5). </li><li> Quando sterili, mettere gli strumenti in un contenitore sterile e mantenere coperta fino al momento dell&#39;uso. Immediatamente prima dissezione, immergere gli strumenti in una soluzione di etanolo al 70%. </li><li> Preparare una piastra di coltura da 6 pozzetti con inserto cultura prima di iniziare la procedura di dissezione aggiungendo 1 ml di terreno di coltura / pozzetto e memorizzare la piastra nella incubatore a 35 ° C unand 5% di CO 2. </li></ol> 2. Disporre strumenti di dissezione in cappa sterile a flusso laminare <ol><li> Spruzzare la cappa a flusso laminare con il 70% di etanolo e rimuovere strumenti di dissezione sterili da alcol. Lasciare gli strumenti per asciugare mentre riposa su una piastra di Petri sterile per evitare il contatto tra l&#39;alcol e il tessuto cerebrale sezionato. </li><li> Deposito 5-7 ml di soluzione di dissezione ghiacciata sterilizzato in 2 grandi piastre sterili. Un piatto sarà raffreddare e pulire la testina (sporca), l&#39;altro per il raffreddamento e il risciacquo del cervello scavato (pulito). Inserire un filtro di carta sterilizzata in uno dei coperchi piatto Petri per sezionare il cervello. </li><li> Inserire un piccolo, carta da filtro sterilizzato in una piccola capsula di Petri coperchio per sezionare l&#39;ippocampo. Deposito 3-5 ml di soluzione di dissezione ghiacciata sterilizzato in 2 piccoli, piastre sterili. Un piatto terrà l&#39;ippocampo scavato, si terrà sezioni durante la separazione delle sezioni di ippocampoal microscopio dissezione. </li><li> Preparare il chopper tessuto con nastro adesivo un pezzo di carta da filtro sterile per la fase di taglio e il montaggio di una lama di rasoio sterile. Bagnare la carta da filtro con una soluzione sterilizzata dissezione. </li><li> Spruzzare un bio-bag pulito con alcool e collocarlo in flusso laminare banco pulito. </li></ol> 3. ippocampale Dissection <ol><li> Spruzzare il ratto pup P7 Sprague Dawley con il 70% di etanolo al di fuori del banco pulito flusso laminare e decapitarlo rapidamente l&#39;animale con grandi forbici chirurgiche sterili all&#39;interno del banco a flusso laminare. Lasciate che la testa cadere nella soluzione dissezione ghiacciata in una delle piastre di Petri. </li><li> Nella capsula di Petri, lavare via il sangue e velocemente trasferire la testa di carta da filtro sterilizzato, lato ventrale verso il basso. </li><li> Usando il bisturi, tagliare lungo la superficie dorsale nel piano sagittale per esporre il cranio sottostante. Tagliare attraverso la pelle, ma non l&#39;osso sottostante, che è morbido e facilmente penetrabili in ratti di tla sua età. Mettere da parte il bisturi &quot;sporco&quot; e non usare su tessuto cerebrale. </li><li> Utilizzando le piccole forbici dissettore (angolato a lato) e pinze, taglio aperto il cranio lungo sutura sagittale del cranio di bregma, il punto anatomica sul cranio dove la sutura coronale è intersecato perpendicolarmente dalla sutura sagittale. Usare pinze per tirare lembi cranio alto e lontano dalla linea mediana del cranio. </li><li> Posizionare il micro cucchiaio sul lato inferiore del cervello, sotto il tronco cerebrale, per sollevare delicatamente il cervello dal cranio. Sollevare il cervello per esporre i nervi ottici e bulbo olfattivo sulla superficie basale del cervello. Tagliare queste strutture con piccole forbici per staccare completamente il cervello da teschio. Rimuovere e trasferire il cervello integro e il loro piatto Petri grande contenente soluzione dissezione ghiacciata. </li><li> Utilizzando il micro cucchiaio, trasferire il cervello di un piccolo coperchio Petri-piatto contenente carta da filtro sterile. Con una pipetta Pasteur sterile, lavare il cervello con qualche goccia di dissecting soluzione per mantenere il tessuto umido. </li><li> Utilizzando un &quot;clean&quot; bisturi tagliare i due emisferi a parte. Trasferire l&#39;emisfero sinistro torna a grande piatto di Petri con micro cucchiaio e posizionare emisfero lato pia dalla soluzione di dissezione ghiacciata per un uso successivo. </li><li> Guarda la faccia mediale dell&#39;emisfero destro e identificare il fornice bordo, una fascia importante della sostanza bianca lungo il bordo mediale dell&#39;ippocampo. Usando un bisturi sterile, fare un taglio sagittale attraverso il fornice, ma prendersi cura perché solo 0,5 cm di punta bisturi saranno sufficienti per tagliare il fornice. </li><li> Usando 2 micro-spatole, rimuovere il primo dell&#39;ippocampo da emisfero destro mettendo la destra-spatola su tronco cerebrale e sollevando la corteccia sovrastante con la spatola sinistra. Sollevare delicatamente la corteccia per rivelare il ventricolo laterale e la superficie mediale dell&#39;ippocampo. Una linea curva bianca, la fimbria, dovrebbe essere visibile. </li><li> Allineate la curvatura della spatola con la curvatura della fimbria e premere delicatamente la spatola sotto la fimbria. Far scorrere la spatola sinistra e corsa lungo l&#39;asse rostrale-caudale e poi sollevare spatola in direzione dorsale per rimuovere ippocampo. </li><li> Trasferire l&#39;ippocampo di un piccolo piatto ° 2 Petri con una soluzione di dissezione ghiacciata. Ripetere la stessa procedura sul emisfero sinistro per rimuovere l&#39;ippocampo sinistro. </li><li> Usando una micro spatola, trasferire accuratamente l&#39;ippocampo alla fase chopper tessuti. Disporre loro adiacenti e parallele fra loro e perpendicolari all&#39;asse della lama chopper. Utilizzare un pennello per posizionare il tessuto e aggiungere qualche goccia della soluzione dissezione sulla parte superiore del ippocampi. </li><li> Tagliare il tessuto in 400 micron fette senza pause per rimuovere singole fette (di solito non si atterranno alla lama). Dopo tutto ippocampo è stato tagliato, utilizzare il pennello per trasferire delicatamente le sezioni per un piccolo piatto ° 2 Petri con una soluzione dissezione. </li><li> Sotto annuncioissecting microscopio, separare accuratamente le fette galleggianti con amicro-spatola e pennello. </li><li> Rimuovere la piastra di coltura pre-preparati con inserto cultura dal termostato e posto in una cappa a flusso laminare. </li><li> Usando una pipetta Pasteur fuoco lucido, disegnare 4-5 fette nelle fette pipetta e trasferire alla superficie apicale della membrana inserto cultura. Quindi, regolare il posizionamento con un pennello e lasciare uno spazio tra le singole sezioni e il bordo dell&#39;inserto cultura. </li><li> Usando una pipetta Pasteur sterile, rimuovere la soluzione dissezione in eccesso dalla superficie apicale di membrana. NOTA: Durante la rimozione soluzione evitare di tracciare sezioni di tessuto nella pipetta. In alternativa, utilizzare una pipetta regolare (P200 o P1000), con punte per pipette sterilizzate per rimuovere lentamente soluzione. </li><li> Posizionare la piastra di coltura con siero contenente terreno di coltura e le fettine di ippocampo posteriore in incubatore a 35 ° C e 5% di CO 2. NOTA: Se le chiamate esperimentoper la generazione di colture da più animali, pulire accuratamente il banco pulito flusso laminare tra dissezioni. Strumenti Torna a soluzione alcolica e sostituire tutti Petri-piatti, filtri di carta e lame di rasoio sterile prima della seconda dissezione. </li></ol> 4. Alimentazione e Mantenimento Organotipica Slices <ol><li> Culture mangimi in un banco pulito flusso laminare sterile. </li><li> Eseguire la prima alimentazione delle sezioni coltivate due giorni dopo la dissezione. Aspirare il terreno vecchia cultura usando pipetta di vetro sterilizzati. </li><li> Utilizzare sterile 5 ml pipetta sierologica aggiungere 1 ml di fresco sterili, medium, il siero contenente ai pozzetti. </li><li> Sostituire delicatamente l&#39;inserto cultura e prendersi cura di eliminare eventuali bolle d&#39;aria che possono essersi formate sotto la superficie della membrana. </li><li> Dopo la prima alimentazione, cambiare media ogni altro giorno. </li></ol> 5. incubazione fette di tessuto con timidina Analoghi etichettare Newborn Neuroni <ol><li> Per studiarela maturazione e l&#39;integrazione dei granuli dentato dell&#39;ippocampo, incubare le fette organotipiche con un analogo della timidina, come ad esempio Bromodeossiuridina (BrdU) o Chlorodeoxyuridine (CldU). Qui, usiamo CldU causa della sua elevata solubilità in soluzione salina. </li><li> Dopo 3DIV, aggiungere 1 ml di 10 mg / ml soluzione madre CldU in soluzione fisiologica a 1 ml di terreno di coltura per una concentrazione finale di 10 mg / ml. Se viene utilizzato BrdU, correggere la concentrazione delle differenze di peso molecolare. Aggiungere mezzo con CldU ai pozzetti cultura e incubare il tessuto con CldU contenenti media per 2 ore a 35 ° C. </li><li> Dopo 2 h di incubazione a 35 ° C, rimuovere il terreno contenente CldU e sostituirlo con il mezzo d&#39;alimentazione normale per riprendere programma di alimentazione normale (descritto sopra). </li></ol> 6. I tessuti Fissazione e bagagli <ol><li> Stabilire un calendario per l&#39;applicazione di trattamenti e fissaggio campioni di tessuto prima di avviare esperimenti di coltura (Figura 2b). </li> &lt;li&gt; A un giorno prestabilito post-dissezione, preparare quanto segue in una cappa di laboratorio: un bicchiere 10-50 ml contenente 4% paraformaldeide (PFA) in tampone fosfato (PBS); un bicchiere vuoto per mezzo di coltura scartato; pinzetta; e una pipetta 1000 ml con punte monouso. </li><li> Rimuovere la piastra di coltura (s) dalla camera di incubazione e trasferimento in una cappa aspirante. Inclinare singoli inserti piastre e con una pinza. Successivamente, utilizzare una pipetta per rimuovere terreno di coltura e trasferirla in un bicchiere di smaltimento. Inclinare la piastra di coltura con un angolo per contribuire a garantire che tutti i medium di coltura viene rimosso. </li><li> Termina rimozione mezzo per una piastra di coltura per volta. Quando medio è stato rimosso, aggiungere 1 ml di 4% PFA per ogni cultura bene e sigillare bene piatto con parafilm. Ripetere l&#39;operazione per il maggior numero di piastre di coltura come piatti necessari e trasferimento in frigorifero a 4 ° C per 24 ore. </li><li> Dopo 24 ore, di preparare le seguenti in una cappa aspirante: 10-50 bicchiere ml contenente 0,1% di sodio azide in PBS; un vuotobecher per scartato PFA (seguire le precauzioni di sicurezza quando scartando sostanze tossiche); pinzetta; e 1.000 ml pipetta con punte monouso. </li><li> Seguire la procedura descritta in 6.4 per l&#39;aggiunta di PFA, ma aggiungere 1 ml di PBS con sodio azide invece. Una volta completamente trasferita, piastre di tenuta e per un utilizzo futuro avvolgendo bordi in parafilm e conservare in frigorifero a 4 ° C. </li></ol> 7. sezionamento del tessuto per immunoistochimica <ol><li> Eseguire il tessuto sezionamento con un vibratome. La seguente serie di passi aiutano a massimizzare la resa di sezioni di tessuto utilizzabili da colture organotipiche. NOTA: Immediatamente dopo dissezione dell&#39;ippocampo e placcatura di fette, il tessuto ha uno spessore di circa 400 micron. Tuttavia, dopo 2-3 settimane nella camera di incubazione, fette di tessuto inizieranno ad appiattirsi, causando uno spessore sezione finale di 150-300 micron. </li><li> Preparare le seguenti voci alla sezione di tessuto coltivato: una # 11 bisturi blade e gestire, una piastra di Petri di vetro contenente ghiaccio freddo PBS, un micro-dissezione forcipe, e una fase di taglio vibratome pulito per montare il tessuto. </li><li> Utilizzare un bisturi per tagliare attentamente lungo il perimetro della membrana inserto circolare in modo che possa essere staccato dalla custodia inserto in plastica. Lasciare ampio spazio tra la fetta colta e il bisturi. </li><li> Trasferire la membrana inserto distaccato di una piastra di Petri contenente PBS. Dopo lavaggio in PBS, utilizzare pinze per trasferire la membrana alla fase di montaggio vibratome. </li><li> Quindi, utilizzare il bisturi per eliminare l&#39;eccesso di membrana che circonda le fette in coltura e tagliare il materiale in eccesso per creare bordi puliti. Questo passaggio garantirà la membrana è piana e può facilmente aderire alla superficie di taglio. </li><li> Mettere 1-2 gocce di colla sul palco taglio vibratome e diffondere in strato uniforme con un ago 22 G. Distribuire adesivo in una forma rettangolare, con il lato lungo parallelo alla lama di taglio del vibratome. Eseguire questo passaggiorapidamente, per evitare che l&#39;adesivo si secchi. </li><li> Utilizzare pinze per trasferire la membrana rifilato contenente fettine di ippocampo per la fase di taglio e delicatamente posizionare la membrana sulla colla e assicurarsi che non vi siano bolle d&#39;aria. </li><li> Come la supercolla asciuga, trasferire il palco vibratome con la membrana incollato torna alla piastra di Petri contenente PBS. Preparare la lama vibratome e 48 pozzetti contenente azide di sodio per memorizzare sezioni di tessuto. </li><li> Utilizzare il vibratome di generare 30 micron sezioni del tessuto fetta organotipica e trasferimento in un 48 pozzetti contenente sodio azide per lo stoccaggio e la successiva colorazione immunoistochimica. </li><li> Fare riferimento alle pre-esistenti protocolli per la colorazione immunoistochimica 26,29. </li></ol>

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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.

Dopo CldU (o BrdU) amministrazione, la linea temporale di applicazione di agenti farmacologici può essere scelto a bersaglio DGCS neonato durante particolari finestre di sviluppo. Ad esempio, un agente ipotetico può essere applicato durante la seconda settimana post-iniezione CldU, che si propone di coincidere con l&#39;età di neuroni immaturi che sono in fase di sviluppo in cui viene depolarizzante GABA. Futuri studi che utilizzano questo protocollo potrebbe adattare l&#39;agente farmacologico e la finestra di espos...

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

Qui si descrive una tecnica per lo studio dell&#39;ippocampo la neurogenesi postnatale nel cervello dei roditori utilizzando la tecnica organotipica cultura fetta. Questo metodo mantiene la morfologia caratteristica topografica dell&#39;ippocampo pur consentendo l&#39;applicazione diretta di agenti farmacologici per le sviluppo giro dentato dell&#39;ippocampo. Inoltre, culture fetta possono essere mantenuti fino a 4 settimane e, quindi, permettono di studiare il processo di maturazione dei neuroni granulari neonati. Culture fetta consentono efficace manipolazione farmacologica di fettine ippocampali escludendo variabili complesse quali incertezze relative alla posizione anatomica profonda dell&#39;ippocampo e la barriera ematoencefalica. Per queste ragioni, abbiamo cercato di ottimizzare culture fetta organotipiche specificamente per la ricerca neurogenesi postnatale.

Determinare se colture organotipiche sarebbe adatto per la ricerca neurogenesi adulta richieste che soddisfano due criteri principali: 1) che le fette di mantenere le peculiari caratteristiche morfologiche delle fettine di ippocampo dopo 10-21 giorni in vitro (DIV), e 2) che DGCS neonati possono essere quantificati utilizzando tecniche di immunoistochimica standard, comunemente impiegati nella ricerca neurogenesi adulta. Per quanto riguarda il primo criterio, Figure 1A e 1B evi...

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

Qui si descrive una tecnica per lo studio dell&#39;ippocampo la neurogenesi postnatale utilizzando la tecnica organotipica cultura fetta. Questo metodo permette la manipolazione in vitro di neurogenesi adulta e permette l&#39;applicazione diretta di agenti farmacologici per l&#39;ippocampo in coltura.

Colture organotipiche Slice di studi di post-natale neurogenesi

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 Views.  University of Toronto.  Qui si descrive una tecnica per lo studio dell'ippocampo la neurogenesi postnatale utilizzando la tecnica organotipica cultura fetta. Questo metodo permette la manipolazione in vitro di neurogenesi adulta e permette l'applicazione diretta di agenti farmacologici per l'ippocampo in coltura. 

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.

Ex utero elettroporazione e Organotipica Fetta Cultura del mouse ippocampale Tissue

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

Ricerca

Biologia dello sviluppo

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.

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

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.

Generazione di primaria fibroblasti culture da orecchio e coda tessuti di topo

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.

Imaging Azzerato embrionale e postnatale Cuori a risoluzione di una singola cella

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.

La visualizzazione delle variazioni del ciclo cellulare e la determinazione di nucleazione in postnatali cardiomiociti

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.

Dissezione e preparazione coronale fetta di sviluppare la ghiandola pituitaria del Mouse

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.

Colture organotipiche tridimensionale degli organi sensoriali uditivi e vestibolari

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.

Analisi trascrittomica unicellulare delle cellule Endocrine del pancreas del Mouse

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.

Neurogenesi con cellule carcinoma embrionale P19

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.

Risonanza in tempo reale sulla bioluminescenza delle dinamiche di segnalazione di nota durante la neurogenesi di Murine

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