Ringraziamo il Dott. Yoko Nabeshima dell&#39;Università di Kyoto per il suo insegnamento sulla tecnica di dissezione e la signora Aki Miyakawa a Fujita University Health per il suo sostegno al cinema. Questo lavoro è stato sostenuto dal Programma per la promozione di studi fondamentali in Scienze della Salute dell &#39;Istituto Nazionale per l&#39;Innovazione Biomedica, una sovvenzione in aiuti per la ricerca scientifica sulle Settori prioritari-Integrativa Brain Research (Shien) - da MEXT in Giappone, e da una borsa-in-Aid dal CREST di Scienze Giappone e Agenzia Technology.

Dissezione di giro dentato dell&#39;ippocampo <ol><li> In un mouse profondamente anestetizzati, accuratamente sezionare il cervello fuori dal cranio e il luogo in ghiacciata tampone fosfato salino (PBS). </li><li> In una capsula di Petri contenente ghiaccio PBS freddo, tagliare il cervello lungo la fessura longitudinale del cervello utilizzando un bisturi, e tagliare le regioni posteriori a lambda (mesencefalo, romboencefalo, e cervelletto). </li><li> Posizionare il lato mediale dell&#39;emisfero cerebrale e, con cautela il numero del diencefalo (talamo e ipotalamo) al microscopio di dissezione. Questo esporre il lato mediale dell&#39;ippocampo, consentendo la visualizzazione del giro dentato. Il giro dentato è distinguibile dal corno di Ammone da parte del gap tra di loro. Lesioni all&#39;ippocampo o nell&#39;area circostante renderà più difficile isolare il giro dentato. </li><li> Inserire un forte ago-punta (ad esempio, 27-gauge) in ogni lato del giro dentato (confini del giro dentato e corno di Ammon; Figura 1), e far scorrere gli aghi superficialmente lungo il setto-temporale asse di ippocampo di isolare il giro dentato. </li><li> Sollevare il giro dentato isolato usando un ago o pinze e collocarlo in una provetta. Il così-dentato ottenuto campione di tessuto giro può essere utilizzato immediatamente per qualsiasi saggio o conservati in un congelatore per un uso successivo. </li><li> Isolare il giro dentato dall&#39;altro emisfero cerebrale utilizzando lo stesso metodo. </li></ol> Quantitativa real-time PCR Il giro dentato è stato isolato utilizzando il metodo sopra menzionato e l&#39;ippocampo rimanente è stato sezionato come esempio il corno di Ammone da topi wild-type. PCR in tempo reale di beta-actina, TDO2, DSP, Mrg1b e Tyro3 sono stati eseguiti con il giro dentato e campioni di corno di Ammon come descritto in precedenza 1. Primer 5&#39;-CTGGCGAGATCACGATGACG e 5&#39;-AAGCTACGCTGTTGTCTAACC sono stati utilizzati per Mrg1b e GCCTCCAAATTGCCCGTCA e 5&#39;-CCAGCACTGGTACATGAGATCA per Tyro3. Microarray analisi Esperimenti di microarray sono stati effettuati con maschi topi wild-type e topi eterozigoti per l&#39;alfa-isoforma di calcio / calmodulina-dipendente delle proteine ​​chinasi II (alpha-CaMKII + / - topi) come descritto in precedenza 1. In breve, l&#39;RNA isolato da tutto l&#39;ippocampo o giro dentato di topi wild-type e mutante è stato ibridato con un mouse Genome Array 430 2.0 (Affymetrix, Santa Clara, CA), e ogni GeneChip è stato analizzato da un Affymetrix GeneChip scanner 3000 (GCS3000) . GeneChip analisi è stata eseguita con Microarray Analysis Suite versione 5.0.

<ol>
	<li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K., Toyama, K., Kanzaki, K., Fukunaga, K., Sudo, Y., Ichinose, H., Ikeda, M., Iwata, N., Ozaki, N., Suzuki, H., Higuchi, M., Suhara, T., Yuasa, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Mol. Brain. 1, 6-6 (2008).</li><li>Insausti, A. M., Megias, M., Crespo, D., Cruz-Orive, L. M., Dierssen, M., Vallina, I. F., Insausti, R., Florez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+neuronal+number+in+Ts65Dn+mice:+a+murine+model+of+Down+syndrome.">Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome.</a> Neurosci. Lett. 253, 175-175 (1998).</li><li>Redwine, J. M., Kosofsky, B., Jacobs, R. E., Games, D., Reilly, J. F., Morrison, J. H., Young, W. G., Bloom, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+gyrus+volume+is+reduced+before+onset+of+plaque+formation+in+PDAPP+mice:+a+magnetic+resonance+microscopy+and+stereologic+analysis.">Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: a magnetic resonance microscopy and stereologic analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1381-1381 (2003).</li><li>Lein, E. S., Zhao, X., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+molecular+atlas+of+the+hippocampus+using+DNA+microarrays+and+high-throughput+in+situ+hybridization.">Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization.</a> J. Neurosci. 24, 3879-3879 (2004).</li><li>Matsuo, N., Yamasaki, N., Ohira, K., Takao, K., Toyama, K., Eguchi, M., Yamaguchi, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+changes+underlying+the+working+memory+deficit+in+alpha-CaMKII+heterozygous+knockout+mice.">Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.</a> Front. Behav. Neurosci. 3, 20-20 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

Gli esperimenti su animali sono stati eseguiti in conformità con il National Institute of Health Guide di cura e dell&#39;uso degli animali da laboratorio, e approvato dal comitato di cura degli animali e l&#39;uso di Fujita University Health.

Il giro dentato occupa circa il 25% al 30% del volume della formazione dell&#39;ippocampo 2,3. Ha una composizione unica delle cellule e svolge un ruolo fondamentale nelle funzioni cerebrali diverse. Pertanto, le tecniche per isolare il giro dentato sono utili per analizzare gli eventi che si verificano in particolare in questa regione. Qui, abbiamo dimostrato una procedura per sezionare in modo efficiente il giro dentato dell&#39;ippocampo di topo adulto e ha confermato la precisione della tecnica. In primo luogo, lo studio istologico ha rivelato che il giro dentato era separato senza contaminazione da altre regioni (Figura 1), che indica che un campione puro giro dentato possono essere preparati. In secondo luogo, abbiamo confermato che il tessuto è sezionato giro dentato conducendo real-time PCR del giro dentato geni specifici, TDO2 e DSP, e corno di Ammon geni arricchito, Mrg1b e Tyro3 4 (Figura 2). Le espressioni mRNA di TDO2 (p = 0,000023; n E = 4 e 4, rispettivamente) e DSP (p = 0,0000030, n E = 4 e 4, rispettivamente) nei campioni giro dentato Sono state rinvenute, ovviamente, più elevati, mentre Mrg1b (p = 0.000080, n E = 4 e 4, rispettivamente) e Tyro3 (p = 0,00017, n E = 4 e 4, rispettivamente) sono stati più bassi, rispetto a quelli nei campioni corno di Ammon. Beta-actina livelli di espressione non differivano in questi campioni (p = 0,11, n E = 4 e 4, rispettivamente). Così, abbiamo potuto verificare se il giro dentato è stato accuratamente sezionato da condurre tale semplici esperimenti in tempo reale PCR. In terzo luogo, per valutare l&#39;utilità di questo metodo di dissezione, abbiamo confrontato il livello di espressione di mRNA di ippocampo tutto con quella del giro dentato. Ippocampo intero e giro dentato ottenuto da wild-type (n = 9 e 4, rispettivamente) e alfa-CaMKII + / - topi (n è = 18 e 4, rispettivamente) sono stati processati per l&#39;analisi microarray, e per tutti i geni segnato, la piega- cambiamento è stato calcolato dividendo il valore mutante dalla wild-type di valore. I risultati hanno indicato che i cambiamenti di espressione dell&#39;mRNA, in particolare di giro dentato specifiche molecole come DSP e TDO2, sono stati rilevati fino ad un aumento di 5 volte nella sensibilità nei campioni giro dentato rispetto ai campioni di tutto dell&#39;ippocampo (Tabella 1). Abbiamo precedentemente dimostrato che l&#39;alfa-CaMKII + / - topi mostrano comportamenti relativi alla umano disturbi psichiatrici come deficit di memoria di lavoro e un ritmo esagerato infradiani 1,5. Inoltre, le caratteristiche morfologiche ed elettrofisiologiche dei neuroni nel giro dentato topi mutanti sono sorprendentemente simili a quelle di neuroni immaturi giro dentato nei roditori normali, indicando che i neuroni di questi topi mutanti non riescono a svilupparsi fino alla scadenza 1. L&#39;immaturo espressione dentato giro e down-regolato di Dsp e TDO2 mRNA in alpha-CaMKII + / - i topi sono in linea con la constatazione che Dsp e TDO2 possono essere utilizzati come marcatori delle cellule dei granuli maturi nel giro dentato (Ohira et al, non pubblicata. dati). Nel loro insieme, questa tecnica dissezione comodo e preciso in modo attendibile utilizzato per studi si sono concentrati sul giro dentato. Tessuto giro dentato ottenuto con questo metodo è applicabile ad altri tipi di analisi pure, comprese le analisi proteomica e di biologia cellulare. <img src="/files/ftp_upload/1543/1543fig1.jpg" alt="figura 1" /> Figura 1. Verifica del giro dentato isolati dallo studio istologico. Una sezione coronale del cervello dopo aver isolato giro dentato è stato elaborato per la colorazione di Nissl (riquadro a sinistra), e un diagramma schematico tratto dal cervello di topo atlas6 rappresenta circa lo stesso livello della sezione mostrata nel pannello di sinistra (pannello di destra). Le frecce indicano le direzioni dell&#39;ago punta di inserimento. Scala grafica, 1 mm. <img src="/files/ftp_upload/1543/1543fig2.jpg" alt="figura 2" /> Figura 2. Verifica del giro dentato isolata dal real-time PCR. Il giro dentato e il corno di Ammone s ottenuto da quattro topi wild-type sono stati trattati per PCR in tempo reale di beta-actina, TDO2, DSP, Mrg1b e Tyro3. I risultati sono presentati come media ± SEM. Per l&#39;analisi statistica, test t di Student s è stato impiegato, e valori di p sono seguiti: beta-actina, p = 0,11; TDO2, p = 0,000023 (** 1); Dsp, p = 0,0000030 (** 2); Mrg1b, p = 0.000080 (** 3), e Tyro3, ​​p = 0,00017 (** 4). Tabella 1. . Analisi microarray di dell&#39;ippocampo tutto e giro dentato geni differenzialmente espressi nel giro dentato dell&#39;ippocampo e tutta l&#39;alpha-CaMKII + / - i topi sono stati determinati calcolando il fold-change da quello rilevato in topi wild-type. I dati sono stati analizzati per la significatività statistica utilizzando il test t di Student s tra wild-type e alfa-CaMKII + / - topi. Tra i geni la cui espressione esposti p &lt;0,05 inil giro dentato di alpha-CaMKII + / - topi rispetto a quella di topi wild-type, i primi 50 geni sono elencati. Si noti che il numero di campioni per giro dentato sono molto meno di quelli per intero ippocampo. AffyID, Affymetrix sonda identificatore; CKII, alpha-CaMKII + / - topi; WT, topi wild-type. <table border="1"><tbody><tr><td></td><td></td><td></td><td></td><td colspan="2"> Giro dentato (p &lt;0,05) WT: n = 4, CKII + / -: n = 4 </td><td></td><td colspan="2"> Ippocampo tutto WT: n = 9, CKII + / -: n = 18 </td></tr><tr><td> Gene Titolo </td><td> Alle banche di geni </td><td> AffyID </td><td></td><td> Fold change </td><td> valore di p </td><td></td><td> Fold change </td><td> valore di p </td></tr><tr><td> desmoplakina </td><td> AV297961 </td><td> 1435494_s_at </td><td></td><td> 0,011018913 </td><td> 7.02694E-06 </td><td></td><td> 0,037021003 </td><td> 1.86126E-13 </td></tr><tr><td> desmoplakina </td><td> AV297961 </td><td> 1435493_at </td><td></td><td> 0,014369734 </td><td> 7.86747E-06 </td><td></td><td> 0.04232106 </td><td> 1.00579E-12 </td></tr><tr><td> triptofano 2,3-diossigenasi </td><td> AI098840 </td><td> 1419093_at </td><td></td><td> 0,020986484 </td><td> 5.23546E-09 </td><td></td><td> 0,101037776 </td><td> 4.14823E-13 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452106_at </td><td></td><td> 0,075479901 </td><td> 1.05191E-08 </td><td></td><td> 0,234001154 </td><td> 1.66301E-15 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452107_s_at </td><td></td><td> 0,079457767 </td><td> 1.40433E-07 </td><td></td><td> 0,177974715 </td><td> 3.9758E-12 </td></tr><tr><td> tireotropina rilasciando recettore ormonale </td><td> M59811 </td><td> 1449571_at </td><td></td><td> 0,103105815 </td><td> 0,003093796 </td><td></td><td> 0,801412732 </td><td> 0,283994361 </td></tr><tr><td> recettore della rianodina 1, del muscolo scheletrico </td><td> X83932 </td><td> 1427306_at </td><td></td><td> 0,104825517 </td><td> 3.38513E-07 </td><td></td><td> 0,650685017 </td><td> 0,000308462 </td></tr><tr><td> ignorante anello elica elica 1 </td><td> NM_010916 </td><td> 1419533_at </td><td></td><td> 9.431896 </td><td> 6.7979E-06 </td><td></td><td> 4,078815314 </td><td> 5.27E-11 </td></tr><tr><td> copine familiare IX </td><td> BB274531 </td><td> 1454653_at </td><td></td><td> 9.159157 </td><td> 7.99492E-06 </td><td></td><td> 1,797304153 </td><td> 0,000296375 </td></tr><tr><td> doublecortin-like chinasi 3 </td><td> BB326709 </td><td> 1436532_at </td><td></td><td> 0,109336662 </td><td> 1.95278E-07 </td><td></td><td> 0.56697229 </td><td> 2.62633E-08 </td></tr><tr><td> calpaina 3 </td><td> AF127766 </td><td> 1426043_a_at </td><td></td><td> 0,111269769 </td><td> 8.07053E-06 </td><td></td><td> 0,370956608 </td><td> 2.04421E-14 </td></tr><tr><td> Adulti di sesso maschile corpo striato cDNA, RIKEN full-length arricchito biblioteca, clone: ​​C030023B07 prodotto: inclassificabile, inserire la sequenza completa </td><td> BB357628 </td><td> 1460043_at </td><td></td><td> 0,118712341 </td><td> 6.16926E-07 </td><td></td><td> 0,682339204 </td><td> 2.33001E-06 </td></tr><tr><td> collagene e calcio vincolante FEG settori 1 </td><td> AV264768 </td><td> 1437385_at </td><td></td><td> 0,124043978 </td><td> 3.65669E-05 </td><td></td><td> 0,488394112 </td><td> 4.05538E-06 </td></tr><tr><td> precursore della proteina beta amiloide vincolante, la famiglia A, membro 2 legame con le proteine ​​(A4) </td><td> AK013520 </td><td> 1431946_a_at </td><td></td><td> 7.7986307 </td><td> 1.2098E-06 </td><td></td><td> 2,099164713 </td><td> 1.67047E-06 </td></tr><tr><td> calbindin-28K </td><td> BB177770 </td><td> 1456934_at </td><td></td><td> 0,130255444 </td><td> 3.32186E-06 </td><td></td><td> 0,572605751 </td><td> 1.99157E-10 </td></tr><tr><td> Locus trascritto </td><td> AV328597 </td><td> 1443322_at </td><td></td><td> 0,133290835 </td><td> 5.43583E-06 </td><td></td><td> 0,562767164 </td><td> 7.56544E-06 </td></tr><tr><td> neuropeptide Y recettori Y2 </td><td> NM_008731 </td><td> 1417489_at </td><td></td><td> 0,135319609 </td><td> 0,000113407 </td><td></td><td> 0,781498474 </td><td> 0.00394504 </td></tr><tr><td> ras proteine ​​elemento reattivo legame 1 </td><td> BE197381 </td><td> 1428657_at </td><td></td><td> 0,138235114 </td><td> 7.93691E-07 </td><td></td><td> 0,651220705 </td><td> 2.94209E-05 </td></tr><tr><td> linea cellulare gliale neurotrofico derivato della famiglia dei recettori fattore alpha 2 </td><td> BB284482 </td><td> 1433716_x_at </td><td></td><td> 0,139062563 </td><td> 2.35371E-06 </td><td></td><td> 0,669544709 </td><td> 0,000214146 </td></tr><tr><td> preproenkephalin 1 </td><td> M13227 </td><td> 1427038_at </td><td></td><td> 6.9850435 </td><td> 2.39074E-08 </td><td></td><td> 1,766018828 </td><td> 0,000250501 </td></tr><tr><td> RIKEN gene cDNA 1810010H24 </td><td> BI729991 </td><td> 1428809_at </td><td></td><td> 6.8658915 </td><td> 1.88516E-05 </td><td></td><td> 2.77573142 </td><td> 6.81865E-09 </td></tr><tr><td> recettore della rianodina 1, del muscolo scheletrico </td><td> BG793713 </td><td> 1457347_at </td><td></td><td> 0,151364292 </td><td> 3.35612E-05 </td><td></td><td> 0,503144617 </td><td> 4.32907E-05 </td></tr><tr><td> Protocadherin 21 </td><td> NM_130878 </td><td> 1418304_at </td><td></td><td> 0,152671849 </td><td> 8.57783E-06 </td><td></td><td> 0,670714726 </td><td> 1.56309E-05 </td></tr><tr><td> cornichon omologo 3 (Drosophila) </td><td> NM_028408 </td><td> 1419517_at </td><td></td><td> 0,153724144 </td><td> 8.90755E-06 </td><td></td><td> 0.95780695 </td><td> 0,661055608 </td></tr><tr><td> harakiri, BCL2 proteine ​​interagenti (contiene solo BH3 dominio) </td><td> BQ175572 </td><td> 1439854_at </td><td></td><td> 0,154284407 </td><td> 2.0118E-05 </td><td></td><td> 0.56516812 </td><td> 4.86925E-09 </td></tr><tr><td> carboidrati (N-acetilgalattosamina 4-0) sulfotransferasi 9 </td><td> AK017407 </td><td> 1431897_at </td><td></td><td> 0,155238951 </td><td> 5.37423E-06 </td><td></td><td> 1.14910007 </td><td> 0,215637733 </td></tr><tr><td> calpaina 3 </td><td> AI323605 </td><td> 1433681_x_at </td><td></td><td> 0,160871988 </td><td> 1.07655E-05 </td><td></td><td> 0,477164757 </td><td> 1.33753E-11 </td></tr><tr><td> dito di zinco, dominio CCHC contenente 5 </td><td> BQ126004 </td><td> 1437355_at </td><td></td><td> 0,161812078 </td><td> 3.08262E-06 </td><td></td><td> 0,421252632 </td><td> 0.01152969 </td></tr><tr><td> loricrin </td><td> NM_008508 </td><td> 1448745_s_at </td><td></td><td> 0,165129967 </td><td> 1.86362E-05 </td><td></td><td> 0,639733409 </td><td> 0,000729772 </td></tr><tr><td> spondina 1, (f-spondina) di proteine ​​della matrice extracellulare </td><td> BC020531 </td><td> 1451342_at </td><td></td><td> 0,168035879 </td><td> 6.67867E-07 </td><td></td><td> 0,821042412 </td><td> 0,023650765 </td></tr><tr><td> RIKEN cDNA A930035E12 gene </td><td> AV348640 </td><td> 1429906_at </td><td></td><td> 5.9086795 </td><td> 1.747E-07 </td><td></td><td> 1,470383201 </td><td> 0,104085454 </td></tr></tbody><tbody><tr><td> BB247294 RIKEN full-length arricchito, 7 giorni neonato Mus musculus cervelletto clone cDNA A730018G18 3 &#39;, sequenza di mRNA. </td><td> BB247294 </td><td> 1447907_x_at </td><td></td><td> 5.9047494 </td><td> 1.04931E-05 </td><td></td><td> 1,968147585 </td><td> 0.00010636 </td></tr><tr><td> Dominio FERM contenente 3 </td><td> BB099015 </td><td> 1437075_at </td><td></td><td> 5.860216 </td><td> 0,000345581 </td><td></td><td> 2,780297178 </td><td> 1.83072E-06 </td></tr><tr><td&gt; Neuronale pentraxina 2 / / / proteine ​​ipotetico LOC100044234 </td><td> NM_016789 </td><td> 1420720_at </td><td></td><td> 5.7568517 </td><td> 1.34227E-06 </td><td></td><td> 2,652516957 </td><td> 0,000206279 </td></tr><tr><td> Sequenze trascritte </td><td> BG076361 </td><td> 1460101_at </td><td></td><td> 5.657735 </td><td> 2.5015E-06 </td><td></td><td> 1,296248831 </td><td> 0.22870031 </td></tr><tr><td> spondina 1, (f-spondina) di proteine ​​della matrice extracellulare </td><td> BC020531 </td><td> 1424415_s_at </td><td></td><td> 0.17783576 </td><td> 1.01658E-06 </td><td></td><td> 0,836181248 </td><td> 0,001380141 </td></tr><tr><td> calbindin-28K </td><td> BB246032 </td><td> 1448738_at </td><td></td><td> 0,180317904 </td><td> 1.35961E-05 </td><td></td><td> 0,647334052 </td><td> 4.12268E-09 </td></tr><tr><td> Marcks-come 1 </td><td> AV110584 </td><td> 1437226_x_at </td><td></td><td> 0,186235935 </td><td> 1.47067E-06 </td><td></td><td> 0,499291387 </td><td> 2.34984E-08 </td></tr><tr><td> matrilin 2 </td><td> BB338441 </td><td> 1455978_a_at </td><td></td><td> 0,187783528 </td><td> 6.19122E-05 </td><td></td><td> 0.8967688 </td><td> 0,282337853 </td></tr><tr><td> matrilin 2 </td><td> BC005429 </td><td> 1419442_at </td><td></td><td> 0,188195795 </td><td> 0,000105295 </td><td></td><td> 0,915528892 </td><td> 0.35282097 </td></tr><tr><td> spondina 1, (f-spondina) di proteine ​​della matrice extracellulare </td><td> BQ175871 </td><td> 1442613_at </td><td></td><td> 0,189956563 </td><td> 9.41195E-06 </td><td></td><td> 0,861033222 </td><td> 0.1394266 </td></tr><tr><td> arrestina 3, della retina </td><td> NM_133205 </td><td> 1450329_a_at </td><td></td><td> 5.2130346 </td><td> 2.90599E-05 </td><td></td><td> 3,944218329 </td><td> 1.07437E-07 </td></tr><tr><td> RIKEN cDNA A330050F15 gene </td><td> AV325555 </td><td> 1457558_at </td><td></td><td> 0.19186781 </td><td> 0,000119342 </td><td></td><td> 0,660282035 </td><td> 2.47342E-05 </td></tr><tr><td> contactin 3 </td><td> BB559510 </td><td> 1438628_x_at </td><td></td><td> 0,194404608 </td><td> 4.08641E-07 </td><td></td><td> 0,918742591 </td><td> 0,022545297 </td></tr><tr><td> calbindin-28K </td><td> BB246032 </td><td> 1417504_at </td><td></td><td> 0,196381321 </td><td> 2.24182E-05 </td><td></td><td> 0,619305124 </td><td> 3.6222E-06 </td></tr><tr><td> gastrina rilasciando peptide </td><td> BC024515 </td><td> 1424525_at </td><td></td><td> 4.9436426 </td><td> 3.00588E-05 </td><td></td><td> 2,752845903 </td><td> 5.72954E-07 </td></tr><tr><td> sortilin legati VPS10 dominio contenente recettore 3 </td><td> AK018111 </td><td> 1425111_at </td><td></td><td> 4.885766 </td><td> 1.03645E-05 </td><td></td><td> 1.29051599 </td><td> 0,029733649 </td></tr><tr><td> recettore della dopamina D1A </td><td> BE957273 </td><td> 1455629_at </td><td></td><td> 4.869493 </td><td> 3.77525E-05 </td><td></td><td> 1,815881979 </td><td> 0,000516498 </td></tr><tr><td> proprotein convertasi subtilisina / kexin tipo 5 </td><td> BB241731 </td><td> 1437339_s_at </td><td></td><td> 0,210528027 </td><td> 7.83039E-05 </td><td></td><td> 0,574126078 </td><td> 9.15496E-05 </td></tr></tbody><tbody><tr><td> interleuchina 1 recettore di tipo I </td><td> NM_008362 </td><td> 1448950_at </td><td></td><td> 0,210572243 </td><td> 9.64524E-06 </td><td></td><td> 0,241135352 </td><td> 2.79816E-08 </td></tr></tbody></table>

dissection of hippocampal dentate gyrus from adult mouse

L&#39;ippocampo è una delle zone più studiati nel cervello a causa del suo importante ruolo funzionale nella elaborazione della memoria e l&#39;apprendimento, la sua notevole plasticità delle cellule neuronali, e il suo coinvolgimento in epilessia, malattie neurodegenerative, e disturbi psichiatrici. L&#39;ippocampo è composto da diverse regioni, il giro dentato, che comprende principalmente i neuroni dei granuli, e corno di Ammon, che comprende principalmente i neuroni piramidali, e le due regioni sono collegate da entrambi i circuiti anatomici e funzionali. Molti mRNA differenti e le proteine ​​sono selettivamente espresse nel giro dentato, e il giro dentato è un sito di neurogenesi adulta, cioè nuovi neuroni sono continuamente generati nel giro dentato adulti. Per studiare l&#39;espressione di mRNA e proteine ​​specifiche per il giro dentato, microdissezione laser capture viene spesso utilizzata. Questo metodo ha alcune limitazioni, tuttavia, come la necessità di apparecchiature particolari e complicate procedure di gestione. In questo video-registrato protocollo, abbiamo dimostrato una tecnica di dissezione per la rimozione del giro dentato da topo adulto sotto uno stereomicroscopio. Campioni giro dentato preparato con questa tecnica sono adatte ad ogni test, tra cui trascrittomica, proteomica e analisi di biologia delle cellule. Abbiamo confermato che il tessuto è sezionato giro dentato conducendo PCR in tempo reale di giro dentato geni specifici, triptofano 2,3-diossigenasi (TDO2) e desmoplakina (Dsp), e Ammon corno geni arricchito, Meis legati 1b gene (Mrg1b ) e TYRO3 proteina tirosin-chinasi 3 (Tyro3). Le espressioni di mRNA TDO2 e Dsp nei campioni giro dentato Sono state rinvenute, ovviamente, più elevati, mentre Mrg1b e Tyro3 erano più bassi livelli, rispetto a quelle dei campioni del corno di Ammone. Per dimostrare il vantaggio di questo metodo, abbiamo effettuato delle analisi del DNA microarray utilizzando campioni di ippocampo intero e giro dentato. L&#39;espressione di mRNA TDO2 e DSP, che sono espressi selettivamente nel giro dentato, nell&#39;ippocampo tutto di alfa-CaMKII + / - topi esposti 0,037 e 0,10 volte i cambiamenti rispetto a quella di topi wild-type, rispettivamente. Nel giro dentato isolato, però, queste espressioni esposti 0,011 e 0,021 volte modifiche rispetto a quella di topi wild-type, dimostrando che i cambiamenti dell&#39;espressione genica in giro dentato possono essere rilevati con maggiore sensibilità. Nel loro insieme, questa tecnica dissezione comodo e preciso in modo attendibile utilizzato per studi si sono concentrati sul giro dentato.

Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Una tecnica di dissezione per la rimozione del giro dentato da topo adulto allo stereomicroscopio è stata dimostrata in questo video-registrato di protocollo.

Dissezione di giro dentato ippocampale di topo adulto

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Research

JoVE Journal

Biology

82.2K Views.  Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST). Una tecnica di dissezione per la rimozione del giro dentato da topo adulto allo stereomicroscopio è stata dimostrata in questo video-registrato di protocollo.

Video: Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissezione di 6,5 embrioni di topo dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Ricerca

Biologia

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissezione di organi dal Zebrafish adulti

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissezione di Oenocytes da adulti Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissezione del rene Zebrafish adulti

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caratterizzazione e differenziamento di cellule staminali cardiache dal cuore di topo adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e coltura di cheratinociti orali primari dal palato del topo adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.