Ringraziamo il Dott. Shirin Bonni per la fornitura del pSil-GFP costrutto, Dr. Alper Uzun per l&#39;illustrazione della figura 1, e la struttura Bioimmagini Leduc per la microscopia confocale. EMM è sostenuta dal Premio alla Carriera per la scienza medica dalla Burroughs Wellcome Fund, un NARSAD Premio Giovani Ricercatori e NIH NCRR COBRE RR018728 P20-01. SBL è supportato dal NIH NCRR COBRE RR018728 P20-01, ed ha ricevuto il sostegno PHS NRSA 5T32MH019118-20.

1. Preparazione Solutions cultura e dei media (non in video) <ol><li> Preparare 1 litro di soluzione salina equilibrata di Hank completa (HBSS) contenente HBSS 1x, 2,5 mM di HEPES (pH 7,4), 30 mM D-glucosio, 1 mM CaCl 2, 1 mM MgSO 4 e 4 mM NaHCO 3. Aggiungere acqua bidistillata (DDH 2 O). Filtrare sterilizzare con un filtro 0,2 micron e conservare a 4 ° C. </li><li> Preparare terreno di coltura sezione utilizzando 35 ml di Basal Medio Aquila supporti, 12,9 ml di HBSS completi, 20 mM D-glucosio (1,35 mL di soluzione 1 M), 1 mM Glutamax (0,25 mL di una soluzione 200 mM), 0,5 ml di penicillina -streptomicina magazzino 100x. Filtrare sterilizzare con un filtro da 0,2 um-, quindi aggiungere inattivato al calore siero di cavallo ad una concentrazione finale del 5%. </li><li> Preparare la soluzione laminina lavorare facendo un 1 mg / mL di soluzione stock Laminin con acqua distillata sterile deionizzata. Preparare aliquote di 100 microlitri di 0.5 ml provette Eppendorf e congelare a -80 ° C. </li><li> Preparare Poly-L-lisina di lavoro cosìluzione aggiungendo 5 ml di H 2 O sterile a 5 mg di poli-L-lisina per fare un 1 mg / ml di soluzione concentrata. Preparare aliquote di 1 ml e congelare a -20 ° C. </li><li> Preparare la soluzione di rivestimento diluendo 1 ml di poli-L-lisina e 100 pl di laminina ad un volume finale di 12 mL con acqua sterile. Rendere questa soluzione fresca ogni volta. </li></ol> 2. Preparazione Inserti Slice organotipiche (non in video) <ol><li> Preparare due a sei pozzetti con un inserto per la cultura e l&#39;uso di pinze sterili. Aggiungere 2 ml di soluzione sterile ddh 2 O sotto gli inserti culturali. </li><li> Aggiungere 1 mL della soluzione di rivestimento sulla parte superiore della membrana facendo attenzione a non forare la membrana. Incubare per una notte in un incubatore umidificato a 37 ° C e 5% CO 2. </li><li> Rimuovere i supporti di rivestimento e lavare la membrana con sterili H 2 0 tre volte. Lasciate che inserisce a secco prima dell&#39;uso. Aggiungere 1,8 mL di terreno fetta cultura e luogo nel 37 ° C incubatore. Avvolgere le piastre utilizzate con Parafilme conservare a 4 ° C per un massimo di 4 settimane. </li></ol> 3. Preparazione per l&#39;elettroporazione (non in video) <ol><li> Preparare costrutti RNAi per l&#39;elettroporazione. Doppia elica inserti forcina RNA sono stati clonati in un vettore pSilencer. Le Contiene plasmide: 1) un promotore che pilota l&#39;U6 doppio filamento RNA generazione, e 2) una cassetta di espressione GFP guidata dal promotore CMV 8,9. Questo plasmide è stato precedentemente descritto da Konishi e colleghi 9 e altri 7,8. I plasmidi sono stati purificati utilizzando una maxi-prep kit Qiagen, e usato ad una concentrazione di 1 mg / mL. </li><li> Al fine di visualizzare il DNA mentre iniettando, preparare una soluzione 0,5% veloce colorante verde e utilizzarlo a 1:20 con il DNA da iniettare (di solito 20 pl di DNA con 1 pl di verde veloce). Rimasto DNA-veloce miscela colorante verde può essere conservato a -20 ° C per una settimana. </li><li> Dissezione Pulire l&#39;area, dissectistrumenti ng e vasi vibratomo con il 70% di etanolo. Raffreddare HBSS completi in modo che sia ghiacciato. Freddo nave vibratomo da imballaggio ghiaccio intorno all&#39;interno della vibratomo con un pò d&#39;acqua per il raffreddamento rapido. Preparare 3% agarosio a basso punto di fusione con HBSS completi. Microonde per 1 min. Evitare di ebollizione. Conservare in un bagnomaria a 42 ° C fino al momento dell&#39;uso. </li><li> Parametri di elettroporazione sono fissati come segue. Per un embrione E15 uso 35 V, 5 impulsi di lunghezza, 100 ms, 900 ms intervallo tra gli impulsi. Per animali più vecchi, per garantire elettroporazione, utilizzare una tensione più elevata fino a 50 V o aumentare il numero di impulsi massimo di 8 impulsi. Per evitare di danneggiare il tessuto in animali giovani, utilizzare impulsi o meno o fino a 2 legumi e bassa tensione fino a 25 V. Questi parametri possono essere variati e determinata empiricamente in base all&#39;età dell&#39;animale. </li></ol> 4. Dissezione ed elettroporazione (in video) <ol><li> Dopo l&#39;eutanasia una femmina incinta, sezionare embrioni fuori nel freddo ghiaccio HBSS completi. Keep ogni embrione nei loro sacchi individuali placentari. </li><li> Dissect embrione fuori e tagliare la testa dopo la prima vertebra. Tenere in ghiaccio HBSS freddi completi. </li><li> Per l&#39;iniezione, posizionare la testa su un pezzo di Parafilm su una capsula di Petri. Utilizzando misura Hamilton siringa (vedi tabella I) iniettare circa 6 a 8 microlitri di DNA: veloce mix colorante verde attraverso il terzo ventricolo al fine di colmare in entrambi i ventricoli laterali nelle vescicole corticali. Alternativamente, l&#39;iniezione può essere fatto direttamente in ciascun ventricolo laterale. </li><li> Per l&#39;elettroporazione in vivo ex, utilizzare BTX-pinzette elettrodi di platino. Posizionare l&#39;elettrodo positivo verso il lato della corteccia che si desidera elettroporare all&#39;inizio cioè della testa per corteccia dorsale. </li><li> Dopo elettroporazione, incubare le teste sul ghiaccio per almeno 5 minuti prima dissezione. </li><li> Dissect cervello in HBSSby ghiacciata una piccola incisione sul lato della testa e pelatura la pelle fuori dai lati della testa. Successivamente, conpinza sottile delicatezza staccarsi la pia dal cervello. Togliere il cervello intatto dal cranio, facendo attenzione a non danneggiare la corteccia. </li></ol> 5. Incorporare e sezionamento delle cortecce elettroporate (in video) <ol><li> Trasferire 3% agarosio a basso punto di fusione in uno stampo grande posti su ghiaccio. Il fondo dello stampo inizia a solidificare rapidamente che impedirà cervelli di affondare fino al fondo dello stampo. Delicatamente, trasferire il cervello con le pinze sottili ad uno ad uno dopo aver tolto il tampone in eccesso con un Kimwipe o carta da filtro. Utilizzare una punta di 10 microlitri pipetta a girare il cervello all&#39;interno dello stampo per garantire la massima interfaccia tra il agarosio e tessuto cerebrale. </li><li> Orientare i cervelli per garantire che tutti i cervelli sono nello stesso orientamento e circa allo stesso livello del agarosio. Lasciate che l&#39;agarosio solidificare per circa 5 min. Utilizzare incollaggio (crazy colla) per fissare i blocchi di agarosio in modo che i bulbi olfattivi sono in piedi. Una volta che i blocchi sono collegati, subito aggiungere ice HBSS freddo e tagliare il agarosio per assicurarsi che le singole sezioni sono ottenuti per ogni cervello. </li><li> Per tagliare i blocchi, impostare la velocità della vibratomo a una bassa velocità (circa la metà del massimo) e impostare la frequenza di vibrazione della lama alla massima regolazione. Genera fette spesse 250 micron coronali. Recuperare le fette con una spatola piegata fine e trasferirli ai pozzi dei tessuti con un pennello fine o forcipe. </li><li> Nel cofano di coltura di tessuti, trasferire fette in inserti patinati. Aggiungere 500 microlitri di terreni di coltura porzione per ogni inserto per rendere il facile trasferimento. Fino a 5 fette possono essere inseriti per inserto. Rimuovere supporti in eccesso dalla parte superiore delle fette e incubare a 37 ° C in incubatore umidificato. </li></ol> 6. Cultura e Analisi delle fette organotipiche (in video) <ol><li> Per mantenere fette sani, mezzi freschi deve essere aggiunto almeno ogni altro giorno sotto la membrana sostituendo metà dei mezzi ogni volta. </li><li> Per analizzare fette dopo la desired giorni in coltura, fissare le fette nella membrana. Lavare con 1x tampone fosfato salino (PBS) a 37 ° C per tre volte per 10 minuti ogni volta. Successivamente, fissare con 4% paraformaldeide (PFA) notte a 4 ° C o per 1 ora a temperatura ambiente. </li><li> Fette possono essere analizzati con diversi marcatori cellulari o colorati con Hoechst solo per visualizzare cellule elettroporate e non elettroporata. Permeabile e bloccare fette per 2 ore a temperatura ambiente con siero di capra 10%, 0,1% Triton in PBS 1x con agitazione delicata. </li><li> Colorazione con Hoechst per 1 ora a temperatura ambiente, lavate 3 volte con PBS 1x 10 minuti ogni volta con agitazione delicata. </li><li> Per montare fette tagliare la membrana con un bisturi, e utilizzare una pinza sottile per trasferire la membrana contenente fette di un vetro smerigliato scorre in una camera d&#39;acqua. Fino a 5 fette può essere posizionato per vetrino. Eliminare l&#39;acqua in eccesso, e aggiungere una goccia di soluzione Flourmount ad ogni fetta cervello. Posizionare delicatamente un coprioggetto sopra le fettine di cervello e di rimuovere uny bolle d&#39;aria. Analizzare fette utilizzando un microscopio confocale. </li></ol> 7. Paraffina Embedding Alternative di Slices organotipiche (non in video) <ol><li> Fette organotipiche può anche essere incorporato per paraffina per l&#39;analisi morfologica più fine, a tal fine la membrana contenente le fette organotipiche possono essere fissati in 4% PFA come descritto sopra. </li><li> Le fette fissi sono incorporati in% agarosio 1 (pre-riscaldato a 37 ° C), e solidificato in ghiaccio per 30 min. I blocchi di agarosio possono quindi essere post-fissate in PFA 4% a 4 ° C per 30 min. </li><li> Il blocco agarosio contenente la fetta organotipica verrà quindi inclusi in paraffina e trattati per immunofluorescenza come descritto in precedenza 10. </li></ol> 8. Risultati rappresentativi Una rappresentazione schematica del elettroporazione di corteccia murina e cultura di fette organotipiche è mostrato in Figura 1. Questo metodo è utile per una strategia rapid valutazione della funzione di geni coinvolti nello sviluppo neuronale 11. A seconda della quantità di DNA elettroporata e lo stadio embrionale elettroporazione, l&#39;efficienza di transfezione varierà. Fette inizierà esprimere GFP almeno 8 ore post-elettroporazione e le cellule saranno sottoposti alla normale sequenza di eventi neurogenica (proliferazione, la migrazione, e all&#39;inizio la differenziazione neuronale) nella cultura. La figura 2 mostra una fetta elettroporate cervello che sta esprimendo un controllo pSilencer-GFP vettoriale e si può osservare progenitori neuronali, i neuroni che migrano e neuroni differenziati in slice. Fette organotipiche mantengono la loro morfologia finché sono mantenute in un buon supporto aria interfaccia e le membrane possono essere utilizzate fino ad almeno 5 giorni di coltura. <img alt="Figura 1" src="/files/ftp_upload/3621/3621fig1.jpg" /> Figura 1. Illustrazione di elettroporazione in vivo e ex fetta organotipicala cultura del saggio. E14.5 embrioni sono sezionati, e individualmente iniettati con DNA mescolato con veloci colorante verde per visualizzare il sito di iniezione. Il DNA può essere iniettato in entrambi i ventricoli laterali come illustrato nella figura o nel terzo ventricolo per riempire i ventricoli laterali. Dopo l&#39;iniezione, i cervelli sono elettroporate con un elettroporatore onda quadra, ponendo l&#39;elettrodo positivo sul lato desiderato del cervello. Brains sono incorporati in 3 agarosio a basso punto di fusione% e sezionato usando un vibratomo. 250 fettine di cervello micron sono immessi sul inserti 0,4 micron e coltivati ​​fino ad una settimana. GFP può essere osservato dopo 8 ore dopo la trasfezione. <img alt="Figura 2" src="/files/ftp_upload/3621/3621fig2.jpg" /> Figura 2. Analisi delle fettine di cervello elettroporate. Fettine di cervello elettroporate sono state colorate per Hoescht. Corteccia dorsale mostra elettroporate progenitori neuronali nella zona ventricolare (VZ). I neuroni in cortical piastra (cp) sono delimitate dalla zona marginale (MZ). Le frecce bianche indicano i neuroni che migrano. In questo caso, i cervelli sono stati iniettati a E15.5 e elettroporata con un vettore pSilencer controllo GFP. Le sezioni rappresentano espianti corticali 4 giorni dopo l&#39;elettroporazione. Scale bar 100 micron. Risoluzione dei problemi: <ol><li> Bassa efficienza di trasfezione: Regolare la concentrazione del DNA usato per almeno 1 mg / pl. Usare sempre il DNA molto pulito da una preparazione maxi se necessario utilizzare uno endo-free Quiagen kit per purificare il DNA. </li><li> Cellule trasfettate in un&#39;area del cervello diversa da quella desiderata: garantire che gli elettrodi siano posizionate correttamente con la posizione di elettrodo positivo verso il lato del cervello da elettroporazione. </li><li> Fette organotipiche perdere morfologia: i media cambiano ogni giorno e assicurare le fette non sono galleggianti in media </li><li> Fette staccarsi agarosio mentre vengono tagliati in vibratome: Assicurarsi che una buona interfaccia è fatto quando si incorpora i cervelli in agarosio a basso punto di fusione. </li></ol>

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	<li>Angevine, J. B., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+study+of+cell+migration+during+histogenesis+of+cerebral+cortex+in+the+mouse.">Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse.</a> Nature. 192, 766-766 (1961).</li><li>Kriegstein, A. R., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neuronal+migration+in+the+embryonic+cortex.">Patterns of neuronal migration in the embryonic cortex.</a> Trends Neurosci. 27, 392-392 (2004).</li><li>Barnes, A. P., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+axon-dendrite+polarity+in+developing+neurons.">Establishment of axon-dendrite polarity in developing neurons.</a> Annu. Rev. Neurosci. 32, 347-347 (2009).</li><li>Haydar, T. F., Bambrick, L. L., Krueger, B. K., 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=Organotypic+slice+cultures+for+analysis+of+proliferation,+cell+death,+and+migration+in+the+embryonic+neocortex.">Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex.</a> Brain Res. Brain Res. Protoc. 4, 425-425 (1999).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal.</a> Sci. STKE. 2002, pl9-pl9 (2002).</li><li>Guerrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-BAR+domain+of+srGAP2+induces+membrane+protrusions+required+for+neuronal+migration+and+morphogenesis.">The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.</a> Cell. 138, 990-990 (2009).</li><li>Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+regulation+of+axonal+morphogenesis+by+the+Cdh1-APC+target+SnoN.">Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN.</a> Neuron. 50, 389-389 (2006).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PLoS Genet. 4, e1000111-e1000111 (2008).</li><li>Konishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303, 1026-1026 (2004).</li><li>Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+paraffin-embedding+of+mouse+tissues+for+GFP+visualization.">Fixation and paraffin-embedding of mouse tissues for GFP visualization.</a> Cold Spring Harb Protoc. 2009, 5298-5298 (2009).</li><li>Hand, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Neurogenin2+specifies+the+migration+properties+and+the+dendritic+morphology+of+pyramidal+neurons+in+the+neocortex.">Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.</a> Neuron. 48, 45-45 (2005).</li><li>Taniguchi, Y., Young-Pearse, T., Sawa, A., Kamiya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Electroporation+as+a+Tool+for+Genetic+Manipulation+in+Vivo+to+Study+Psychiatric+Disorders:+From+Genes+to+Circuits+and+Behaviors.">In Utero Electroporation as a Tool for Genetic Manipulation in Vivo to Study Psychiatric Disorders: From Genes to Circuits and Behaviors.</a> Neuroscientist. 18, 169-179 (2012).</li></ol>

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Questi metodi che comportano l&#39;elettroporazione ex vivo di plasmidi che codificano RNA a doppio filamento tornanti 8 e cultura della organotipiche 4 fette di fornire diversi vantaggi. In primo luogo, questi metodi consentono una rapida valutazione di RNAi derivati ​​fenotipi. L&#39;inclusione di una codifica GFP cassetta di espressione nel vettore pSilencer stesso che contiene il promotore U6 che guida la forcina RNA a doppio filamento consente una rapida identificazione e caratter...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Hamilton siringa </td><td> HAMILTON </td><td> 80008 </td><td> 31 gauge, 0,5 centimetri di lunghezza, PT-4 (livello di smussatura punto), volume 10 pl </td></tr><tr><td> Platinum tweezertrodes </td><td> BTX </td><td> 45-0489 </td><td> Size 5 millimetri </td></tr><tr><td> ECM830 elettroporatore </td><td> BTX </td><td> 45-0002 </td><td></td></tr><tr><td> BTX Footswitch </td><td> BTX </td><td> 45-0208 </td><td> Per l&#39;utilizzo con ECM830 elettroporatore </td></tr><tr><td> Vibrante microtomo blade </td><td> LEICA </td><td> VT1000 S </td><td></td></tr><tr><td> 6 - piatto bene da utilizzare con inserti </td><td> FALCON </td><td> 353502 </td><td> Contiene tacche per adattarsi inserti </td></tr><tr><td> Coltura tissutale inserti </td><td> FALCON </td><td> 353090 </td><td> 0,4 micrometri </td&gt; </tr><tr><td> Fast Green </td><td> SIGMA </td><td> F7252 </td><td></td></tr><tr><td> Bassa fusione Agarose </td><td> FISHER </td><td> BP165-25 </td><td> DNA grade </td></tr><tr><td> Laminina </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Poly-L-lisina </td><td> Sigma-Aldrich </td><td> P5899 </td><td></td></tr><tr><td> Basale medio Aquila </td><td> Sigma Aldrich </td><td> B-1522 </td><td></td></tr><tr><td> HBSS 10x senza Ca e Mg </td><td> GIBCO </td><td> 14180-046 </td><td></td></tr><tr><td> HEPES acid-free </td><td> Sigma-Aldrich </td><td> H4034 </td><td></td></tr></tbody></table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

La corteccia cerebrale dirige funzioni cognitive superiori. Questa sei struttura a strati è generata in un dentro-prima, fuori-ultimo modo, in cui i neuroni primi nati rimanere più vicino al ventricolo, mentre gli ultimi neuroni nati migrare passato i primi neuroni nati verso la superficie del cervello 1. Oltre alla migrazione neuronale 2, un processo chiave per la normale funzione corticale è la regolazione della morfogenesi neuronale 3. Mentre morfogenesi neuronale può essere studiato in vitro in colture primarie, c&#39;è molto da imparare dal modo in cui questi processi sono regolati in ambienti tessuti. Descriviamo le tecniche per analizzare la migrazione neuronale e / o morfogenesi in fette organotipiche della corteccia cerebrale 4,6. Un vettore pSilencer modificato viene utilizzato, che contiene sia un promotore U6 che guida l&#39;RNA a doppio filamento tornanti e una cassetta di espressione separato che codifica la proteina GFP guidato bya CMV promoter 7-9. Il nostro approccio consente per la valutazione rapida dei difetti nella crescita dei neuriti su knockdown specifico di geni candidati ed è stato utilizzato con successo in uno schermo per i regolatori di crescita dei neuriti 8. Poiché soltanto un sottoinsieme di cellule esprimono i costrutti RNAi, le fette organotipiche permettere un&#39;analisi mosaico dei fenotipi potenziali. Inoltre, poiché questa analisi è fatta in approssimazione nei pressi del ambiente in vivo, si fornisce un basso costo e rapida alternativa alla generazione di animali transgenici o knockout per geni di funzione sconosciuta corticale. Infine, in confronto con la tecnologia di elettroporazione in vivo, il successo di esperimenti ex vivo elettroporazione non dipende sviluppo abile capacità chirurgico e può essere eseguita con un tempo più breve di formazione e di abilità.

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

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

Per condurre una rapida valutazione della funzione dei geni nello sviluppo della corteccia cerebrale, si descrivono i metodi che comportano l&#39;<em&gt; Ex vivo</emElettroporazione&gt; plasmidi di co-espressione inibitoria RNA (RNAi) e GFP in murino corteccia embrionale. Questo protocollo è suscettibile allo studio dei vari aspetti della sviluppo neurologico, quali la neurogenesi, la migrazione neuronale e la morfogenesi neuronale compresi dendrite e l&#39;estensione assonale.

Metodi per lo studio della morfogenesi neuronale:<em&gt; Ex vivo</em&gt; Elettroporazione RNAi in Embryonic Cerebral Cortex Murine

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

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

11.7K Views.  Brown University. Per condurre una rapida valutazione della funzione dei geni nello sviluppo della corteccia cerebrale, si descrivono i metodi che comportano l' Ex vivo plasmidi di co-espressione inibitoria RNA (RNAi) e GFP in murino corteccia embrionale. Questo protocollo è suscettibile allo studio dei vari aspetti della sviluppo neurologico, quali la neurogenesi, la migrazione neuronale e la morfogenesi neuronale compresi dendrite e l'estensione as...

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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Neuroscienze

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Elettroencefalografia simultanea, in tempo reale misura della concentrazione di lattato e la manipolazione Optogenetic di attività neuronale nella corteccia cerebrale Rodentia

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

Elettroporazione in utero approcci per studiare l&#39;eccitabilità neuronale sottopopolazioni e connettività single-cell

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Induzione della ricombinazione di Cre-lox nella corteccia cerebrale del Mouse attraverso elettroporazione In Utero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

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

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

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

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

Ex Utero Elettroporazione e colture Organotypic fetta di cervelli embrionali del Mouse per vivere-Imaging di migrazione interneuroni GABAergici

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