Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Etica Dichiarazione: Il trattamento dei topi e le procedure sperimentali sono state condotte in conformità agli orientamenti europei, nazionali e istituzionali per la cura degli animali. 1. In Utero Elettroporazione Nota: In utero elettroporazione è stato eseguito come precedentemente pubblicato 12,14. Pertanto, il metodo è descritto brevemente nel seguito (figura 1). <ol><li> Preparativi <ol><li> Preparare veloce verde soluzione priva di endotossine color avanzato trasfezione qualità plasmide (contenente 1,5 pCAGGS) come descritto in precedenza 12. </li><li> Tirare e macinare (35 ° angolo) capillari di vetro borosilicato (0,8-0,9 diametro) per preparazioni iniettabili. </li><li> Sterilizzare gli strumenti. </li></ol></li><li> Chirurgia <ol><li> Somministrare analgesici (carprofen, 4 mg / kg di peso corporeo, sc, 24 ore) deposito di almeno 30 minuti prima di iniziare l&#39;intervento chirurgico. </li><li> Mantenere la lunghezza dellaanestesia ad un massimo di 35-45 min (Fase III - fase dell&#39;anestesia chirurgici secondo Guedel 15 - massima 25-30 min). </li><li> Anestetizzare topi cronometrati in gravidanza con isoflurano (induzione in una camera a 2,8%, la chirurgia tramite maschera: 2,5%). Confermare l&#39;anestesia da non responsività ai piedi-pizzico. </li><li> Applicare pomata occhio per prevenire la secchezza oculare durante l&#39;anestesia. </li><li> Sterilizzare l&#39;area chirurgica con 7,5% di soluzione Providone-iodio e coprire il mouse con garza sterile (umido con una soluzione allo 0,9% di alcool benzilico soluzione fisiologica), con solo l&#39;area chirurgica esposta. </li><li> Tagliare aprire la cavità addominale con un bisturi (incisione cutanea: 1,5-2 cm, incisione muscolo: 1-1,5 cm). </li><li> Preservare la cavità addominale aperta da secchi da inumidire con soluzione salina riscaldata alcool benzilico allo 0,9% o altra soluzione isotonica sterile, come indicato dal personale veterinario o del comitato. </li><li> Estrarre attentamente corna uterine (con pinze ad anello, non toccare l&#39;utero con le ditaS). </li></ol></li><li> L&#39;iniezione di DNA ed elettroporazione. Nota: I dettagli esatti per il targeting regioni specifiche sono indicati nella sezione 2. <ol><li> Iniettare lentamente la soluzione di DNA colorato nel ventricolo come indicato nella figura 1 e la sezione 2 tramite i capillari borosilicato preparati. </li><li> Applicare la tensione appropriata per il corrispondente stadio embrionale (ad es., 36-38 V per E14) tramite elettrodi specializzati forcipe tipo platino (ciclo intervallo di lunghezza 50 msec, intervallo di pausa 950 msec). Pulse le palette elettroporazione durante l&#39;applicazione della tensione per minimizzare i danni della parete uterina. </li></ol></li><li> Messaggio elettroporazione / assistenza post-operatoria <ol><li> Ricollocare accuratamente le corna uterine nella cavità addominale. </li><li> Suturare il muscolo e l&#39;incisione cutanea separatamente. </li><li> Lasciate che il resto del mouse su un dispositivo di supporto per il recupero termico. </li><li> Sorvegliare il mouse durante il recupero (comportamento, alimentazione econsumo di acqua), 4 ore, 24 ore e 48 ore dopo l&#39;intervento chirurgico. Se necessario, applicare analgesici supplementari (carprofen, 4 mg / kg di peso corporeo, 24 ore deposito) per altri 2 giorni. </li></ol></li></ol> 2. Trasfezione di aree cerebrali specifiche Nota: La posizione del polo positivo è mostrato come l&#39;angolo tra la linea centrale verticale attraverso il ventricolo riempito con la soluzione di DNA (ventricolo destro o terzo) e la posizione del polo positivo. Durante il riempimento del ventricolo sinistro le posizioni sono speculare. In Figura 4 sono mostrate le posizioni primordiale dell&#39;orecchio, che forniscono importanti punti di riferimento. <ol><li> Trasfezione di aree corticali (Figura 2A, Tabella 1). <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;elettroporazione in utero di aree corticali utilizzare embrioni E14. </li><li> Iniettare 1,5-2 microlitri DNA-soluzione contenente 4 mg di DNA nel ventricolo laterale destroattraverso il capillare borosilicato preparati. <ol><li> Posizionare accuratamente l&#39;embrione utilizzando una pinza ad anello in modo che non ci sono vasi visibili sopra il sito di puntura. </li><li> Iniettare lentamente il DNA-soluzione circa 0,75 mm coronale dalla sutura lambdoidal e 0,5 mm lateralmente dalla sutura sagittale (Figura 3). Assicurarsi che la profondità di inserimento è di 1,2 mm (E14, misurata dalla parete uterina). Verificare la presenza di una colorazione blu-verde con una demarcazione tagliente. </li></ol></li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo pala 3 o 5 mm. </li><li> Utilizzare una tensione compresa 36-38 V. </li><li> Posizionare il centro degli elettrodi solo anteriori dei primordi dell&#39;orecchio. </li><li> Per trasfettare gli angoli uso corteccia retrosplenial 330-0 °, per trasfettare la corteccia motoria 0-40 °, per trasfettare la corteccia somatosensoriale 40-95 °, per trasfettarela corteccia piriforme 95-110 ° (Figura 2A). </li></ol></li><li> Eseguire elettroporazione / assistenza post-operatoria dopo come descritto nel paragrafo 1.4. Sacrifica i topi e analizzare gli embrioni 4 giorni dopo l&#39;elettroporazione (E18), come descritto nella sezione 3. </li></ol></li><li> Transfection dell&#39;ippocampo (Figura 2B, Tabella 1). <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;elettroporazione in utero di formazione dell&#39;ippocampo utilizzare embrioni E15. </li><li> Iniettare 2 microlitri DNA-soluzione contenente 4 mg di DNA nel ventricolo laterale destro attraverso il capillare borosilicato preparati. </li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo pala 3 o 5 mm. </li><li> Utilizzare una tensione compresa 38-40. </li><li> Posizionare il centro del polo negativo appena anteriore del primordio orecchio e il centro of il polo positivo al centro della primordium orecchio. </li><li> Per trasfettare giro dentato utilizzano angoli 190-210 °, per trasfettare il Ammonis cornu (CA) 3 210-230 °, per trasfettare il CA2 230-250 °, per trasfettare il CA1 250-275 ° (Figura 2B). </li></ol></li><li> Eseguire elettroporazione / assistenza post-operatoria dopo come descritto nel paragrafo 1.4. Eseguire l&#39;analisi dopo la formazione dell&#39;ippocampo completa (dopo la nascita, p21), come descritto nella sezione 3. </li></ol></li><li> Transfection del laterali nucleo settale e striato (Figura 2C, tabella 1) Nota: Gli angoli si sovrappongono con quelli transfecting l&#39;ippocampo. Per trasfettare efficacemente il nucleo settale laterale e striato senza indesiderate ippocampo transfezione fasi precedenti embrionali (E12) devono essere utilizzati. <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;elettroporazione in utero del nucleo settale laterale e striatum utilizzare embrioni E12. </li><li> Iniettare 1 ml di DNA-soluzione contenente 4 mg di DNA nel ventricolo laterale destro attraverso il capillare borosilicato preparati. </li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo 0,5 mm. </li><li> Utilizzare 33 V. </li><li> Posizionare il centro degli elettrodi a livello dei primordi dell&#39;orecchio. </li><li> Per trasfettare le uso angoli del setto nucleo laterali 100-170 °, per trasfettare striato 170-240 ° (Figura 2C). </li></ol></li><li> Eseguire elettroporazione / assistenza post-operatoria dopo come descritto nel paragrafo 1.4. Sacrifica i topi e analizzare gli embrioni 6 giorni dopo l&#39;elettroporazione (E18), come descritto nella sezione 3. </li></ol></li><li> Trasfezione del talamo e ipotalamo (Figura 2D, Tabella 1) <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;in utero elettroporazione delle talamo e ipotalamo uso E12-E13 embrioni. </li><li> Iniettare 1-1.5-DNA microlitri soluzione contenente 4 mg DNA nel ventricolo laterale (sinistro o destro) attraverso il capillare borosilicato preparati. Attendere 2-3 minuti, fino a quando la soluzione viene diffuso nel terzo ventricolo. In alternativa iniettare direttamente nel terzo ventricolo. Nota: Questo migliora la specificità, ma potrebbe causare un tasso di sopravvivenza ridotta (alto rischio di danni). </li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo 0,5 o 3 mm. </li><li> Utilizzare una tensione compresa 33-35 V. </li><li> Posizionare il centro degli elettrodi appena posteriore i primordi dell&#39;orecchio. </li><li> Per trasfettare gli angoli uso talamo 25-40 °, per trasfettare l&#39;ipotalamo 90-180 ° (Figura 2D). </li></ol></li><li> Eseguire posta elettroporazione / assistenza post-operatoria come described nella sezione 1.4. Sacrifica i topi e analizzare gli embrioni 6 giorni dopo l&#39;elettroporazione (E18), come descritto nella sezione 3. </li></ol></li></ol> 3. Fissazione perfusione <ol><li> Preparativi <ol><li> Warm 4% paraformaldeide (PFA) a 37 ° C. </li><li> Riempire una siringa da 50 ml con PBS (4 ° C) e una seconda siringa con riscaldato PFA. Evitare le bolle. </li><li> Collegare le siringhe a un rubinetto a tre vie. Collegare il terzo fine di un set di infusione alato (farfalla). </li><li> Lavare il set di infusione alato con PBS. </li></ol></li><li> Perfusione <ol><li> Profondamente anestetizzare topi (gravidanza o postnatale trasfettate) con l&#39;applicazione sottocutanea di ketamina cloridrato (60 mg / kg) e xilazina (7,5 mg / kg). </li><li> Confermare la fase chirurgica tolleranza refrattarietà ai piedi-pizzico. </li><li> Aprire la cavità addominale appena sotto la gabbia toracica. </li><li> Tagliare con cautela la membrana. </li><li> Aprire con cautela la gabbia toracica mediante un taglio sull&#39;esito ach fino alla clavicola. </li><li> Inserire la punta della farfalla nella camera del cuore sinistro (partendo dalla punta del cuore). </li><li> Tagliare l&#39;appendice atriale sinistra. </li><li> Lentamente profumato il mouse con PBS (circa 10 ml). </li><li> Accendere il rubinetto a tre vie. </li><li> Profumato il mouse con 5-10 ml 4% PFA. </li><li> Decapitare il mouse utilizzando le forbici e sezionare il cervello a partire dalla forame magno. Eseguire la dissezione come precedentemente pubblicato 16. </li><li> Postfixate il cervello per 2 giorni a 4% PFA. Mantenere il cervello a 4 ° C al buio. </li></ol></li></ol> 4. immunoistochimica <ol><li> Genera 70 sezioni micron (ad esempio, con un vibratome). </li><li> Opzionale: incrementare la fluorescenza con l&#39;anticorpo colorazione. <ol><li> Bloccare le sezioni per 1 ora a RT (0,1-0,2% Triton X-100, siero di capra normale 5%) </li><li> Incubare O / N con l&#39;anticorpo appropriata (anti-GFP, 1: 1.000) </li><li> Lavare 3-5 Volte con tampone fosfato 0,1 M. </li><li> Incubare 3-5 ore con l&#39;appropriato anticorpo secondario fluorescenza coniugato (Alexa Fluor 488 coniugato anti-coniglio IgG, 1: 1.000). </li><li> Opzionale: Controcolorare con 4-6-diaminodino-2-fenilindolo (DAPI, 0,2 g / ml in tampone fosfato 0,1 M) per 1 min. </li><li> Analizzare fluorescenza con un microscopio a fluorescenza appropriata. </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific regions and therefore the detailed information needed for specific transfections is only scarcely standardized. 
The position and the size of the electrode as well as the applied voltage for the corresponding embryonic stage, which are the critical steps for effective and specific transfections, are precisely described. The provided angle-map helps to orientate the electrodes for specific electroporation. The locations of the electrodes are additionally shown on an embryo in the uterus, an embryo in the amniotic sac and also on a pure, dissected embryo. All of this is intended to help in identifying the correct positions of the electrodes. 
Usage of the appropriate voltage is a central aspect in the provided protocol. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Voltage recommendations given in this protocol are specific for C57BL/6 mice and slightly differ from these for CD12,3,12. CD1 mice tolerate higher voltages than C57BL/6 mice and therefore higher voltages might be used for transfecting CD1 mice12. No data exist for other mouse strains, but the standards might need some modifications. 
A further central point is the size of the electrode. The usage of a smaller electrode, rather than a bigger one, is always recommended for experiments where specificity is important. Smaller electrodes enhance specificity4,12. If the specificity of the transfection is not the main point, especially for inexperienced experimenters, bigger electrodes enhance the chance of hitting the desired region. Thereby one has to keep in mind that electrodes covering too much of the embryo, might affect the heart rhythm and lead to reduced survival. For example a 10 mm electrode covers the whole E12 embryo and therefore is not recommended for transfecting E12 embryos, even if specificity is not the primary goal. 
The stages used in this protocol were chosen exemplary. Considering the fact that progenitors that are transfected on a certain time point give rise to a particular type of neuron the embryonic stages for transfection can be adapted to the specific aim of the study. For instance during cortical development, neurons are born at different time points, forming the six cortical layers in a sequential manner. Hence, depending of the time point of transfection the transfected progenitors give rise to neurons of a specific layer2,17.
For a successful transfection the following points also should be observed. First, the careful preparation and injection of the glass capillaries play a crucial role for the survival of the embryos and therefore for effective transfection. Second, the selection of an appropriate DNA concentration can also be very important and might have to be specifically adapted for different vectors18. Third, application of high voltages affects neighboring embryos, and therefore transfection of neighboring embryos might reduce the survival of these embryos. Further it has also been recommended to avoid the embryos closest to the vaginal duct, because the damage of these embryos might lead to abortion18.
Besides great advantages the technique of in utero electroporation has its drawbacks, which should be taken into consideration while designing experiment. First the regions, which can be efficiently transfected, are manly restricted to areas surrounding the ventricles. This is due to the fact that the plasmid-solution has to be injected into a ventricle. Further, even with all the details provided in this protocol it is not possible to transfect all cells out of a specific region. Even the number of transfected cells is hardly controllable. The analysis of the gene function is not restricted to embryonic stages. Adult mice can also be analyzed because the transfection of postmitotic neurons lasts at least up to 4 months13, but is not passed to the next generation. 
Further specifications, such as specifically electroporating thalamic and hypothalamic nuclei or temporally addressing cortical layers can additionally broaden the scope. The combination of genetically engineered mice, or inducible systems in with the in utero electroporation also additionally enhances possible applications. 
With all the given facts, this protocol facilitates specific transfections and therefore reduces the number of animal used. Standardization of the protocol additionally enhances the comparability of the obtained results.

Dalla prima descrizione nel 2001 da tre gruppi indipendenti 1-3 i n utero elettroporazione è diventato uno strumento standard ampiamente utilizzato per analizzare l&#39;espressione genica nel sistema nervoso centrale dei roditori. Rispetto alla generazione di topi knockout, che è, nonostante le tecniche di continuo miglioramento, ancora di tempo e denaro che consumano, i ricorsi in utero elettroporazione a causa della sua semplicità. Così, in utero elettroporazione consente GAIN- veloce ed efficiente e la perdita di funzione studi 4. Per trasfettare le regioni cerebrali, la soluzione contenente il plasmide carica negativa viene iniettato in un ventricolo. Durante l&#39;impulso elettrico, il DNA carico negativamente migra verso il polo positivo e quindi la regione trasfettate è selezionabile semplicemente modificando la posizione del polo positivo. E &#39;stato spesso dimostrato che numerose regioni del sistema nervoso centrale possono essere targeted 3,5-8. Ad esempio, recenti studi dimostrano trasfezioni specifiche del ippocampo, la corteccia piriforme o striato 9-11. Tuttavia, le informazioni sulle posizioni appropriate sono spesso solo poco standardizzato e non sono sempre facili da trasferire a diversi ceppi di topi. Trasfezione di alcune fasi embrionali è tutt&#39;altro che banale. Molti fattori influenzano devono essere presi in considerazione quando si sceglie il set-up per specifici elettroporazione in utero. In primo luogo, per trasfettare in modo ottimale i rispettivi stadi embrionali, è necessaria la conoscenza delle tensioni del caso. Tensioni elevate riducono il tasso di sopravvivenza, mentre basse tensioni riducono l&#39;efficienza di trasfezione 2,3,12. Anche le dimensioni del paddle dell&#39;elettrodo gioca un ruolo fondamentale, perché l&#39;uso di piastre di elettrodi che sono troppo grandi risultati a ridotta specificità o possono causare la morte per affetto del ritmo cardiaco 4,12,13. La applicatatensione e la dimensione e la posizione della pala dell&#39;elettrodo sono le caratteristiche più importanti da considerare, ma ci sono anche altri fattori che influenzano il risultato della elettroporazione, come la quantità applicata di DNA-soluzione. Abbiamo sviluppato un protocollo dettagliato che consente trasfezione veloce ed efficiente di varie regioni cerebrali del C57BL / 6 del mouse 12. In questo protocollo informazioni dettagliate sulle tensioni da utilizzare e la dimensione del paddle dell&#39;elettrodo per una maggiore specificità è disponibile. Inoltre, informazioni riguardanti il ​​ventricolo da riempire con raccomandazioni per la quantità di soluzione plasmide e la posizione dell&#39;elettrodo è fornito. L&#39;indicazione delle informazioni dettagliate sulla posizione in una cartina e l&#39;ulteriore visualizzazione di queste posizioni permette specifico semplice ed efficiente in utero elettroporazione della corteccia retrosplenial, la corteccia motoria, la corteccia somatosensoriale, la corteccia piriforme, tegli cornu Ammonis 1-3, il giro dentato, lo striato, il nucleo settale laterali, il talamo e l&#39;ipotalamo.

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

La figura 2 mostra esempi di specifica in utero elettroporazione delle regioni in via di sviluppo nella corteccia retrosplenial, la corteccia motoria, la corteccia somatosensoriale, la corteccia piriforme, il cornu Ammonis 1-3, il giro dentato, lo striato, il nucleo settale laterali , il talamo e l&#39;ipotalamo. I risultati delle trasfezioni sono confrontate con l&#39;angolazione consigliata (Figura 2). Per una migliore visualizzazione degli angoli in vivo la posizione dell&#39;elettrodo (0,5 mm) sono anche mostrato un embrione (Figura 5, embrione in utero, embrione sacco amniotico, embrione sezionato). Al fine di fornire una tabella panoramica 1 ha riassunto i fatti rilevanti per la trasfezione specifica. Inoltre, è stato dimostrato che la dimensione dell&#39;elettrodo gioca un ruolo cruciale per la specificità (Figura 6). Più grande dimensione delle pale elettrodi comporta larger aree trasfettate, esemplare mostrato nella Figura 6. Inoltre, gli elettrodi che sono troppo grandi copertura troppo dell&#39;embrione, migliorando così il rischio di influenzare il ritmo cardiaco embrioni (Figura 7). Ad esempio un elettrodo a pale 10 mm copre l&#39;intero embrione E12 (Figura 7). <img alt="Figura 1" src="/files/ftp_upload/53303/53303fig1.jpg" /> Figura 1. In utero elettroporazione. Step di base delle prestazioni della elettroporazione in utero sono presenti. (A) mostra l&#39;intero setup della chirurgia, con il mouse posizionato su una piastra già anestetizzati con isoflurano. (B) La disinfezione dell&#39;area chirurgica è un passo fondamentale prima di aprire la cavità addominale (C) del mouse. (D) La soluzione contenente DNA è colorata con Fast Green, che consente la visualizzazione di iniezione di successo (E, F). Iniezione di successo in un ventricolo laterale si traduce in punto a forma di mezzaluna. (G) Dopo l&#39;iniezione della tensione appropriata è applicata mediante elettrodi di platino tipo pinze. (H) Dopo aver sostituito accuratamente l&#39;utero nella cavità addominale della ferita chirurgica viene suturata. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53303/53303fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53303/53303fig2.jpg" /> Figura 2. Angle-map per elettroporazione di aree specifiche nella corteccia murino, ippocampo, laterale settale nucleo striato, talamo e ipotalamo. Le posizioni appropriate per il polo positivo e negativo, è indicata nel disegno schematico. Per (AC </stro<html> ng&gt;) le posizioni sono indicate per l&#39;iniezione del DNA-soluzione nel ventricolo destro. Per le iniezioni nel ventricolo sinistro, gli angoli sono speculare. Le trasfezioni specifici sono stati istologicamente verificati. (A) mostra la posizione del polo positivo e negativo per trasfettando aree corticali (la corteccia retrosplenial (RSC), la corteccia motoria (MC), la corteccia somatosensoriale (SSC) e la corteccia piriforme (PC). (B) Indica la posizioni del polo positivo e negativo per la trasfezione le formazioni dell&#39;ippocampo (la cornu Ammonis 1-3 (CA1, CA2, CA3) e il giro dentato (DG). (C) mostra le posizioni del polo positivo e negativo per trasfezione striato (STR) e il nucleo settale laterale (LS). (D) Mostra le appropriate posizioni degli elettrodi per transfecting il talamo (TH) e l&#39;ipotalamo (HY). Adattato da Baumgart J. &amp; Grebe N. 2015 12.<html> ps: //www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53303/53303fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 3" src="/files/ftp_upload/53303/53303fig3.jpg" /> Figura 3. puntura. La posizione ottimale per l&#39;iniezione della soluzione di DNA è circa 0,75 mm coronale dalla sutura lambdoidal e 0,5 mm laterale dalla sutura sagittale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53303/53303fig3large.jpg" target="_blank">Fate clic qui per visualizzare una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53303/53303fig4.jpg" /> Figura 4. Posizione del primordium orecchio. La posizione del primordium orecchio è chiaramente definito (freccia).nk &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 5A" src="/files/ftp_upload/53303/53303fig5A.jpg" /><img alt="Figura 5B" src="/files/ftp_upload/53303/53303fig5B.jpg" /><img alt="Figura 5C" src="/files/ftp_upload/53303/53303fig5C.jpg" /><img alt="Figura 5D" src="/files/ftp_upload/53303/53303fig5D.jpg" /> Figura 5. Visualizzazione degli angoli di un embrione. Per una migliore visualizzazione delle posizioni appropriate per il polo positivo (indicato con un segno più blu) e il negativo per trasfezioni specifiche delle aree corticali (corteccia retrosplenial, corteccia motoria, corteccia somatosensoriale, Piriform corteccia), le aree dell&#39;ippocampo (Cornu Ammonis (CA) 1-3, giro dentato (DG)),il nucleo settale laterali, lo striato, il talamo e ipotalamo vengono visualizzati su un embrione in utero, un embrione nel sacco amniotico e su un embrione sezionato (tutto E15,5). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53303/53303fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 6" src="/files/ftp_upload/53303/53303fig6.jpg" /> Figura 6. Specificità trasfezione seconda delle dimensioni dell&#39;elettrodo. Aumento delle dimensioni della pala dell&#39;elettrodo riduce la specificità della trasfezione. Ciò è particolarmente evidente nelle fasi embrionali giovani (E12, in alto), ma anche visibile in fase embrionale tardiva (E17, in basso). Adattato da Baumgart J. &amp; Grebe N., 2015 12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53303/53303fig6large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a><img alt="Figura 7" src="/files/ftp_upload/53303/53303fig7.jpg" /> Figura 7. Dimensione del paddle dell&#39;elettrodo rispetto alla dimensione di un embrione E12. Elettrodi che sono troppo grandi copertura troppo dell&#39;embrione e quindi aumentano il rischio di affetto del ritmo cardiaco. Ad esempio, un elettrodo 10 mm (a destra) copre l&#39;intero embrione E12 e pertanto non è raccomandato, anche se la specificità non è l&#39;obiettivo primario. Adattato da Baumgart J. &amp; Grebe N., 2015 12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53303/53303fig7large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <table fo:keep-together.within-page="1"><tbody><tr><td> Regione di destinazione </td><td> Angolo </td><td colspan="2"> Posizione dell&#39;elettrodo </td><td> Età di embrione </td><td> Amou DNAnt / concentrazione </td><td> Voltaggio </td><td> Dimensioni dell&#39;elettrodo </td></tr><tr><td> Cortex retrosplenial </td><td> 330 ° - 0 ° </td><td colspan="2"> Centro degli elettrodi solo anteriore del primordi orecchio </td><td> E14 (E11-E17) </td><td> 1.5- 2 ml / 2.7- 2 mg / mL </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Corteccia motoria </td><td> 0 ° - 40 ° </td><td colspan="2"> Centro degli elettrodi solo anteriore del primordi orecchio </td><td> E14 (E11-E17) </td><td> 1.5- 2 ml / 2.7- 2 mg / mL </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Corteccia somatosensoriale </td><td> 40 ° - 95 ° </td><td colspan="2"> Centro degli elettrodi solo anteriore del primordi orecchio </td><td> E14 (E11-E17) </td><td> 1.5- 2 ml / 2.7- 2 mg / mL </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Piriform Cortex </td><td> 95 ° - 110 ° </td><td colspan="2"> Centro degli elettrodi solo anteriore del primordi orecchio </td><td> E14 (E11-E17) </td><td> 1.5- 2 ml / 2.7- 2 mg / mL </td><td> 36-3 8 </td><td> 3- 5 mm </td></tr><tr><td> Cornu Ammonis (CA) 1 </td><td> 250 ° - 275 ° </td><td colspan="2"> Centro del polo positivo a metà del primordio orecchio </td><td> (E14-) E15 </td><td> 2 microlitri / 2 mg / mL </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> CA2 </td><td> 230 ° - 250 ° </td><td colspan="2"> Centro del polo positivo a metà del primordio orecchio </td><td> (E14-) E15 </td><td> 2 microlitri / 2 mg / mL </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> CA3 </td><td> 210 ° - 230 ° </td><td colspan="2"> Centro del polo positivo a metà del primordio orecchio </td><td> (E14-) E15 </td><td> 2 microlitri / 2 mg / mL </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> Giro dentato </td><td> 190 ° - 210 ° </td><td colspan="2"> Centro del polo positivo a metà del primordio orecchio </td><td> (E14-) E15 </td><td> 2 microlitri / 2 mg / mL </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> Striatum </td><td> 170 ° - 240 ° </td><td colspan="2"> Centro degli elettrodi a livello del primordia orecchio </td><td> E12 </td><td> 1 ml / 4 mg / mL </td><td> 33 </td><td> 0,5 mm </td></tr><tr><td> Laterale setto Nucleus </td><td> 100 ° - 170 ° </td><td colspan="2"> Centro degli elettrodi a livello del primordia orecchio </td><td> E12 </td><td> 1 ml / 4 mg / mL </td><td> 33 </td><td> 0,5 mm </td></tr><tr><td> Talamo </td><td> 25 ° - 40 ° </td><td colspan="2"> Centro delle electodes solo posteriormente il pri orecchiomordia </td><td> E12- E13 </td><td> 1-1,5 ml / 4-2,7 mg / mL </td><td> 33- 35 </td><td> 0,5- 3 millimetri </td></tr><tr><td> Ipotalamo </td><td> 90 ° - 180 ° </td><td colspan="2"> Centro delle electodes solo posteriormente il primordia orecchio </td><td> E12- E13 </td><td> 1-1,5 ml / 4-2,7 mg / mL </td><td> 33- 35 </td><td> 0,5- 3 millimetri </td></tr></tbody></table> Tabella 1:. Sintesi dei risultati Inclusi in questa tabella è tutte le informazioni utili per specifiche trasfezione delle aree corticali descritte. Esso contiene dettagli sulla regione di destinazione, l&#39;angolo, la posizione dell&#39;elettrodo, stadio embrionale, la quantità applicata e la concentrazione della soluzione di DNA, la tensione e la dimensione dell&#39;elettrodo.

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex, Hippocampus-, Thalamus-, Hypothalamus-, laterale del setto Nucleus- e Striatum specifici<em&gt; In Utero</em&gt; Elettroporazione in C57BL / 6 del mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

27.9K Views.  University Medical Center, JGU Mainz. This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Video: Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

Ex utero elettroporazione e Organotipica Fetta Cultura del mouse ippocampale Tissue

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

Ricerca

Biologia dello sviluppo

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero iniezione intraventricolare e Elettroporazione di E15 embrioni di topo

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biologia

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Gene consegna efficiente in più territori SNC utilizzo In Utero Elettroporazione

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Neuroscienze

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Ampliamento della embrionali e cellule staminali neurali adulte per<em&gt; In Utero</em&gt; Elettroporazione o Viral iniezione stereotassica

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

La manipolazione genetica del topo sviluppo dell&#39;ipotalamo attraverso<em&gt; In utero</em&gt; Elettroporazione

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

Doppia elettroporazione in utero per colpire le popolazioni di cellule temporali e separate spazialmente

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

In Vivo Targeting di cellule progenitrici neurali in Ferret Neocortex di In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Cortex, Hippocampus-, Thalamus-, Hypothalamus-, laterale del setto Nucleus- e Striatum specifici

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