Siamo grati a R. Gutiérrez e A. Morales per la loro eccellente assistenza tecnica e di LA Weiss per la modifica. CGB è finanziato dagli spagnoli Ministerio de Ciencia e Innovación (MICINN), FPI-BES-2012-056011. Questo lavoro è stato finanziato da una sovvenzione da BBVA Foundation e SAF2014-58598-jin (MINECO) a M. Navarrete e da una sovvenzione della Fondazione Areces e concede SAF2014-52119-R e BFU2014-55738-REDT (da MINECO) a Ramon M. Nieto.

<ol>
	<li>Dehorter, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+fast-spiking+interneuron+properties+by+an+activity-dependent+transcriptional+switch.">Tuning of fast-spiking interneuron properties by an activity-dependent transcriptional switch.</a> Science. 349 (6253), 1216-1220 (2015).</li><li>Rodriguez-Tornos, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cux1+Enables+Interhemispheric+Connections+of+Layer+II/III+Neurons+by+Regulating+Kv1-Dependent+Firing.">Cux1 Enables Interhemispheric Connections of Layer II/III Neurons by Regulating Kv1-Dependent Firing.</a> Neuron. 89 (3), 494-506 (2016).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> J Vis Exp. (54), (2011).</li><li>Sonego, M., Zhou, Y., Oudin, M. J., Doherty, P., Lalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+postnatal+electroporation+and+time-lapse+imaging+of+neuroblast+migration+in+mouse+acute+brain+slices.">In vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices.</a> J Vis Exp. (81), (2013).</li><li>Baumgart, J., Baumgart, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortex-,+Hippocampus-,+Thalamus-,+Hypothalamus-,Lateral+Septal+Nucleus-+and+Striatum-specific+In+Utero+Electroporation+in+the+C57BL/6+Mouse.">Cortex-, Hippocampus-, Thalamus-, Hypothalamus-,Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse.</a> J Vis Exp. (107), (2016).</li><li>Petros, T. J., Rebsam, A., Mason, C. 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+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> J Vis Exp. (31), (2009).</li><li>Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. <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+followed+by+primary+neuronal+culture+for+studying+gene+function+in+subset+of+cortical+neurons.">In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.</a> J Vis Exp. (44), (2010).</li><li>Woodworth, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctip1+Regulates+the+Balance+between+Specification+of+Distinct+Projection+Neuron+Subtypes+in+Deep+Cortical+Layers.">Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers.</a> Cell Rep. 15 (5), 999-1012 (2016).</li><li>Wickersham, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+restriction+of+transsynaptic+tracing+from+single,+genetically+targeted+neurons.">Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.</a> Neuron. 53 (5), 639-647 (2007).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc Natl Acad Sci U S A. 104 (3), 1027-1032 (2007).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc Natl Acad Sci U S A. 101 (1), 16-22 (2004).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protoc. 1 (3), 1552-1558 (2006).</li><li>Bullmann, T., Arendt, T., Frey, U., Hanashima, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transportable,+inexpensive+electroporator+for+in+utero+electroporation.">A transportable, inexpensive electroporator for in utero electroporation.</a> Dev Growth Differ. , (2015).</li><li>Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+rat+visual+cortex.+I.+A+quantitative+study+of+Golgi-impregnated+pyramidal+neurons.">Maturation of rat visual cortex. I. A quantitative study of Golgi-impregnated pyramidal neurons.</a> J Neurocytol. 10 (5), 859-878 (1981).</li><li>Miller, M., Peters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+rat+visual+cortex.+II.+A+combined+Golgi-electron+microscope+study+of+pyramidal+neurons.">Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons.</a> J Comp Neurol. 203 (4), 555-573 (1981).</li><li>Cubelos, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cux-2+controls+the+proliferation+of+neuronal+intermediate+precursors+of+the+cortical+subventricular+zone.">Cux-2 controls the proliferation of neuronal intermediate precursors of the cortical subventricular zone.</a> Cereb Cortex. 18 (8), 1758-1770 (2008).</li><li>Kang, J. Y., Kawaguchi, D., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Control+of+a+Neuronal+Protein+Using+a+Genetically+Encoded+Unnatural+Amino+Acid+in+Neurons.">Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons.</a> J Vis Exp. (109), (2016).</li><li>Mathis, D. M., Furman, J. L., Norris, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+acute+hippocampal+slices+from+rats+and+transgenic+mice+for+the+study+of+synaptic+alterations+during+aging+and+amyloid+pathology.">Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology.</a> J Vis Exp. (49), (2011).</li><li>Maravall, M., Stern, E. A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+intrinsic+properties+and+excitability+of+layer+2/3+pyramidal+neurons+during+a+critical+period+for+sensory+maps+in+rat+barrel+cortex.">Development of intrinsic properties and excitability of layer 2/3 pyramidal neurons during a critical period for sensory maps in rat barrel cortex.</a> J Neurophysiol. 92 (1), 144-156 (2004).</li><li>Karadottir, R., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+patch-clamping+of+cells+in+brain+slices+with+immunocytochemical+labeling+to+define+cell+type+and+developmental+stage.">Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage.</a> Nat Protoc. 1 (4), 1977-1986 (2006).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annu Rev Physiol. 46, 455-472 (1984).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li></ol>

Gli autori dichiarano conflitti di interesse.

Questo protocollo descrive in dettaglio come etichettare i neuroni della corteccia somatosensoriale di C75BL / 6 topi al fine di analizzare la loro connettività e la loro eccitabilità. Rispetto ai metodi esistenti, visualizza aspetti discriminanti connettività, come il numero di rami assonali per neurone, la loro precisa topografia, e la loro posizione anatomica. Modificando la posizione degli elettrodi, è possibile indirizzare altre popolazioni neuronali, come la corteccia del cingolo (mantenere lo stesso angolo tr...

Lo sviluppo di strutture dendritiche e assonale è un aspetto importante della regolazione circuito nel sistema nervoso, compreso nella corteccia cerebrale. Essa svolge un ruolo critico durante il cablaggio selettivo delle diverse sottopopolazioni neuronali. Un certo numero di rapporti recenti hanno dimostrato che, oltre alla connettività, la diversità molecolare dei neuroni è riflessa dalla acquisizione di modalità altamente specifiche di cottura. Tuttavia, i meccanismi che determinano l&#39;eccitabilità e la connettività dei sottotipi neuronali distinti durante lo sviluppo, così come il loro grado di coordinamento, sono ancora poco conosciuti 1, 2. In vivo in perdita e guadagno di funzione analizza consentire lo studio del rapporto tra il livello di espressione di geni specifici e la loro influenza nello sviluppo del circuito. In utero elettroporazione (IUE) è una tecnica ampiamente utilizzata per lo studiola funzione di un gene di interesse in specifiche popolazioni neuronali e studiare gli schemi generali della loro connettività. Tuttavia, per determinare le caratteristiche morfologiche degli assoni e dendriti in strati corticali in topi vivi, è essenziale per etichettare i neuroni scarsamente. Un sistema di ricombinazione Cre combinato con IUE può essere usato per marcare una scarsa popolazione di neuroni in una sufficientemente bassa densità per risolvere le proiezioni emessi dalle singole celle delle lamine corticali identificati. Questo metodo etichetta un numero sufficiente di neuroni per corteccia di ottenere dati quantitativi dopo l&#39;analisi dei numeri ragionevoli di cervelli elettroporate (Figura 1). Questo manoscritto presenta un metodo per tale analisi multa di connettività. Presenta inoltre una strategia simile per l&#39;analisi, in esperimenti separati, le proprietà elettriche dei neuroni eseguendo registrazioni current-clamp su verde proteina fluorescente (GFP) -electroporated cellule da fette corticali acute. questi protocoli sono versatili e possono essere applicati allo studio dell&#39;eccitabilità e la connettività dei neuroni di animali WT e transgenici, e anche di neuroni in cui perdite e guadagni di funzione vengono introdotti da plasmidi addizionali durante IUE. Sebbene questo protocollo descrive l&#39;elettroporazione di topi a giorno embrionale (E) 15.5, questa tecnica può essere eseguita a qualsiasi età tra E9.5 3 al giorno postnatale (P) 2 4. Mentre elettroporazione nelle fasi iniziali si rivolge neuroni e precursori del talamo e strati profondi della corteccia, i marchi di elettroporazione in seguito allo stadio strati più superficiali (ad esempio, neuroni E15.5 IUE obiettivi strato II-III). In sintesi, la combinazione di IUE con l&#39;analisi morfologica singola cellula ed elettrofisiologia è uno strumento utile per chiarire i meccanismi molecolari alla base della enorme diversità strutturale e funzionale dei neuroni nel sistema nervoso.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>pCAG-Cre</td>
			<td>Addgene</td>
			<td>13775</td>
		</tr>
		<tr>
			<td>pCALNL-GFP</td>
			<td>Addgene</td>
			<td>13770</td>
		</tr>
		<tr>
			<td>pCAG-DsRed2</td>
			<td>Addgene</td>
			<td>15777</td>
		</tr>
		<tr>
			<td>pCAG-GFP</td>
			<td>Addgene</td>
			<td>11150</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Carl Roth</td>
			<td>301.1</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
		</tr>
		<tr>
			<td>Carprofen (Rimadyl)</td>
			<td>Pfizer GmbH</td>
			<td>1615 ESP</td>
		</tr>
		<tr>
			<td>Isoflurane (IsoFlo)</td>
			<td>Abbott (Esteve)</td>
			<td>1385 ESP</td>
		</tr>
		<tr>
			<td>Ketamine (Imalgene)</td>
			<td>Merial</td>
			<td>2528-ESP</td>
		</tr>
		<tr>
			<td>Xylazine (Xilagesic)</td>
			<td>Calier</td>
			<td>0682-ESP</td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Meda</td>
			<td>694109.6</td>
		</tr>
		<tr>
			<td>Eye Ointment (Lipolac)</td>
			<td>Angelini</td>
			<td>65.277</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Gibco by Life Technologies</td>
			<td>24020-091</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma -Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>Scalpel Handle #3 - 12cm</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
		</tr>
		<tr>
			<td>Scalpel Blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
		</tr>
		<tr>
			<td>Adson Forceps-Serrated - Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors - Straight &#160; 11 cm</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
		</tr>
		<tr>
			<td>Scissors Mezenbaum-Nelson Curved L=14,5cm</td>
			<td>Teleflex</td>
			<td>PO143281</td>
		</tr>
		<tr>
			<td>Thin curved tips - Style 7 Dumoxel</td>
			<td>Dumont</td>
			<td>0303-7-PO</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
		</tr>
		<tr>
			<td>Mathieu Needle Holder - Serrated</td>
			<td>Fine Science Tools</td>
			<td>12010-14</td>
		</tr>
		<tr>
			<td>AutoClip Applier</td>
			<td>Braintree scientific, Inc</td>
			<td>ACS&#160;APL</td>
		</tr>
		<tr>
			<td>9mm AutoClips</td>
			<td>MikRon Precision, Inc.</td>
			<td>205016</td>
		</tr>
		<tr>
			<td>Sutures - Polysorb 6-0</td>
			<td>Covidien</td>
			<td>UL-101</td>
		</tr>
		<tr>
			<td>Electric Razor&#160;</td>
			<td>Panasonic</td>
			<td>ER 240</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (100mm, 1.0/0.58 Outer/Inner diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma -Aldrich</td>
			<td>A5177-5EA</td>
		</tr>
		<tr>
			<td>Gauze (Aposan)</td>
			<td>Laboratorios Indas, S.A.U.</td>
			<td>C.N. 482232.8</td>
		</tr>
		<tr>
			<td>Cotton Swabs (Star Cott)</td>
			<td>Albasa</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Needle 25G (BD Microlance 3)</td>
			<td>Becton, Dickinson and Company</td>
			<td>300600</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma -Aldrich</td>
			<td>S0389</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>158127</td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 100 x 20 mm</td>
			<td>Falcon</td>
			<td>353003</td>
		</tr>
		<tr>
			<td>GFP Tag Polyclonal Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11122</td>
		</tr>
		<tr>
			<td>Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Electroporator ECM 830&#160;</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0002</td>
		</tr>
		<tr>
			<td>Platinum electrodes 650P 7 mm</td>
			<td>Nepagene</td>
			<td>CUY650P7</td>
		</tr>
		<tr>
			<td>Microscope&#160;for Fluorescent Imaging - MZ10F</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>VIP 3000 Isofluorane Vaporizer</td>
			<td>Matrx</td>
			<td>-</td>
		</tr>
		<tr>
			<td>TCS-SP5 Laser Scanning System</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Axiovert 200 Microscope</td>
			<td>Zeiss</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cryostat - CM 1950</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
		</tr>
		<tr>
			<td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td>
			<td>Molecular Devices</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Acquisition software (MultiClamp 700B Amplifier)</td>
			<td>Molecular Devices</td>
			<td>DD1440A</td>
		</tr>
		<tr>
			<td>Motorized Micromanipulator + Rotating Base&#160;</td>
			<td>Sutter Instrument</td>
			<td>MP-225</td>
		</tr>
		<tr>
			<td>Air Table</td>
			<td>Newport</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Miniature Peristaltic Pumps</td>
			<td>WPI</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity

Il sistema nervoso è composto da una vasta gamma di tipi neuronali distinti. Queste sottopopolazioni neuronali sono caratterizzati da, tra le altre caratteristiche, le loro morfologie dendritiche distinti, i loro modelli specifici di connettività assonale, e le loro risposte di cottura selettive. I meccanismi molecolari e cellulari responsabili di questi aspetti di differenziazione durante lo sviluppo sono ancora poco conosciuti. Qui, descriviamo protocolli combinati per l&#39;etichettatura e caratterizzare la connettività strutturale e l&#39;eccitabilità dei neuroni corticali. Modifica del protocollo di elettroporazione in utero (IUE) consente l&#39;etichettatura di una popolazione sparsa di neuroni. Questo, a sua volta, consente l&#39;identificazione e la manutenzione dei dendriti e assoni dei singoli neuroni, la precisa caratterizzazione della posizione laminare di proiezioni assonale e analisi morfometrica. IUE può anche essere usato per studiare variazioni l&#39;eccitabilitàwild-type (WT) o neuroni geneticamente modificati attraverso la combinazione con la registrazione a cellula intera da fettine acute di cervelli elettroporate. Queste due tecniche contribuiscono ad una migliore comprensione dell&#39;accoppiamento di connettività strutturale e funzionale dei meccanismi molecolari che controllano diversità neuronale durante lo sviluppo. Questi processi di sviluppo hanno importanti implicazioni sul cablaggio assonale, la diversità funzionale dei neuroni, e la biologia dei disturbi cognitivi.

Per caratterizzare i cambiamenti morfologici dei neuroni nel dettaglio e in tutto lo sviluppo, è essenziale per etichettare i neuroni scarsamente. Un sistema Cre-ricombinasi diluita permette l&#39;espressione di un gene di interesse in una popolazione sparsa di neuroni, in modo che solo i neuroni che incorporano questo enzima esprimono GFP (Figura 1A). Usando questa strategia, strato di II-III è mirato ed etichettato da IUE a E15.5. CAG-DsRed2 a 1 mg / mL, è co-elettr...

Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

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

Questo manoscritto fornisce protocolli che utilizzano in utero elettroporazione (IUE) per descrivere la connettività strutturale dei neuroni a livello di singola cellula e l&#39;eccitabilità dei neuroni fluorescente. Istologia è utilizzato per caratterizzare dendritiche e le proiezioni assonali. registrazione a cellula intera a fette acuta viene utilizzato per studiare l&#39;eccitabilità.

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, ...

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JoVE Journal

Neuroscience

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

9.5K Views.  Centro Nacional de Biotecnología (CNB-CSIC).  Questo manoscritto fornisce protocolli che utilizzano in utero elettroporazione (IUE) per descrivere la connettività strutturale dei neuroni a livello di singola cellula e l'eccitabilità dei neuroni fluorescente. Istologia è utilizzato per caratterizzare dendritiche e le proiezioni assonali. registrazione a cellula intera a fette acuta viene utilizzato per studiare l'eccitabilità. 

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

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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Neuroscienze

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 ...

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 ...

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

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

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

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

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 ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Generazione di topi transgenici per uso topico<em&gt; In utero</em&gt; Elettroporazione e<em&gt; In vivo</em&gt; Screening bioluminescenza

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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 ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analizzando dendritiche Morfologia in colonne e strati

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...