Name: in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity
Uploaded: 2017-02-15T13:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

Biz onların mükemmel teknik yardım ve düzenleme için LA Weiss R. Gutiérrez ve A. Morales minnettarız. CGB İspanyol Ministerio de Ciencia e INNOVACIÓN (MICINN), FPI-BES-2012-056011 tarafından finanse edilmektedir. Bu çalışma, M. Navarrete BBVA Vakfı ve SAF2014-58598-Jin (MINECO) bir hibe ile ve Ramón Areces Vakfı ve hibe SAF2014-52119-R ve (MINECO itibaren) BFU2014-55738-REDT için bir hibe ile finanse edildi M. Nieto.

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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>

Yazarlar herhangi bir çıkar çatışması beyan ederim.

Bu protokol onların bağlantı ve heyecanlanma analiz etmek amacıyla C75BL / 6 farelerinin somatosensoriyel korteks nöronları etiketlemek için nasıl ayrıntılı olarak anlatılmaktadır. Mevcut yöntemlere göre, bu tür nöron başına aksonal şube sayısı, bunların kesin topografya ve anatomik konumu gibi bağlantı ayrımcı yönlerini görüntüler. Elektrotların pozisyonlarını değiştirerek, tür singulat korteks gibi diğer nöron popülasyonları, hedef (elektrotlar ve beyin arasındaki aynı açıy...

dendritik ve aksonal yapıların geliştirilmesi serebral korteks olmak üzere, sinir sisteminde devre düzenleme önemli bir yönüdür. Bu farklı nöronal alt popülasyonlar seçici kablolama sırasında kritik bir rol oynamaktadır. son raporlar bir dizi bağlantının yanı sıra, sinir hücrelerinin molekül çeşitliliği ateşleme oldukça spesifik modları satın tarafından yansıtılır, göstermiştir. Ancak, gelişim sırasında farklı nöron alt tipleri heyecanlanma ve bağlantı belirleyen mekanizmaları yanı sıra koordinasyon dereceleri, hala tam, 2 1 anlaşılmaktadır. Vivo kaybı: ve kazanç fonksiyon-ifadesi spesifik genlerin düzeyi ve devrenin gelişimindeki etkisi arasındaki ilişkinin incelenmesi için izin analiz eder. Utero elektroporasyon (İEÜ) yaygın incelemek için kullanılan bir tekniktirspesifik sinir hücresi popülasyonlarını daki bir genin işlevi ve bunların bağlantı biçimin çalışma. Bununla birlikte, canlı farelerde kortikal katmanları aksonların ve dendritlerin morfolojik özelliklerini belirlemek için, seyrek nöronlar etiketlemek için gereklidir. İEÜ ile birleştirilmiş Cre rekombinasyon sistemi tespit kortikal laminaların tek tek hücreler tarafından yayılan projeksiyonları çözmek için yeterince düşük bir yoğunlukta nöronların seyrek popülasyonu işaretlemek için de kullanılabilir. Bu yöntem, elektroporasyona beyinleri (Şekil 1) arasında uygun sayılar analiz sonra niceliksel veri elde etmek için korteks başına nöronların yeterli sayıda etiket. Bu yazıda bağlantı, örneğin ince analizi için bir yöntem sunulur. Bu durum, ayrı deneylerde analiz etmek için benzer bir strateji sunulur, yeşil floresan proteini (GFP) üzerine akım kelepçe kayıtları gerçekleştirerek nöronların elektriksel özellikleri, akut korteks dilimlerinin hücreleri -electroporated. bu proprotokollere‖ kayıpları ve fonksiyon kazancı İEÜ sırasında ek plazmidlerle dahil edildikleri nöronların zamanda çok yönlü ve WT ve transjenik hayvanların nöronların uyarılabilirliğinin ve bağlantı çalışma uygulanabilir ve. Bu protokol, embriyonik gün (E) 15.5 farelerin elektroporasyon açıklar, ancak bu teknik E9.5 3 ve doğum sonrası güne (P) 2 ile 4 arasındaki herhangi bir yaşta uygulanabilir. Erken dönemde elektroporasyon nöronlar ve talamus öncülerini ve korteksin derin katmanlannda daha sonra aşamalı elektroporasyon işaretleri daha yüzeysel tabakalar (örneğin, E15.5 IUE hedefleri tabaka II-III nöronlar) hedef ise. Özetle, tek hücreli morfolojik analiz ve elektrofizyoloji ile İEÜ kombinasyonu sinir sisteminde nöron muazzam yapısal ve işlevsel çeşitlilik altında yatan moleküler mekanizmaları aydınlatmak için yararlı bir araçtır.

<table><tbody><tr><td>pCAG-Cre</td><td>Addgene</td><td>13775</td><td></td></tr><tr><td>pCALNL-GFP</td><td>Addgene</td><td>13770</td><td></td></tr><tr><td>pCAG-DsRed2</td><td>Addgene</td><td>15777</td><td></td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td></td></tr><tr><td>Fast Green</td><td>Carl Roth</td><td>301.1</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>1615 ESP</td><td></td></tr><tr><td>Isoflurane (IsoFlo)</td><td>Abbott (Esteve)</td><td>1385 ESP</td><td></td></tr><tr><td>Ketamine (Imalgene)</td><td>Merial</td><td>2528-ESP</td><td></td></tr><tr><td>Xylazine (Xilagesic)</td><td>Calier</td><td>0682-ESP</td><td></td></tr><tr><td>Povidone Iodine</td><td>Meda</td><td>694109.6</td><td></td></tr><tr><td>Eye Ointment (Lipolac)</td><td>Angelini</td><td>65.277</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Gibco by Life Technologies</td><td>24020-091</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma -Aldrich</td><td>P4333</td><td></td></tr><tr><td>Scalpel Handle # 3 - 12 cm</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Blades # 10</td><td>Fine Science Tools</td><td>10010-00</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>Hardened Fine Scissors - Straight 11 cm</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Scissors Mezenbaum-Nelson Curved L=14.5 cm</td><td>Teleflex</td><td>PO143281</td><td></td></tr><tr><td>Thin curved tips - Style 7 Dumoxel</td><td>Dumont</td><td>0303-7-PO</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>Mathieu Needle Holder - Serrated</td><td>Fine Science Tools</td><td>12010-14</td><td></td></tr><tr><td>AutoClip Applier</td><td>Braintree scientific, Inc</td><td>ACS&amp;nbsp;APL</td><td></td></tr><tr><td>9 mm AutoClips</td><td>MikRon Precision, Inc.</td><td>205016</td><td></td></tr><tr><td>Sutures - Polysorb 6-0</td><td>Covidien</td><td>UL-101</td><td></td></tr><tr><td>Electric Razor&amp;nbsp;</td><td>Panasonic</td><td>ER 240</td><td></td></tr><tr><td>Borosilicate glass capillaries (100 mm, 1.0/0.58 Outer/Inner diameter)</td><td>World Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma -Aldrich</td><td>A5177-5EA</td><td></td></tr><tr><td>Gauze (Aposan)</td><td>Laboratorios Indas, S.A.U.</td><td>C.N. 482232.8</td><td></td></tr><tr><td>Cotton Swabs (Star Cott)</td><td>Albasa</td><td>-</td><td></td></tr><tr><td>Needle 25 G (BD Microlance 3)</td><td>Becton, Dickinson and Company</td><td>300600</td><td></td></tr><tr><td>Sucrose</td><td>Sigma -Aldrich</td><td>S0389</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma -Aldrich</td><td>158127</td><td></td></tr><tr><td>OCT Compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Tissue Culture Dish 100 x 20 mm</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>GFP Tag Polyclonal Antibody</td><td>Thermo Fisher Scientific</td><td>A-11122</td><td></td></tr><tr><td>Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10270106&amp;nbsp;</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>Electroporator ECM 830&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td>45-0002</td><td></td></tr><tr><td>Platinum electrodes 650P 7 mm</td><td>Nepagene</td><td>CUY650P7</td><td></td></tr><tr><td>Microscope&amp;nbsp;for Fluorescent Imaging - MZ10F</td><td>Leica</td><td>-</td><td></td></tr><tr><td>VIP 3000 Isofluorane Vaporizer</td><td>Matrx</td><td>-</td><td></td></tr><tr><td>TCS-SP5 Laser Scanning System</td><td>Leica</td><td>-</td><td></td></tr><tr><td>Axiovert 200 Microscope</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Cryostat - CM 1950</td><td>Leica</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>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>Acquisition software (MultiClamp 700B Amplifier)</td><td>Molecular Devices</td><td>DD1440A</td><td></td></tr><tr><td>Motorized Micromanipulator + Rotating Base&amp;nbsp;</td><td>Sutter Instrument</td><td>MP-225</td><td></td></tr><tr><td>Air Table</td><td>Newport</td><td>-</td><td></td></tr><tr><td>Miniature Peristaltic Pumps</td><td>WPI</td><td>-</td><td></td></tr></tbody></table>

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

sinir sistemi farklı nöron türlerinden büyük bir aralık oluşmaktadır. Bu nöronal alt popülasyonlar diğer özellikleri, onların farklı dendritik morfolojileri, aksonal bağlantı kendi özel desenler ve bunların seçici ateşleme yanıtları arasında, ile karakterize edilir. gelişimi sırasında farklılaşma bu yönleri sorumlu moleküler ve hücresel mekanizmalar hala tam olarak anlaşılamamıştır. Burada, etiketleme için kombine protokolleri tanımlar ve kortikal nöronların yapısal bağlantı ve eksitabilite karakterize. Utero elektroporasyon (İEÜ) protokolünün Modifikasyon nöronların seyrek nüfus etiketleme sağlar. Bu da, dendritler ve bireysel nöronlar, aksonal projeksiyonlar laminer konumu hassas karakterizasyonu ve morfometrik analizi akson belirlenmesini ve izlenmesini sağlar. İEÜ ayrıca eksitabilite değişiklikleri araştırmak için kullanılabilirvahşi tip (WT) ya da elektroporasyon beyinleri akut kesitlerden bütün hücre kaydı ile birleştirerek genetik olarak modifiye edilmiş nöronlar. Bu iki teknik yapısal ve işlevsel bağlantı kuplajın daha iyi anlaşılmasına ve gelişme sırasında nöronal çeşitliliği kontrol eden moleküler mekanizmaların katkıda bulunur. Bu gelişimsel süreçler aksonal kablolama, nöronların fonksiyonel çeşitlilik ve bilişsel bozuklukların biyolojisi üzerinde önemli etkileri vardır.

detaylı bir şekilde ve gelişimi boyunca nöronların morfolojik değişikliklerini karakterize etmek için, seyrek nöronlar etiketlemek için gereklidir. Bu enzim dahil yalnızca nöronlar GFP (Şekil 1A) ifade için bir Cre rekombinaz seyreltildi sistemi, nöronların seyrek popülasyonunda ilgi konusu bir genin sentezlenmesi için izin verir. Bu strateji kullanılarak, tabaka II-III hedefli ve E15.5 İEÜ tarafından etiketlenmiştir. CAG-DsRed2 / ml 1 ug, CO-elekt...

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

Bu yazının tek hücre düzeyinde ve floresan etiketli nöronların eksitabilite nöronların yapısal bağlantı tanımlamak için utero elektroporasyon (İEÜ) kullanmak protokolleri sağlar. Histoloji dendritik ve aksonal projeksiyonlar karakterize etmek için kullanılır. Akut dilim tam hücreli kayıt eksitabilitesini araştırmak için kullanılır.

Rahimde Elektroporasyon Nöronal alt popülasyonlar heyecanlanma ve Tek hücreli Bağlantı Eğitim Yaklaşımları

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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Neuroscience

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

9.6K Görüntüleme.  Centro Nacional de Biotecnología (CNB-CSIC).  Bu yazının tek hücre düzeyinde ve floresan etiketli nöronların eksitabilite nöronların yapısal bağlantı tanımlamak için utero elektroporasyon (İEÜ) kullanmak protokolleri sağlar. Histoloji dendritik ve aksonal projeksiyonlar karakterize etmek için kullanılır. Akut dilim tam hücreli kayıt eksitabilitesini araştırmak için kullanılır. 

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 Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

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

Nörobilim

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.

Verimli kullanarak birden çok CNS Topraklar&#39;da Gen Teslimat In Utero Elektroporasyon

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

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.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

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 ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Eski utero Elektroporasyon ve Tüm Yarımküre Eksplantlarından: Erken Kortikal Kalkınma Çalışmaları İçin Basit Bir Deney Yöntemi

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Yenidoğan Pial Yüzey Elektroporasyon

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Kortikal internöronlar alt tip-selektif Elektroporasyon

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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

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

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

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

Eski Utero Elektroporasyon ve embriyonik fare beyni canlı görüntüleme, GABAergic Interneurons geçiş için Organotypic dilim kültürleri

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

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.

Geçici ve Mekansal Olarak Ayrılmış Hücre Popülasyonlarını Hedef lemek için Utero Elektroporasyonunda Çift

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.

Utero Elektroporasyon in Ferret Neokorteks nöral progenitor hücrelerinin Vivo Hedefleme

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

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

Rahimde Elektroporasyon Nöronal alt popülasyonlar heyecanlanma ve Tek hücreli Bağlantı Eğitim Yaklaşımları

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In utero Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

Verimli kullanarak birden çok CNS Topraklar'da Gen Teslimat In Utero Elektroporasyon

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon

Eski utero Elektroporasyon ve Tüm Yarımküre Eksplantlarından: Erken Kortikal Kalkınma Çalışmaları İçin Basit Bir Deney Yöntemi

Yenidoğan Pial Yüzey Elektroporasyon

Kortikal internöronlar alt tip-selektif Elektroporasyon

Eski Utero Elektroporasyon ve embriyonik fare beyni canlı görüntüleme, GABAergic Interneurons geçiş için Organotypic dilim kültürleri

Geçici ve Mekansal Olarak Ayrılmış Hücre Popülasyonlarını Hedef lemek için Utero Elektroporasyonunda Çift

Utero Elektroporasyon in Ferret Neokorteks nöral progenitor hücrelerinin Vivo Hedefleme

Murine In Utero Electroporation