Biz teknik yardım için Lihong Yin minnettarız. NVD bir NARSAD Genç Araştırmacı Ödülü bir alıcı ve aynı zamanda NIH (5 K99 MH095825-02) hibe tarafından desteklenmektedir. Fishell laboratuarında Araştırma, Ulusal Sağlık Enstitüsü, Ruh Sağlığı (5 R01 MH095147-02, 5 R01 MH071679-09) Ulusal Enstitüsü, Nörolojik Bozukluklar ve İnme (5 R01 NS081297-02 Ulusal Enstitüsü tarafından desteklenen 1 P01 NS074972 -01A1) ve Simons Vakfı.

Tüm hayvanlar Tıp NYU Okulu Kurumsal Hayvan Bakım ve Kullanım Kurulu yönetmelik ve yönergelere uygun olarak tedavi edildi. Fare Suşlar Taconic tarafından sağlanan İsviçre Webster dişi farelerine, bu deneylerde kullanıldı. Özellikle yüzeysel katman hedef nöronları için, embriyolar E15.5 kullanılmıştır. Not: standart klonlama teknikleri kullanılarak elde edilen bu çalışma (Dlx5 / 6.eGFP plazmid 3 ug / ml) kullanılan plazmid. EGFP cDNA Dlx5 / 6-Pmin-Poli A plazmidi içine klonlandı. Bu plazmid istek (demarn02@nyumc.org) üzerine mevcuttur. Mikroenjeksiyon Pipetler hazırlanması 1. <ol><li> 860 çektirmesi Isı 2. ayarlayın. </li><li> Yerleştirin ve filamanın yakın cam kılcal sabitleyin. Pipet dış çapı (OD) kabaca 30 mikron olup olmadığını kontrol edin. </li><li> &quot;Çekme&quot; düğmesine basın. </li><li> Dikkatlice kılcal çıkarın. Taban kısmı, bir iğne. Üst kısmını atın. </li></ol> 2. Anestezi Prosedürü <ol><li> Izofluran içeren odayı hazırlayın. Not: İsofluranın nedeniyle hızlı hareket ve yan etki azlığı için tercih edilen anestezi olduğunu. Pentobarbital başka bir seçenektir; Bununla birlikte, bu ilaç tolerans değişkendir ve izofluran daha yüksek bir ölüm oranına neden olabilir. </li><li> 0.5-1 L / dk O 2% 4-5 kadar izofluran akışını ayarlayın. </li><li> Kafa adaptörü için vana kapalı iken odası için vanasının açık olduğundan emin olun. </li><li> Odacık içinde hamile fare yerleştirin. </li><li> Fare anestezi etkisi altında gitmek için izin verin. Bu yaklaşık 2-3 dakika sürer. Solunum hızını kontrol edin. Fare ım başlarsa, izofluran dozunu. </li><li> Fare anestezi sonra derhal, bölmeden çıkarın. Isıtma yastığı ventral tarafta yerleştirin ve iameliyat boyunca izofluran tedarik etmeye devam edecek maskesi kafasını nsert. Kafa adaptörü bağlantı parçasını boru odacıktan izofluran akışını değiştirmek için emin olun. Her gözün göz yağlayıcı bir damla koyun ve gözlerinizi kapatın. </li><li> Hipotermi en aza indirmek için 36 ° C dereceye ısıtma yastığı ayarlayın. </li><li> Arka pençeleri ve kuyruk kısma ayak ve kuyruk reflekslerin yokluğu kontrol edin. </li></ol> 3. Cerrahi <ol><li> % 70 etanol ile karın kapsayan cildi temizlemek. </li><li> Yukarı cilt çekmek için forseps kullanabilir. Her ameliyat sonrası, deterjan ve su ile tüm enstrümanları yıkayın ve 72 ° CO / N sterilize. </li><li> Meme bezlerinin üçüncü ve dördüncü çiftleri arasındaki yarıda başlayan orta hat boyunca küçük bir düşey kesi (yaklaşık 2 cm) olun. Ince cerrahi makas kullanın. Karın duvarına zarar değil dikkat edin. </li><li> Yavaşça periton cilt ayırmak. </li><li> Arterleri görselleştirmekve karın duvarı üzerinde damarlar. Karın boşluğuna maruz (damarları kaçınarak) karın zarı aracılığıyla başka bir 2 cm kesi yapın. </li><li> Arterleri sıkma kaçınarak her tarafta deri ve karın duvarı Kelepçe. Not: arterler yanlışlıkla delinirse, servikal çıkık veya izofluran doz aşımı yaparak hayvanı hemen kurban. </li><li> 37 ° C&#39;ye kadar ısınmaya öncesi steril PBS. Kim mendil nemli PBS ile kelepçeleri örtün. </li><li> PBS ile maruz karın boşluğuna nemlendirir. Ameliyat yaparken bu adımı birkaç kez tekrarlayın. </li><li> Yuvarlak forseps kullanarak, yavaşça uzak boşluğundan embriyonik zinciri (E15.5) çekin. Islak mendillerin üzerindeki zincir ömür. Birinci rahim yarısını ortaya başlayın. Içinde embriyolar elektroporasyona sonra,, içine geri getirmek ardından ikinci yarıda çalışır. </li></ol> 4. Elektroporasyon <ol><li> Embriyolar işlemek için yuvarlak forseps kullanmaya devam edin. </li><li> Viyanal ventriküller sualize. Orta hat (Şekil 1b) boyunca çalıştırın lateral ventriküller. </li><li> Hızlı Yeşil (Stok:% 10 ağ / hac) eklemek için, bir 01:20 karışım için DNA solüsyonuna enjeksiyon sırasında görselleştirmek için. Damıtılmış su içinde standart bir maksi-hazırlık saflaştırma protokolü elde edilen pelet eritilerek DNA solüsyonu hazırlayın. </li><li> Hızlı Yeşil DNA çözeltisi (Dlx5 / 6-eGFP, 3 ug / ml) 1 ml enjekte etmek için bir piston ile önceden çekilmiş mikropipetler kullanın. Pipet yüklemeden önce bir iğne ucunun sonuna omuz başlangıcından 6 mm uzunluk bırakmak için iyi bir forsepsle # 5 (Dumont) ile mikropipet ucu kesilir. Yuvarlak cımbız ile embriyo baş tutun. Orta hat yakınında bulunan lateral ventrikül amaçlayan bir 45 ° açıyla pipet ile beyin girin. İğne ventrikül ile nüfuz Eğer DNA enjekte pistonu kullanabilirsiniz gibi, yavaş yavaş pipet çekin. Ventrikül yeşile ne zamançözüm doldurmak başlar. Bu noktada, pipet hareket durdurmak ve DNA 1 ml enjekte edilir. Yavaşça, pipet çekilme. </li><li> 950 msn ile aralıklı 50 milisaniye beş 45V bakliyat CUY21 elektrogözenekleme ayarlayın. </li><li> E15.5 embriyolar 5 mm raket elektrotları kullanın. Etkili akım iletimi sağlamak için PBS içinde elektrotlar batırın. </li><li> DNA çözümü içeren ventrikül olumlu kürek ile embriyonun başının etrafında elektrot kürekler. Biraz ganglionik tubera ile ventral akım yaratmak için negatif bir saygı ile pozitif raket indirin. Bu manipülasyon piramidal hücrelerinde ektopik ifadesini minimize edecektir. </li><li> Elektrotlar yerleştirerek sonra, bir kez ayak pedalına basarak bir darbe teslim. </li><li> Elektrotları çıkarın ve temiz silin. Tekrar her bir embriyo ile 4,6-4,9 adımları. </li><li> Tüm embriyolar electroporating sonra, karın boşluğuna PBS 3 ml dökün ve karın boşluğunda onları geri. </li><li>Üretim dikiş iğnesi ve kavisli bir 5-0 ipek dikiş ipliği daha sonra deri ile karın duvarı. Yaranın üzerine Lidokain (% 4) uygulayın. Analjezi için Aspirin içi periton 120 mg / kg yönetmek. Not: Bütün işlem 20 dakika veya daha kısa yapılmalıdır. Bu yeni başlayanlar sadece kısa çalışma süresini tutmak için bir kaç embriyolar electroporate önerilir. Uzun süreli ameliyatlar embriyoların hayatta kalma oranını azaltacaktır. </li><li> Anestezi borudan hayvan çıkarın ve bir kağıt üzerinde ısıtma yastığı kurtarma silin sağlar. </li><li> , Kafes hayvan dönün 37 ° C&#39;de ısıtma yastığı üzerinde tutmak ve sağlık durumunu izlemek. Hayvanlar genellikle ameliyat iyi tolere ve anestezi tüp çıkarılır kısa bir süre sonra yavaş yavaş hareket etmeye başlar. Aşırı kanama ya da uyuşukluk görülürse, IACUC gibi servikal dislokasyon veya anestezi doz aşımı gibi ötenazi prosedürleri onaylanmış gerçekleştirerek hemen hayvan kurban. </li><li> Viva kafesi dönrium. Dişiler daha doğum doğal verecektir. </li></ol>

<ol>
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D., 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=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., 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=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, A., 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=The+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, A., 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=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. 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Yazarlar ifşa hiçbir şey yok.

Tekniğin Sınırlamalar Bu teknik, hücre süreçlerinin hücre özerk analizi için sağlar, bu popülasyon analizi için uygun değildir. Electroporations beyin başına elektropore az bin hücreleri ile çok seyrek. Bunun bir sonucu olarak, bu teknik CGE türetilmiş internöron genetik manipülasyon kaynaklanan davranış sonuçlarını değerlendirmek için kullanılamaz. Elektroporasyon E13.5-E14.5 hedef MGE türetilmiş alt tiplerinde üzerinden taşınan...

Kortikal GABAerjik internöronlar toplu medial ve kaudal ganglion eminences (sırasıyla MGE ve JMO) adında iki geçici embriyonik yapılarından kaynaklanan 4. Parvalbumin ve somatostatin ifade internöron internöron JMO&#39;ya kaynaklanan ifade Kalretinin ise MGE (Cr), Vasointestinal peptid (VIP) ve Reelin (Re) köken. Bu interneuron alt tipler kendi doğum günlerinin ile ayırt edilebilir. MGE türetilmiş alt tipler embriyonik gün 9.5 (E9.5) ve e16.5 5,6 arasında doğarlar. Buna karşılık, JMO türetilmiş internöron üretim E15.5 6 seviyesinde zirve ile E18.5 yoluyla E12.5 doğar. Bu geç doğdu nüfusun genetik hedefleme, ancak, zor kalır. Kemirgen distal-az (Dlx) genler sadece gelişmekte olan ventral önbeyin 3 olarak ifade edilmiştir. GABAerjik internöron ve striatum projeksiyon nöronları değil, kortikal piramidal hücreleri Dlx1 ifade, Erken gelişim evrelerinde 3 de 2,5, ve 6 genler. Gerçekten de, dlx genlerinin hepsi GABA&#39;erjik atalarda MGE ve CGE subventricular (SVZ&#39;una) olarak ifade edilmiştir. Bu genlerin sentezlenmesi postmitotik aşamalarında 7-9 de alt tiplerini belirlemek için kısıtlanır. Önceki deneysel kanıtlar Dlx5 / 6 arttırıcı bir eleman transgenik fare modellerinde 2 GABAerjik soy seçici hedefleme için izin verdiğini gösterdi. Bu, gelişmekte olan fare beyin epizomal ifade bağlamında bu zenginleştirici elemanların birinin kullanılmasını test edilmiştir. Bir minimal promotör ve Bluescript (BS) omurga plasmid olarak gelişmiş yeşil floresan protein (EGFP) (Şekil 1) ile birlikte Dlx5 / 6 güçlendirici elemanın alt klonlandı. Burada özellikle Cr, VIP hedef E15.5 rahimde elektroporasyon suretiyle plazmid tanıtıldı ve Yeniden 3,8,10 alt tip. Bizim teknik kolaylaştırır seyrek elektroporasyon, sağlarateşten hücrelerin morfolojik özellikleri yeniden. Buna ek olarak, kortikal nöronlarda GABAerjik gen ekspresyonu son derece yüksek seviyelerde fonksiyonel çalışmalarda da sağlar. Biz kaybı ve çeşitli yabani tip ve dominant negatif genler 11 ile fonksiyon testleri kazancını gerçekleştirilir.

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subtype-selective electroporation of cortical interneurons

Merkezi sinir sistemi (CNS) olgunlaşma çalışma sinirsel genetik hedefleme dayanır. Bununla birlikte, özel nöron alt tiplerine ilgi genlerinin ekspresyonunu kısıtlama görev nedeniyle spesifik promoter elementlerinin nispeten kıt olması oldukça zor olduğu kanıtlanmıştır. GABA&#39;erjik internöron geniş genetik ve morfolojik çeşitliliği olan bir nöronal nüfus oluşturmaktadır. Gerçekten de, GABAerjik internöron fazla 11 farklı alt tipi fare korteksi 1 ile karakterize edilmiştir. Burada GABAerjik nüfus seçici hedefleme için uyarlanmış bir protokol mevcut. Bu homeobox transkripsiyon enhanser elemanını kullanarak, GABAerjik internöron alt tip seçici olarak hedeflenmesini elde utero elektroporasyon spesifik genlerin ekspresyonunu ile sürücü ve Dlx5 Dlx6, Drosophila uzak-az (DLL) geni 2,3 homologlarını, faktörüdür.

Biz olgunlaşan nöronların hücre tipi özel hedefleme ulaşmak için utero elektroporasyon tekniği adapte. CGE türevi internöronlarda eGFP ekspresyonunu tahrik etmek için, Dlx5 / 6 güçlendirici eleman ikinci ve E15.5 için enjeksiyon sınırlı aşama CGE türetilmiş internöronlar çoğunluğu oluştuğunda. Biz de P8 analizi gerçekleştirilir ve P15 11 (Şekiller 1 ve 2). Biz E15.5 eşit molar konsantrasyonlarda CAG-mCherry ve Dlx5 ...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

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 ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.8K Görüntüleme.  New York University School of Medicine. 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.

Video: Subtype-selective Electroporation of Cortical Interneurons

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 ...

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In vivo Electroporation of Developing Mouse Retina

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

In vivo Electroporation of Developing Mouse Retina

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

Gelişmekte olan Fare Retina in vivo Elektroporasyon olarak

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

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Fare Kortikal astrositlerde İzolasyon ve Kültür

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Yenidoğan subventricular Bölge Elektroporasyon

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- ve nörokimyasal tanımlanan hipokampal internöronlardan gelen tüm hücre Patch-kelepçe Kayıtlar

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A Guide<em&gt; In vivo</emOptogenetically Belirlenen Kortikal inhibitör dan&gt; Tek birim Kayıt

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</em&gt; Işık Uyaranların için Fotoreseptörler ve internöronlar<em&gt; İn Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Görselleştirme düzleştirilmiş memeli Cortices'deki kortikal modülleri

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...