我们非常感谢尹立红提供技术援助。 NVD是一个NARSAD青年研究者奖的获得者，也从美国国立卫生研究院（5 K99 MH095825-02）支持补助。研究在Fishell实验室由卫生心理健康（5 R01 MH095147-02，5 R01 MH071679-09）的神经疾病和中风（5 R01 NS081297-02研究所研究所，研究所，支持，1 P01 NS074972 -01A1）和西蒙斯基金会。

所有的动物都按照医学纽约大学的机构动物照顾及使用委员会的规例和指引处理。 品系小鼠 由TACONIC提供瑞士韦伯斯特雌性小鼠用于这些实验。为了特异性靶向表面层的interneurons，E15.5胚胎被使用。 注意:该作品（DLX5 / 6.eGFP质粒3微克/微升），使用标准的克隆技术生成中使用的质粒。 绿色荧光蛋白的cDNA克隆到DLX5 / 6-p最小-聚腺苷酸质粒。该质粒可应要求提供（demarn02@nyumc.org）。 1，准备显微注射移液器<ol><li>将牵引热火＃2到860。 </li><li>放置并固定在附近的长丝玻璃毛细管。检查该移液管的外径（OD）为约30微米。 </li><li>按"拉"按钮。 </li><li>小心地取出毛细管。底部的部分是针。弃顶部。 </li></ol> 2，麻醉过程<ol><li>准备一个含有异氟醚室。注:异氟醚是首选的麻醉剂，由于其快速的行动和副作用发生率低。苯巴比妥是另一种选择;然而，这种药物的耐受性是可变的，并可能导致死亡率比异氟烷更高。 </li><li>在0.5-1升/分钟O 2的设置异氟醚4-5％的流量。 </li><li>确保阀的腔室是打开的，而阀的头部适配器被关闭。 </li><li>将怀孕小鼠中的腔室中。 </li><li>让鼠标在麻醉状态下的作用去。这将需要大约2-3分钟。检查呼吸速率。如果鼠标开始换气过度，低级异氟醚的剂量。 </li><li>之后鼠标麻醉，迅速将其从室内除去。把它放在腹侧的加热垫，我nsert头中，将继续在整个手术提供异氟烷掩模。确保从室切换的异氟醚的流动连接头转接件管材。放在每只眼睛一滴眼润滑剂和瞑。 </li><li>设定加热垫36°C度，以减少体温过低。 </li><li>按捏后爪和尾检查没有脚和尾巴反射。 </li></ol> 3手术<ol><li>清洁皮肤覆盖，用70％乙醇的腹部。 </li><li>使用镊子拉扯皮肤向上。每次手术后，清洗所有工具用清洁剂和水，灭菌在72℃CO / N。 </li><li>做一个小切口垂直（2厘米左右）沿着第三和第四对乳腺腺体之间的中途开始的中线。用细的手术剪刀。小心不损坏腹壁。 </li><li>轻轻分开腹膜皮肤。 </li><li>可视化的动脉和腹壁上的静脉。使通过腹膜另外2厘米的垂直切口（避开血管）以暴露腹腔。 </li><li>夹紧每侧避免夹住动脉皮肤和腹壁。注意:如果动脉被意外刺破，通过进行颈椎脱位或异氟过量，立即牺牲的动物。 </li><li>预暖无菌PBS至37℃。封面用PBS夹湿抹布金。 </li><li>滋润暴露腹腔，用PBS。在执行手术重复这一步骤数次。 </li><li>用圆钳，轻轻一拉胚链（E15.5）离腔。让我们在湿巾链条休息。首先露出半个子宫第一。一旦它的胚胎被电穿孔，将其带回里面，然后工作在下半年。 </li></ol> 4，电穿孔<ol><li>继续用圆钳操纵胚胎。 </li><li>六sualize侧脑室。沿中线（ 图1b）上运行的侧脑室。 </li><li>添加固绿（股票:10％W / V）为1:20混合的DNA溶液的注射过程中形象化了。通过将来自于蒸馏水中的标准加厚制备型纯化方案得到的粒料制备的DNA溶液。 </li><li>使用预拉带微量柱塞注射1毫升DNA溶液（DLX5 / 6-EGFP，3微克/微升）与固绿的。切微量的前端用细钳子＃5（蒙特）离开6毫米长度从肩部到针的尖端的端部的开始装入吸管之前。保持胚胎的头圆镊子。进入大脑的吸管以45度角瞄准位于中线附近侧脑室。如果针穿过心室，慢慢缩回吸管，所使用的活塞注入的DNA。脑室应该变成绿色时，溶液开始填充它。此时，停止移动的移液管和注1毫升的DNA。轻轻地，撤回吸管。 </li><li>设置CUY21电穿孔五45V脉冲50毫秒由950毫秒间隔。 </li><li>使用5毫米桨电极的E15.5胚胎。浸在PBS的电极，以保证高效的电流传输。 </li><li>周围放置胚胎的头部的电极极板与上含有该DNA溶液的心室正桨。略低的积极桨与尊重的负一层通过神经节隆起创建腹电流。该操作会减少异位表达的锥体细胞。 </li><li>将电极放置后，通过按下脚踏开关一次提供一个脉冲。 </li><li>取下电极和擦拭干净。重复，每个胚胎的步骤4.6-4.9。 </li><li>电穿孔所有的胚胎后，倒入3mL的PBS的进入腹腔，并将它们返回到腹腔。 </li><li>首先缝合腹壁有弯针和5-0丝线缝合，然后在皮肤上。对伤口应用利多卡因（4％）。辖120毫克/千克的阿司匹林腹膜内镇痛。 注意:整个过程应在20分钟或更少的时间内完成。可取的做法是初学者电穿孔只有几个胚胎，以保持手术时间短。延长手术会降低胚胎存活率。 </li><li>从麻醉导管取出的动物，并允许恢复对纸张加热垫擦拭。 </li><li>返回动物笼子，把它加热垫在37℃和监控健康状况。动物通常耐受手术以及与开始缓慢移动它们从麻醉导管取出后不久。如果出血过多或嗜睡观察，通过执行IACUC批准的安乐死程序，如颈椎脱位或麻醉过量，立即牺牲的动物。 </li><li>回到笼子里的VIVArium。女性会生出自然。 </li></ol>

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	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. 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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A. <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+a+member+of+a+new+family+of+murine+Distal-less+homeobox+genes+in+the+developing+forebrain.">Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.</a> The New biologist. 3, 1183-1194 (1991).</li><li>Close, J., 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=Satb1+is+an+activity-modulated+transcription+factor+required+for+the+terminal+differentiation+and+connectivity+of+medial+ganglionic+eminence-derived+cortical+interneurons.">Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.</a> J Neurosci. 32, 17690-17705 (2012).</li><li>De Marco Garcia, N. 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作者什么都没有透露。

该技术的局限性尽管该技术允许对细胞过程的细胞中自主分析，它是不适合于人口分析。在电穿孔是非常稀疏的不到一千细胞每大脑电穿孔。因此，该技术不能被用来评估从CGE衍生的interneurons的遗传操作而产生的行为后果。 而电穿孔进行在E13.5-E14.5目标MGE衍生的亚型，效率低10。我们推测，DLX5 / 6增强剂是在有丝分裂后的MGE亚型活性较低，?...

从命名的内侧和尾神经节隆起（MGE和专家咨询小组，分别）2瞬态胚胎结构的皮质GABAergic中间神经元的散装起源4。小白蛋白和生长抑素表达的interneurons起源于梅兰日兰，而钙结合蛋白（CR），Vasointestinal肽（VIP）和络丝（RE）表达的interneurons从专家咨询小组发起。这些interneuron亚型可以通过他们的出生日期来区分。梅兰日兰派生亚型胚胎9.5天（E9.5）和E16.5 5,6间出生。相比之下，CGE衍生的interneurons从出生E12.5 E18.5，通过与他们的生产高峰在E15.5 6。这么晚出生人口的遗传定位，但是，仍然遥遥无期。 鼠远侧少（DLX）基因只表达在显影腹侧前脑3。 GABAergic中间神经元及纹状体投射神经元，但不皮质锥体细胞表达Dlx1，2,5，和6个基因在早期发育阶段3。事实上，DLX基因被表达在MGE和CGE脑室下区（SVZ）内所有的GABA能祖细胞。这些基因的表达成为限制在有丝分裂后期阶段7-9选择亚型。先前的实验证据表明，DLX5 / 6增强子元件允许GABA能系在转基因小鼠模型2的选择性靶向。我们测试使用在附加型表达在发育中的小鼠大脑中的上下文这些增强子元件中的一个的。我们的子带最小启动子和在BLUESCRIPT（BS）的骨架质粒的增强型绿色荧光蛋白（EGFP）（ 图1）中克隆的DLX5 / 6增强子元件一起。我们推出的质粒在子宫内电在E15.5的方式选择性地靶向CR-，VIP和重新亚型3,8,10。我们的技术可用于稀疏电穿孔，这有利于对辛格的细胞形态特征的重建。此外，极高的水平在皮层GABA能神经元的基因表达可用于功能研究。我们进行了损失，并使用多种野生型和显性负11的基因功能研究的增益。

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			<td style="width:164px;">CUY650P5</td>
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			<td>DCT-15</td>
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			<td>Sutter Instruments</td>
			<td>3282322</td>
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			<td>Roboz</td>
			<td>&#160;RS-6702</td>
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			<td>Roboz</td>
			<td>&#160;RS-5410</td>
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			<td>11031-15</td>
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subtype-selective electroporation of cortical interneurons

中枢神经系统（CNS）的成熟的研究依赖于神经元群体的遗传定位。然而，限制所关心的特定的神经元亚型的基因的表达的任务已被证明非常困难，因为特异性的启动子元件的相对稀缺。 GABAergic中间神经元构成的神经元群具有丰富的遗传和形态多样性。事实上，超过11种不同的GABAergic中间神经元亚型已被鉴定在小鼠皮质1。在这里，我们提出了一个适应协议GABA能居的选择性目标。我们通过使用同源的转录增强子元件来实现GABAergic中间神经元亚型选择性靶向因子DLX5和Dlx6，果蝇远侧少（DLL）基因2,3的同系物，通过以驱动特定基因的表达， 在子宫内的电穿孔。

我们适应了在子宫内电穿孔技术来实现细胞类型特异性成熟神经元的目标。来驱动的eGFP在CGE衍生的interneurons的表达，我们使用了DLX5 / 6增强子元件和受限制了注射至E15.5，在产生阶段的大部分CGE衍生的interneurons。我们所进行的分析，在P8和P15 11（ 图1和2）。我们通过共电穿孔àCAG-mCherry和DLX5 / 6-EGFP质粒等摩尔浓度在E15.5电穿孔证实神经元?...

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.

皮质的interneurons的亚型选择性电穿孔

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 次观看.  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.

视频: 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.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

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

科研

神经科学

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.

在发展中国家小鼠视网膜体内电穿孔

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.

小鼠脑皮层星形胶质细胞的分离和培养

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

新生儿脑室下区电穿孔

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.

前子宫穿孔和整个半球的外植体早期皮质发育的研究:一个简单的实验方法

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.

新生儿软脑膜表面电穿孔

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.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

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.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

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.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</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.

扁平哺乳动物皮层皮质模块的可视化研究

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