This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management.

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The authors have nothing to disclose.

在这里，我们分离使用荧光标记，然后研究以单细胞的RNA-SEQ每个小区由一个异质群体单细胞。我们报告，我们收获并测序活的TH-eGFP的细胞100％的确多巴胺能神经元，是根据以下三个DA相关基因的转录，TH，DDC和SLC6A3的存在。所有TH-EGFP阳性细胞通过RNA-SEQ的评估表达这三个基因。这是一致的与电生理研究显示，每TH-EGFP细胞也呈现出与多巴胺能神经元8,9相关的离子通道的剧目。在这个协议中，我们采用的GENSAT酪氨酸羟化酶-EGFP小鼠品系10作为现场多巴胺能神经元的可靠标志。在WT文化有现查明根据其形态活多巴胺能神经元没有可靠的方法。尽管我们在iDEN网络的丰富经验tifying和多巴胺能神经元记录，在野生型文化中只有三个了收获怀疑多巴胺能细胞的十是多巴胺能神经元。此外，我们观察到，不是所有的为阴性TH的细胞是GABA能。我们定义了基于所述GAD1转录物的存在和不存在对DA相关的转录物的细胞作为GABA能通过RNA-SEQ评定。 由于多巴胺能神经元代表主虚拟机的文化表达的神经元的一小部分，在活的文化可靠地识别这些神经元的能力将提升现有单细胞研究的范围。该协议可以用于神经保护，神经变性，和药理学试验以研究对多巴胺能转录的各种治疗方法的效果。此外，一个可以使用此协议为免疫组织化学，电，生物光子11，并且在原则上，蛋白质组分析。该本身测定是对于帕金森氏病和成瘾研究特别有用。但当然类似协议，上GENSAT小鼠或类似标记的小鼠中，可用于其它疾病状态或用于那些稀有细胞。此外，该协议使异质性的神经元给予亚型中一个新兴的观点。 在以前的研究中，收获了单细胞后，我们把它直接进入PBS;然而，现在我们直接放置在收获单细胞到反应缓冲液。这将导致更高质量的RNA序列库，在每个库检测基因的数量来评估。一个该技术的主要限制是，荧光标记的细胞是必需的。如前所述，不存在在基于形态学的混合培养，以确定一个特定的细胞类型真正可靠的方法。大范围的小区固有荧光或Cre的表达小鼠线现在提供适当标记的细胞在许多情况下。然而，护理笑选择一个合适的荧光鼠标线时，ULD的，因为它们的转基因小鼠品系进行了广泛的研究发现，TH-Cre重组酶基因敲除小鼠表现出线条明显的nondopaminergic小区12转基因表达。 此外，可能需要在以后的实验中被优化的另一个步骤是微量的所需末端的尺寸。如果未来的实验包括使用比多巴胺能神经元的其它细胞类型的，微量移液器尖端大小，可能需要为感兴趣的细胞进行优化。 RNA深度测序比芯片更强大的技术。 RNA测序提供良好的运行可以运行的重复性。检测到的动态范围相当于细胞内的实际转录物丰度。人们可以发现选择性剪接的形式和新的亚型。样品未经参照基因从头分析是可能的;如果一个新的参考基因被发现的数据可以被重新分析了特异性基因（多个），而无需再次13运行湿式工作试验。 总之，我们报告，收获和测序，该活的TH-eGFP的细胞的100％的多巴胺能神经元。该技术扩展了鉴定急性从GENSAT小鼠，第14，15和技术将在神经科学和分子生物学的广泛应用解离或长期培养的多巴胺能神经元的其他报告。

帕金森氏病（PD）是一种不可治愈的，年龄相关的神经变性疾病。此相对常见病症的起因仍然知之甚少。有广泛的神经元损失整个大脑，用在黑质（SNC）多巴胺能神经元的显着的神经元变性，导致运动徐缓，强直，震颤的诊断的临床特征。 含黑质多巴胺能神经元主要混合培养是帕金森氏病尤为重要。腹侧被盖区（VTA）多巴胺能神经元有牵连的奖励和上瘾。腹侧（VM）的主要混合胚胎培养同时包含黑质致密部和腹侧被盖区多巴胺（DA）神经元和GABA能神经元。 VM初级培养物可以是用于神经保护测定法有用并阐明多巴胺能神经元的选择性脆弱性。有识别基于形态学的培养多巴胺能细胞没有可靠的方法。他再我们开发技术来识别和收获单多巴胺能神经元，并构建单细胞，高产出的RNA测序文库。 我们报告单多巴胺和中脑培养物中分离GABA能神经元代表RNA转录组数据。该协议可以用于神经保护，神经变性，和药理学试验来研究在DA / GABA转录各种处理的影响。由于多巴胺能神经元代表主虚拟机的文化表达的神经元的一小部分，可靠地识别活的文化这些神经元的能力将实现单细胞研究的增强范围。这些新颖的技术将有助于理解发生在细胞水平上的机制的进展，并且可以具有应用在神经科学和分子生物学领域的其它地方。

<table><tbody><tr><td>Papain</td><td>Worthington Biochemical Corporation&amp;nbsp;</td><td>LS003126</td><td></td></tr><tr><td>Hanks-balanced Salt Solution (HBSS), 1x</td><td>Gibco&amp;nbsp;</td><td>14175-095</td><td></td></tr><tr><td>Donor Equine Serum</td><td>Thermo Scientific&amp;nbsp;</td><td>SH30074.03</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>A7030-50G</td><td></td></tr><tr><td>Medium: Neurobasal medium</td><td>Gibco&amp;nbsp;</td><td>21103-049</td><td></td></tr><tr><td>Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX</td><td>Gibco&amp;nbsp;</td><td>35050-061</td><td>0.5 mM final concentration.</td></tr><tr><td>L-Ascorbic acid</td><td>Sigma&amp;nbsp;</td><td>A7506</td><td></td></tr><tr><td>B27</td><td>Gibco&amp;nbsp;</td><td>17504-044</td><td>50x stock solution, 1x final concentration.</td></tr><tr><td>35 mm glass bottom dishes</td><td>MatTek&amp;nbsp;&amp;nbsp;</td><td>P35GC-1.5-10-C</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma&amp;nbsp;</td><td>P4957</td><td></td></tr><tr><td>Laminin</td><td>Sigma&amp;nbsp;</td><td>L2020</td><td>Stored at -20 &amp;deg;C in 20 &amp;micro;L aliquots</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Sigma&amp;nbsp;</td><td>DN25</td><td></td></tr><tr><td>Blue pipette tips P1000</td><td>Sorenson Bioscience</td><td>&amp;nbsp;Inc. 10130</td><td></td></tr><tr><td>10 mm shallow Petri dish</td><td>VWR&amp;nbsp;</td><td>25384-324</td><td></td></tr><tr><td>37 &amp;deg;C Water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C, 5% humidity incubator</td><td></td><td></td><td></td></tr><tr><td>16% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>&amp;nbsp;X100-500ML</td><td></td></tr><tr><td>Donkey serum</td><td>Equitech&amp;nbsp;</td><td>SD-0100HI</td><td>100% stock solution, 1% final concentration.</td></tr><tr><td>Standard Pattern surgical scissors</td><td>Fine Science Tools</td><td>14000-14</td><td></td></tr><tr><td>Forceps - 2x3 teeth</td><td>Fine Science Tools</td><td>11022-14</td><td></td></tr><tr><td>Forceps - Dumont #55</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Forceps - Dumont #5</td><td>Fine Science Tools</td><td>11252-23</td><td></td></tr><tr><td>10x PBS, pH 7.4</td><td>Gibco&amp;nbsp;</td><td>70011-044</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-buffered solution (DPBS) 1x</td><td>Gibco</td><td>14190-144</td><td>500 mL</td></tr><tr><td>21 G needle</td><td>BD PrecisionGlide Needle</td><td>305167</td><td>100 needles</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>model P-87</td><td></td></tr><tr><td>Micromanipulator&amp;nbsp;</td><td>Sutter Instrument</td><td>&amp;nbsp;MP-285</td><td></td></tr><tr><td>Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing</td><td>Clontech</td><td>634936</td><td>100 rnxs,incl Advantage2PCR Kit</td></tr><tr><td>oligonucleotide (12 &amp;micro;M): SMARTer IIA oligonucleotide (12 &amp;micro;M)</td><td></td><td></td><td>From the SMARTer Kit&amp;nbsp;</td></tr><tr><td>Reverse transcriptase (100 units)</td><td>SMARTcribe reverse transcriptase</td><td></td><td>From the SMARTer Kit&amp;nbsp;</td></tr><tr><td>Primer 1</td><td>3&amp;rsquo; CDSIIa primer&amp;nbsp;</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>Primer 2</td><td>IS PCR primer</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>50x 2 polymerase mix</td><td>50x Advantage 2 polymerase mix</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>10x PCR buffer</td><td>10x Advantage 2 PCR buffer</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>RNA Spikes</td><td>ThermoFisher</td><td>4456740</td><td></td></tr><tr><td>PCR cooler rack</td><td>IsoFreeze PCR cooler rack</td><td></td><td></td></tr><tr><td>DNA Sample Preparation Kit&amp;nbsp;</td><td>(Illumina/Nextera) Nexter</td><td>FC-121-1030</td><td>24 Samples</td></tr><tr><td>PCR master mix</td><td>Nextera PCR master mix (NPM)</td><td></td><td>From the Nextera Kit&amp;nbsp;</td></tr><tr><td>PCR primer cocktail (PPC)&amp;nbsp;</td><td>Nextera PCR primer cocktail (PPC)&amp;nbsp;</td><td></td><td>From the Nextera Kit</td></tr><tr><td>Fluorometer</td><td>The Qubit ThermoFisher</td><td>Q33216</td><td></td></tr><tr><td>HS DNA BioAnalyzer kit</td><td>Agilent</td><td>5067-4626</td><td>110 rxns</td></tr><tr><td>Electrophoresis system&amp;nbsp;</td><td>Agilent 2100 Bioanalyzer.</td><td>G2938C</td><td></td></tr><tr><td>Magnetic beads: Agencourt AMPure&amp;nbsp;</td><td>Beckman Coulter</td><td>A63880</td><td>5 mL</td></tr><tr><td>RNAse wipe: RNAse Zap wipes</td><td>Ambion</td><td>AM9786</td><td>Size 100 Sheets</td></tr><tr><td>Qubit ds HS Assay Kit</td><td>Molecular probes&amp;nbsp;</td><td>Q32854</td><td>500 assays</td></tr><tr><td>Gel extraction kit: Qiaquick Gel Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>28704</td><td></td></tr><tr><td>Glass capillary tubing</td><td>(Kimax-51, 1.5 - 1.8 mm o.d.)</td><td></td><td></td></tr><tr><td>Microforge</td><td>Narishige (or equivalent)&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sequencer</td><td>Illumina HiSeq instrument</td><td></td><td></td></tr><tr><td>GENSAT tyrosine hydroxylase-eGFP mouse strain&amp;nbsp;</td><td></td><td>MMRRC stock number: 000292-UNC</td><td>Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center</td></tr></tbody></table>

reliable identification of living dopaminergic neurons in midbrain cultures using rna sequencing and th-promoter-driven egfp expression

<html>在帕金森氏病（PD）有贯穿了在黑质致密多巴胺能神经元的显着退化脑中广泛的神经元丧失，导致运动徐缓，强直和震颤。在初级腹侧中脑（VM）的培养活多巴胺能神经元的使用荧光标记的识别提供了一种替代的方法来研究这些神经元的选择性脆弱而不依靠固定的细胞的免疫染色。在这里，我们分离，解离，并培养小鼠VM神经3周。然后，我们确定在使用的eGFP的荧光（由酪氨酸羟化酶（TH）启动子驱动）的培养物的多巴胺能神经元。单个神经元被收集到用玻璃微离心管。接下来，我们裂解收获的细胞，并进行cDNA合成和转座子介导的"tagmentation"，以产生单细胞RNA测序文库1，2，屁股="外部参照"&gt; 3，4，5。传递一个质量控制检查后，单细胞库进行测序和随后的分析进行测量的基因表达。我们报告个人多巴胺和中脑培养物中分离GABA能神经元转录组的结果。我们报告，收获和测序，该活的TH-eGFP的细胞的100％的多巴胺能神经元。这些技术将在神经科学和分子生物学广泛的应用。

此处我们报告来自小鼠表达EGFP由酪氨酸羟化酶启动子序列驱动的培养腹侧中脑神经元，从培养收获个别荧光神经元，并使用RNA-SEQ（ 图1）测量它们的RNA转录的协议。该数据表明，收获和测序的TH-eGFP的阳性细胞100％为多巴胺能神经元，是根据以下三个DA相关基因的转录物的存在下，TH，多巴脱羧酶（DDC）和多巴胺转运DAT （SLC6A3）。所有TH-EGFP阳性细胞通过RNA-SEQ的评估表达这三个基因。在每一个单细胞RNA测序文库6,000 -检测8000蛋白质编码基因（ 图2）。的多巴胺能神经元鲁棒表达除了多巴胺相关的转录到几个烟碱乙酰胆碱受体（nAChR的）亚基（ 表1）。我们观察到，没有•全部为阴性为TH是GABA能细胞;因此，我们定义的基础上，GAD1成绩单的存在细胞的GABA能由RNA-SEQ的评估。我们定义为GABA能神经元不表达多巴胺相关的转录但如前所述确实表达对GABA合成，GAD1的主要基因细胞。单细胞库还透露长的非编码RNA，以及编码通道，受体，核蛋白质，组蛋白，细胞器的蛋白质，和转录因子的转录。 <img alt="图1" src="/files/ftp_upload/54981/54981fig1.jpg" /> 图1. RNA测序在单个神经元，在我们的初级TH-EGFP鼠标中脑培养实验演出。 （1）培养的TH-eGFP的小鼠腹侧神经3周。 （2）通过使用汞光源和FITC / GFP滤波器激动人心的GFP识别荧光单神经元。 （3）无线网络TH下阶段光学相同40X的目标，可视化单个神经元，并正确地放置吸管。 （4 - 5）吸出单个细胞放入玻璃微。图像显示收获后的细胞:（4）40X阶段; （5）eGFP的荧光。 （6）溶解细胞在72℃用寡-dT引物裂解缓冲液。 （7）cDNA合成; 26周期的放大。 （8）转座子介导的cDNA的"tagmentation"。 （9）的cDNA质量控制检查和多路复用测序。采用高顶礼帽，袖扣和Cuffdiff 7（10）后续计算分析。基因表达的相对丰度每百万映射成绩单千碱基片段为单位测量读取（FPKM）。的RNA-SEQ的目标是提供在细胞和它们的相对丰度表达的基因的列表。图（2 - 4）是从当前窝K，（6 - 10）从先前的一份报告1修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54981/54981fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54981/54981fig2.jpg" /> 图 2。 在从TH-eGFP的阳性细胞系生成的单多巴胺RNA测序库检测蛋白质编码基因的数目。在单细胞库进行检测8000个基因的蛋白质 - 6000。 FPKM:每百万映射成绩单千碱基片段阅读（FPKM）。 <a href="http://ecsource.jove.com/files/ftp_upload/54981/54981fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>基因</td><td> DA单细胞N = 10 </td><td> GABA能的单细胞N = 7 </td></tr><tr><td></td><td colspan="2"> FPKM（平均值±SEM）的</td></tr><tr><td> TH </td><td> 4714±764 </td><td> 1.2±0.14 </td></tr><tr><td> DDC </td><td> 779±167 </td><td> 0.8±0.1 </td></tr><tr><td> SLC6A3 / DAT </td><td> 506±144 </td><td> 0.5±0.04 </td></tr><tr><td> DRD2 </td><td> 14±8 </td><td> 0.1±0.004 </td></tr><tr><td> chrna6（α6） </td><td> 174±57 </td><td> 0.0 </td></tr><tr><td> CHRNA4（α4） </td><td> 17±5 </td><td> 26.4±2.3 </td></tr><tr><td> CHRNB2（β2） </td><td> 9±3 </td><td> 17.3±1.2 </td></tr><tr><td> chrnb3（β3） </td><td> 38±13 </td><td> 0.0 </td></tr><tr><td> GAD1 </td><td> 0.0 </td><td> 959.0±70.0 </td></tr><tr><td> Htr3a </td><td> 0.0 </td><td> 0.6±0.01 </td></tr><tr><td> Htr3b </td><td> 0.0 </td><td> 0.0 </td></tr></tbody></table> 表1.多巴胺相关的21天中VentrMidbrain文化与乙酰胆碱受体基因转录本多巴胺和GABA能神经元。对TH-eGFP的阳性和阴性的神经元RNA测序数据被示出。单细胞收集在培养第21天RNA测序数据表明，TH-eGFP的阳性神经元有高水平的DA相关基因的转录，包括酪氨酸羟化酶（TH），多巴脱羧酶（DDC），多巴胺转运DAT（SLC6A3）和烟碱型乙酰胆碱受体（胆碱受体）亚单位包括α6，α4，β2和β3。但是，这些细胞具有GABA能标记基因谷氨酸脱羧酶1（GAD1）和血清素5-HT 3受体A和B亚基（Htr3A和Htr3B）的低的水平。 GABA能神经元具有较低的无水平DA相关基因转录，chrna6，chrnb3和Htr3A / B，但有高水平的GAD1。 RNA-SEQ（Cuffdiff）数据显示多巴胺有关，GABA能，烟碱受体，血清素和成绩单。 FPKM:每百万映射成绩单千碱基片段阅读（FPKM）。

Watch this Scientific Journal Video about Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression at JoVE.com

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

帕金森病（PD），黑质（SNC）多巴胺能神经元退化，导致运动功能障碍。此处我们报告从表达eGFP的小鼠通过酪氨酸羟化酶（TH）的启动子序列驱动的培养腹侧中脑神经元，从培养收获个别荧光神经元，并使用RNA-SEQ测量它们转录的协议。

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

reliable-identification-living-dopaminergic-neurons-midbrain-cultures

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.3K 次观看.  California Institute of Technology (Caltech). 帕金森病（PD），黑质（SNC）多巴胺能神经元退化，导致运动功能障碍。此处我们报告从表达eGFP的小鼠通过酪氨酸羟化酶（TH）的启动子序列驱动的培养腹侧中脑神经元，从培养收获个别荧光神经元，并使用RNA-SEQ测量它们转录的协议。 

视频: Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

科研

遗传学

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

神经科学

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

增强的绿色荧光蛋白测定，用于研究原神经元的神经质生长

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

发育生物学

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

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此视频中的章节

工程神经前体宫内移植,用于Vivo神经结构研究

在完整的视网膜电生理特性表达GFP的细胞群

评估神经退行性表型

增强的绿色荧光蛋白测定，用于研究原神经元的神经质生长

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像