我们承认K.米基克，谁促成了这部电影和M.尼古列斯库为的miR图片的初始阶段。 C.胡贝尔是由Universtitätsklinikum蒂宾根大学的IZKF，A.奥威炳廷阿南德由Universtitätsklinikum图宾根的财富计划的奖学金支持。

1。用于在OVO电要求<ol><li> 制备的载体构建：的miR义和反义引物设计后Ambion公司的指示，退火并连接到的ApaI / EcoRI位消化的pSilencer U6.1载体。成功转型后所产生的克隆人被种植和使用Quiagen公司的midi制备试剂盒的pSilencer质粒分离碱裂解法。 </li><li> 电极：电极可以购买或容易建立13。我们使用铂丝的自制电极（Φ= 0.2，0.25或0.5毫米的直径），或者在某些情况下甚至更小的钨丝电极（Φ=0.1毫米）8取决于我们要电穿孔的区域。为了避免组织炎炎，我们作为一个规则的接近使用到组织中的电极越细。对于脑的广泛电，我们用焊接到铜管/不锈钢管0.5毫米铂丝轴和固定在具有一定距离的3-5毫米（ 图1）的电极保持器。以电穿孔的特定区域中，我们使用0.25毫米铂丝阳极0.2毫米的铂丝或0.1毫米钨为1毫米或更小的近似距离的阴极。 </li><li> 解决方案：有以下解决方案就绪： <ol><li>蒸压鸡铃声，pH为7.5（9.0克氯化钠，0.42克氯化钾0.24克氯化钙，1升溶于蒸馏水）。 </li><li> PBS（8克氯化钠，0.2克氯化钾0.24克KH 2 PO 4，1.44克Na 2 HPO 4，溶于1L蒸馏水H 2 O）。 </li><li>固绿：原液10毫克/毫升蒸馏水H 2 O的</li><li>慢干油墨（虫胶少，从而为胚胎中毒以下）。注：在粤语索莱尔和阿德勒14说明连接到光纤灯蓝色二向色滤光片增强了对比度，能消除需要注入墨水。 </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> 图1。电极和电极座示意图。 （A，B，C）的铂丝焊接到不锈钢的管状轴的一侧。一个洞钻成的管状轴的另一侧因而该引脚插头可以适应。（一）红色和蓝色导线连接到引脚插头。在另一端的导线被安装到与该电穿孔的电插座工作香蕉连接器（A，C）的铂丝被弯曲成90°的天使。指甲油的隔离层的上面弯曲的轴。 2。准备直接在卵内电穿孔<ol><li>准备以下材料： <ol><li>稀释油墨与鸡铃声1:6，添加抗生素，抗真菌溶液（100X从GIBCO）1:100。 </li><li>准备向量解：1-2微克/每特定的DNA中H 2 O的微升辅以固绿（1:20）。 </li><li>消毒镊子，剪刀，钨针和电极，用70％的乙醇。 </li><li>拉微量核酸注入硼硅玻璃毛细管（长度外径：100毫米x0.9毫米）。我们使用了一个简单的牵拉PUL-1（WPI，柏林）与表1中所示的设置。 </li><li>设定电穿孔的参数如表2所示。 </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> 参数 </td><td fo:width="1in"> 值 </td></tr><tr><td>热</td><td> 350 </td></tr><tr><td>拉</td><td> 100 </td></tr><tr><td>速度</t开发&gt; <td> 50 </td></tr><tr><td>延迟</td><td> 200 </td></tr></tbody></table> 表1中。设置PUL-1 <table border="1"><tbody><tr><td fo:width="1.5in"> 参数 </td><td fo:width="1.5in"> 值 </td></tr><tr><td>频率</td><td> 50赫兹</td></tr><tr><td>延迟</td><td> 20 - 50毫秒* </td></tr><tr><td>宽度</td><td> 20毫秒</td></tr><tr><td>电压</td><td> 8-25 V * </td></tr><tr><td>脉冲</td><td> 3 - 5倍</td></tr><tr><td colspan="2"> *根据电极直径和距离。请改变其中一个参数时考虑到这一点。 </td></tr></tbody></table> 表2。设置为脉冲刺激 <ol start="2"><li>准备小鸡胚胎的电穿孔： <ol><li>培养他们的长边受精鸡蛋在37℃，65％湿度下39小时，以收购汉堡和汉密尔顿（HH）之间的胚胎阶段15 9-11。胚胎沉淀在鸡蛋的最上面的部分，所以你应该把它用铅笔线标记。 </li><li>消毒的鸡蛋用70％乙醇。 </li><li>穿透卵子在其更广泛的方面，这里的空气腔应该是。 </li><li>除去1-2毫升蛋清。这将分离的胚胎从蛋壳。 </li><li>大提琴磁带蛋壳，避免外壳碎片掉落到胚胎。用剪刀剪一个小窗口，进入周围的铅笔线的蛋壳。 </li><li>注入一点墨水下方的区域opaca以可视化的胚胎。 </li><li>使一个切成卵黄膜具有削尖的钨针，以达到更好的可访问性的胚胎用于核酸注射和电穿孔。 </li></ol></li></ol> 3。电穿孔和孵化<ol><li>添加几滴温暖林格氏溶液到胚胎。这将降低胚胎，防止过热，电极粘附到组织，并在两个电极之间的电流提供了一个平台。 </li><li>注入载体溶液成神经管在中脑水平的内腔中。如果你的DNA表达载体，不含有报告基因（ 例如绿色荧光蛋白），增加一个略低浓缩报告基因载体（1微克/微升的miR表达载体至0.9微克/微升的报告基因载体）的DNA溶液。这将确保你认识到转染细胞后8。我们使用了所谓的pCAX矢量，一种赠品来回米C.克鲁尔13。 </li><li>对中脑的广泛的转染，将左电极和胚胎的权利在中脑的高度。电极之间的距离应为大约3-5毫米。其他设置为15V，20毫秒脉冲和50毫秒的延迟HH 10。改变两个电极的位置略微沿脑的背腹（DV）的轴线保证细胞的更广泛的转染对DV轴转染。 </li><li>对于腹侧转染，增加一些林格从蛋黄膜抬高头部，并放置在阳极腹侧中脑的下方。如果头不上升推分开蛋黄和胚胎以避免神经管烧焦膜下的电极。阴极应放置在上面的中脑DORSO-横向相对的阳极。请勿触碰神经管。我们转染中脑的腹侧左边一半，以避免与心脏发育的任何困难。电极之间的距离大约是1-0.5毫米，其余设置均10V，20毫秒宽度和50毫秒的延迟。 </li><li>适用于胚胎的顶级另一滴的盐水冷却下来，并除去气泡。 </li><li>密封鸡蛋与磁带孵育至少4小时;需要载体表达的最短时间。 </li></ol> 4。鸡胚胎的固定<ol><li>从他们的鸡蛋取出胚胎。 </li><li>在立体显微镜下，并评估转染用荧光立体显微镜的程度清洁胚胎。 </li><li>固定过夜的胚胎在4％PFA在4°C进行切片和/或染色（ 荧光原位杂交，免疫组织化学）的变化来分析转染的区域。 </li></ol>

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我们什么都没有透露。

该视频演示了转染质粒转化的小鸡脑的特定区域的神经上皮细胞的有效方法。低电压的矩形电脉冲可以将DNA引入小鸡神经管细胞大毛 6,16。但是，DNA的定位精度由宽电场，其上升通过比较大的电极（Φ= 0.5mm）的经常受阻。我们试图通过下面的百濑等 8的指引，使用电极直径小，以解决这个问题。对于腹侧中脑转染，我们将阴极区带蛋白或膜下如果胚胎的年龄允许它正?...

小非编码RNA作为额外的球员基因表达的识别推出了新的复杂的基因组编程/基因调控。不同品种的非编码RNA在神经细胞，包括非编码小分子RNA 1-4功能的重要性。小分子RNA（MIR或miRNA）为例说明在开发大脑5个不同的和不断变化的表达谱。有针对性的鸡胚蛋内电穿孔提供了开发过程中的基因表达和沉默的时间和空间控制一个独特的机会。 本视频演示了不同的步骤，使用在OVO电6-10在鸡脑的特定区域进行miR的异位表达。为了确保在这些细胞中的小非编码RNA的效果持久，miR的DNA序列克隆到单或双顺反子载体。 在OVO电中，miR含v厄克托基是通过使一个小窗口，在蛋壳后露出的胚胎注入到中脑的神经管。转染的脑小加（阳极）和减号（阴极）的特定区域的铂电极被放置在特定的位置上。对于腹侧中脑的转染，所述阳极被置于左侧中脑腹侧和上面的中脑的右半边的阴极施加电流前下方。在蛋壳的开口被封闭，磁带和胚胎孵育只要所需的任何分析。该方法最初是由村松等 6所描述和特定区域的转染改善了由百濑等 8。 <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> 原理概述。 <ol><li>在鸡蛋胚胎是通过切割一个小窗口，进入次曝光Ë蛋壳。 </li><li>溶解的载体（s）是注入到使用微毛细管中脑。 </li><li>两个电极 - 平行放置，或根据与上面的胚胎 - 产生一个脉冲电场。 </li><li>电场在时间上产生毛孔中的细胞膜，从而促进进入细胞由带负电荷的DNA（或RNA）被吸引到阳极11,12。 </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

非编码RNA是另外玩家在调节基因表达。针对在特定领域OVO电提供了空间和时间的控制异位microRNA表达谱的独特工具。不过，腹脑结构，如中脑腹侧是相当难以达成任何操作。在这里，我们展示了使用薄的铂电极电穿孔的miRNA进入中脑腹侧一种有效的方式。这种方法提供了一种可靠的方法转染中脑和体内研究的有用工具的特定区域。

转染了miR中脑区域的范围是可见的，通过GFP的从沿与所述miR表达载体（ 图2）喷射的报告基因载体的表达。使用铂电极的直径为0.5mm且平行放置脑引线的AP轴通常为大面积的转染沿DV-和AP-轴，包括后脑，中脑和间脑有时（ 图2A和B）。的电极的结果在宽的电场的大小施加到大脑区域，因此广泛染区。 它难以精确地定位，比如例如在腹侧中脑区域。降低0.5...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

异位表达的是一种技术来阐明大脑发育的小分子RNA的作用。但是，使用在OVO电针对特定领域是具有挑战性的。这里，我们表明一个有效的方式来选择性电穿孔腹侧和背侧中脑区域。

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

in-ovo-expression-of-microrna-in-ventral-chick-midbrain

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Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.4K 次观看.  University of Tübingen. 异位表达的是一种技术来阐明大脑发育的小分子RNA的作用。但是，使用在OVO电针对特定领域是具有挑战性的。这里，我们表明一个有效的方式来选择性电穿孔腹侧和背侧中脑区域。 

视频: In ovo Expression of MicroRNA in Ventral Chick Midbrain

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

科研

神经科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

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

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

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, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

用于测试胰岛素模拟化合物的蛋模型

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

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 ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...