Name: profiling voltage-gated potassium channel mrna expression in nigral neurons using single-cell rt-pcr techniques
Uploaded: 2011-09-27T13:00:00
Description: Watch this Scientific Journal Video about Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques at JoVE.com

这项工作是支持由NIH资助R01DA021194和R01NS058850。

1。脑片制备<ol><li>青年（15-40天）用于男性和女性的Sprague - Dawley大鼠。 （我们也可以使用这个相同的协议在小鼠。）深乌拉坦麻醉下，动物断头处死，迅速解剖大脑。接着是300微米厚的冠状脑切片中含有的黑质midrostral部分削减徕卡vibratome（VT - 1200S）。片切割过程是在一个冰冷的，含氧高切削液中含有（毫米）蔗糖：220蔗糖，2.5氯化钾，1.25的NaH 2 PO 4 25 2 0.5碳酸氢钠...

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	<li>J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+pacemaker+frequency+of+individual+dopaminergic+neurons+by+Kv4.3L+and+Kchip3.1+transcription.">Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and Kchip3.1 transcription.</a> EMBO J. 20, 5715-5724 (2001).</li><li>Surmeier, D. J., Song, W. J., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+expression+of+dopamine+receptors+in+neostriatal+medium+spiny+neurons.">Coordinated expression of dopamine receptors in neostriatal medium spiny neurons.</a> J. Neurosci. 16, 6579-6591 (1996).</li><li>Zhou, F. W., Matta, S. G., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutively+active+TRPC3+channels+regulate+basal+ganglia+output+neurons.">Constitutively active TRPC3 channels regulate basal ganglia output neurons.</a> J. Neurosci. 28, 473-482 (2008).</li><li>Zhou, F. W., Jin, Y., Matta, S. G., Xu, M., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-short+dopamine+pathway+regulates+basal+ganglia+output+neurons.">An ultra-short dopamine pathway regulates basal ganglia output neurons.</a> J. Neurosci. 29, 10424-10435 (2009).</li><li>Ding, S., Matta, S. G., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv3-like+potassium+channels+are+required+for+sustained+high-frequency+firing+in+Basal+Ganglia+output+neurons.">Kv3-like potassium channels are required for sustained high-frequency firing in Basal Ganglia output neurons.</a> J. Neurophysiol. 105, 554-570 (2011).</li><li>Zhou, F. W., Xu, J. J., Zhao, Y., Ledoux, M. S., Zhou, F. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+functions+of+histamine+H1+and+H2+receptors+and+H3+receptors+in+substantia+nigra+pars+reticulata.">Opposite functions of histamine H1 and H2 receptors and H3 receptors in substantia nigra pars reticulata.</a> J. Neurophysiol. 96, 1581-1591 (2006).</li></ol>

修补脑片钳记录SCRT - PCR技术相结合，我们这里展示提供了一个极好的方法来研究离子通道，受体和个别特征的神经元的神经递质合成的关键酶的mRNA表达谱。这是特别有用的，当问题中的蛋白质不能被检测和本地化使用其他方法，如免疫组化由于表达水平低和/或缺乏合适的抗体（Surmeier等，1996;。Zhou等2009）。 SCRT - PCR的高灵敏度和选择性，允许检测低丰度的mRNA（Surmeier等，1996年）。此外，这个方?...

<img src="/files/ftp_upload/3136/3136table1.jpg" alt="表1" /> 表1主要试剂和仪器<img src="/files/ftp_upload/3136/3136table2.jpg" alt="表2" /> 引物对大鼠神经元Kv3通道，酪氨酸羟化酶（TH）和谷氨酸脱羧酶（GAD1）SCRT - PCR的mRNA 表2。

profiling voltage-gated potassium channel mrna expression in nigral neurons using single-cell rt-pcr techniques

在哺乳动物中枢神经系统中，不同​​类型的神经元具有不同的分子和功能特点是混杂着对方，难以分开，也不易确定其形态。因此，它往往是很难在一个特定的神经元类型的基因表达分析。在这里，我们记录全细胞膜片钳记录技术，它结合了单细胞逆转录聚合酶链反应（SCRT - PCR）分析mRNA的表达在大量的黑质神经元的不同类型的程序。电生理技术最初是用来记录单个神经元的神经生理学和功能特性。然后吸气，单electrophysiologically特点的黑质神经元的胞浆SCRT - PCR分析，以获取神经递质的合成酶，受体，离子通道的mRNA的表达谱。这种方法特别有用时，不能用免疫组织化学由于缺乏合适的抗体或蛋白质表达水平低的高选择性和灵敏度。这种方法也适用于其他脑区的神经元。

Watch this Scientific Journal Video about Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques at JoVE.com

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

electrophysiologically神经元是第一个特点。然后从所记录的神经元的胞浆吸气和遭受反转录- PCR分析，检测神经递质的合成酶，离子通道和受体的mRNA表达。

在使用单细胞RT - PCR技术的黑质神经元电压门控钾通道mRNA表达分析

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Research

JoVE Journal

Neuroscience

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科研

神经科学

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Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

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

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Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

腺相关病毒介导的脊髓基因神经元的转基因表达

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Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

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荧光标记小鼠神经元的单细胞 RNA 序列的人工分选和双体外转录与绝对计数测序 (天后-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...