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3，氯化钙，7 MgCl 2的 10个D -葡萄糖。它是一个混合的95％Ø 2 / 5％的CO 2，保持溶氧，并保持pH值在7.4冒泡。保持切削液冰冷的整个过程。 </li><li>脑片培养在充满含氧人工脑脊液（ACSF）含（毫米）的孵化室：125氯化钠，2.5氯化钾，1.25的NaH 2 PO 4，25碳酸氢钠3，2.5，1.3氯化钙氯化镁2，10 D -葡萄糖。它是一个混合的95％Ø 2 / 5％的CO 2，保持溶氧，并保持pH值在7.4冒泡。孵化温度为34℃，45分钟，然后在室温下，直到大脑切片被转移到录音室。 </li></ol> 2。黑质神经元的电生理指纹图谱<ol><li>一个大脑切片被转移到一个膜片钳录音室连续灌注含氧学联在32 ° C。 </li><li>补丁移液器拉蒸压硼硅（KG - 33）玻璃毛细管（1.10 mm内径，外径1.65毫米，高精密玻璃国王，克莱蒙特，加州），使用的是PC - 10拉马（Narishige，日本东京）和细胞内的解决方案填充准备与DNase的无RNase的水（尔科技）（含​​135毫米氯化钾，EGTA 0.5毫米，10毫米HEPES，1.5毫米氯化镁 2，调整pH至7.3） 。补丁移液器在洗澡2-3MΩ的电阻。 </li><li>黑质是根据其独特的解剖位置（ 图1A1 - 2）确定。配有Nomarski光学和60X水浸泡的镜头，大黑质平坦致密（SNC）和黑质细胞的视频显微镜（奥林巴斯BX51W1）的视觉引导下收杆马丁（SNR）（可能的DA能神经元和GABA神经元）选定进行录制。传统的千兆欧姆密封全细胞配置（图 1B1） 。电流钳和电压钳分析可以执行。为了尽量减少mRNA降解，电生理记录应当简短（≤5分钟）。一种选择是只记录膜和扣球属性为利益的神经元（ 图B2 - 3）电指纹。这个简短的录音时间只有约1分钟。 </li></ol> 3。细胞质的愿望<ol><li> SNR GABA和DA能神经元的电生理指纹和表征后，应用轻柔的口吸吸细胞质。细胞核内也可能会被吸入，造成基因组DNA的污染。密封不应该被打破;否则外碎片，包括mRNA可能补丁吸管吸进污染的单元格的内容。密封的完整性是良好的，只要保持电流在-70 mV的增加不超过100 pA的。 </li><li>然后吸气单元格的内容被驱逐到0.2 mL PCR管中打破吸头和一个小的正压力。收集到一个单元格的内容是一个PCR管。接受该单元格的内容后，收集的PCR管，在冰上预冷，立即置于-20 ° C冰箱中。 </li><li>为了排除污染外碎片，其中可能包含的mRNA，补丁吸管降低到组织实际上没有吸胞浆，然后吸管内容是受到RT - PCR检测。 </li></ol> 4。反转录（RT） <ol><li>收集的合理数量的神经元，通常10-20的单元格的内容后，RT，应当立即开始减少的mRNA的降解。 </li><li>由于从一个单一的神经元细胞的含量的数量太小，RNA分离过程中不被执行。要删除的潜在污染的基因组DNA，DNA酶我（5μL的DNA酶I和5时25分1.6μLDNA酶我缓冲区° C）吸气单元格的内容是先消化。然后我脱氧核糖核酸酶加入1.2μL，25毫米EDTA和孵化在75 ° C 5分钟灭活。 </li><li> cDNA的合成使用标三反向酶为基础的细胞直接cDNA合成试剂盒（Invitrogen公司， 表1）。第一链cDNA合成在50 ° C为50分钟，然后反应是灭活在85 ° C为5分钟和1μL，核糖核酸酶H添加孵育到消化的mRNA（37℃20分钟）。单链cDNA储存于-20 ° C以后的PCR扩增。 </li><li>完成去除基因组DNA的逆转录被忽略，而所有其他反应成分完全相同的RT减去控制验证。 </li></ol> 5。两阶段的PCR扩增<ol><li>设计引物，根据GenBank中的序列。只要有可能，跨越内含子的引物对用于帮助检测基因组DNA的污染。对引物序列列于表2。对引物的有效性，应首先验证了整个大脑的mRNA产生积极的产品。所有引物合成DNA技术综合（Coralville，美国爱荷华州）。 </li><li>在第一阶段，30μL的cDNA 5μL，35个循环扩增引物（ 表2）的存在。热循环热循环的协议（的Mastercycler，Eppendorf公司， 表1），在94 ° C为初始变性，然后15秒35的周期在94 ° C至变性，55章第30 ° C至退火2分钟， 45秒72 ° C至延长，其次是最后一次延长为7分钟。Taq DNA聚合酶，脱氧核苷三磷酸（dNTPs），PCR扩增所需的所有其他组件提供PCR的超级组合（Invitrogen公司，表 1） </li><li>在第二阶段PCR，1μL从第一阶段的PCR扩增产物为模板，并在第一阶段使用的是相同的引物（ 见表 2 ）。运行40个循环延伸时间缩短至30秒同一PCR超级混合使用。 </li><li>从第二阶段扩增的PCR产物2.5％琼脂糖凝胶电泳分离，溴化乙锭（0.05毫克毫升凝胶）或gelgreen紫外光下可视化，并拍照（图 B4） 。阳性条带，然后切出，提取使用一个Qiagen公司提取试剂盒（ 见表1），测序，并与靶基因的序列比较。 </li></ol> 6。代表性的成果： 多巴胺（DA）神经元的黑质致密部和收杆马丁和邻近的黑质投射神经元的GABA收杆马丁有不同的电生理特征（Zhou等人，2006年; 2011年丁等）。。 如图所示。 1B2，SNR GABA的神经元表现出约10赫兹的高频自发扣球。尖峰有一个基地的持续时间约1毫秒。 SNR GABA的神经元后超极化电流注入，显示响应极化电流注入弱极化凹陷， 说明我目前在这些神经元（图 1B2）弱表达。相比之下，黑质DA能神经元具有低频率（约2赫兹），有一个基地的持续时间约3毫秒的自发尖峰。 DA能神经元也显示响应极化电流注入一个明显的凹陷，表示强烈的表达我目前（图 1B3）（Zhou等，2006年。丁等2011 ）。 SCRT - PCR检测谷氨酸脱羧酶1（GAD1，GABA的合成和GABA的神经元标记的关键酶）基因electrophysiologically确定SNR GABA的神经元，但不是在DA能神经元（图1B4，5） 。相比之下，SCRT - PCR检测酪氨酸羟化酶基因（TH DA能神经元，多巴胺的合成和标记的关键酶）electrophysiologically确定SNC和SNR DA能神经元，但没有GABA的神经元，5）（图1B4。所以SCRT - PCR结果证实电生理神经元的鉴定。接下来，SCRT - PCR用于配置文件中的电压激活Kv3通道亚基，Kv3.1，Kv3.2，Kv3.3和Kv3.4的表达。这些亚基有助于形成电压门控K +渠道的多样化属性取决于亚基组成（丁2011年）等。。在这个例子中SNR GABA的神经元如图所示 。 1B4，Kv3.1，Kv3.2，Kv3.3和Kv3.4的mRNA进行检测。在这个例子中的SNR DA神经元在 Fig.1B5所示，只有Kv3.2，Kv3.3和Kv3.4进行检测。 Kv3.1在我们的汇总数据，更频繁地检测SNR GABA的神经元比在黑质DA能神经元，表明较高的Kv3.1的表达水平在快速扣球SNR GABA的神经元（丁2011年）等。 <img alt="图1" src="/files/ftp_upload/3136/3136fig1.jpg" /> 图1。 SNR GABA的神经元和黑质DA神经元的电生理识别答 ：黑质的地区可以通过其独特的解剖位置确定 A1显示SNC和信噪比在日冕的尼氏染色与1X目标捕获部分的位置。盒装地区被捕获ED为10倍的目标，并显示在中间的箭头表示。细胞丰富的SNC和细胞SNR较差都清晰可见。A2显 ​​示SNC和SNR在现场，未染色冠状4倍的目标捕获部分的位置。细胞丰富的SNC和细胞SNR较差也清晰可辨。 VTA，腹侧被盖区，B1显示SNR GABA的神经元在全细胞模式正在修补夹住，B2显示了典型的电生理特性的信噪比神经元GABA电流钳记录模式。这些神经元火自发的高频率持续时间短的动作电位和弱我H -介导的超极化电流注入的凹陷响应。B3显示在电流钳记录模式下的黑质DA神经元的典型的电生理特性。他们火自发的低频率的动作电位，持续时间长，有一个突出的I H -介导的凹陷（箭头）响应极化电流注入。因此，SNR GABA的神经元和黑质多巴胺神经元是明确确定electrophysiologically B4显示了从一个电确定SNR GABA的神经元SCRT - PCR产物的凝胶图片。谷氨酸脱羧酶（GAD1）的mRNA，但没有酪氨酸羟化酶（TH）mRNA的检测。 Kv3.1，Kv3.2，Kv3.3和Kv3.4的mRNA的检测在这个SNR GABA的神经元。B5显示Kv3在SNR DA神经元的神经通道的mRNA SCRT - PCR检测。 TH mRNA表达，但没有GAD1 mRNA的检测在一个electrophysiologically确定的SNR DA神经元。在这个SNR DA神经元。B6的显示汇总数据，Kv3.2，Kv3.3和Kv3.4的mRNA进行检测。 7。潜在的假阴性和假阳性结果根据我们的经验，有几个因素可能会导致假阴性SCRT - PCR结果。这些因素包括在吸足够量的细胞质中，由于修补枪头堵塞，RNase的污染，和低效的PCR引物的失败。补丁枪头堵塞通常可以看到在视频监视器上，以增加获得性表示。 RNase的污染，可避免彻底的失活和戴手套。 PCR引物的有效性应进行测试使用一拳从脑组织中的总RNA（Zhou等2008;。Ding等人2011年）。 由于我们始终使用DNA酶RT我们的样本中第一次治疗前，我们没有遇到假阳性结果。理论上，无DNA酶消化，基因组DNA的污染，因此假阳性检测时可能出现的基因是intronless或引物对不跨内含子区域。

<ol>
	<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>

没有利益冲突的声明。

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, ...

profiling-voltage-gated-potassium-channel-mrna-expression-nigral

Research

JoVE Journal

Neuroscience

15.2K 次观看.  University of Tennessee College of Medicine. 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。

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

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

科研

神经科学

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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, ...

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

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

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

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

腺相关病毒介导的脊髓基因神经元的转基因表达

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

成人脑组织切片中钾离子选择性电极的制备、检测及应用

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)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.

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

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.

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

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

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 ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

使用多重 原位 杂交检测小鼠迷走神经传入神经元中G蛋白偶联受体表达

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...