Name: preparation of acute subventricular zone slices for calcium imaging
Uploaded: 2012-09-19T11:00:00
Description: Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

这项工作是支持由美国国立卫生研究院（DC007681 AB），CT干细胞（AB）资助，：帕蒂基金会（AB），博士前露丝属Kirschstein国家研究服务奖（NRSA），（SZY），和NSF研究所的研究补助奖学金（BL）。 ，我们感谢Bordey实验室的手稿上了宝贵的意见。本发明的材料是基于部分由康涅狄格州康涅狄格州干细胞研究资助计划下的工作。它的内容是完全的责任作者，并不一定代表官方意见的康涅狄格州，康涅狄格州或CT创新国家公共卫生部，注册成立。

1。制备的解决方案，夹层，Vibratome的<ol><li>解，必须在正确的渗透压和pH值（ 表1）制备。人工脑脊液（ACSF）相比，剥离溶液与较低浓度的钠和钙，和更高浓度的镁制备。这是为了最大限度地减少在切割时的兴奋性毒性的影响。 </li><li>是饱和的夹层和学联解决方案都必须用95％O 2/5％CO 2鼓泡至少10分钟，以达到所需的pH值7.3。 </li><li>准备一盘冰浴中。在冰浴中放置一个夹层?...

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SVZ细胞钙成像已被用于研究在神经母细胞10，在两个4,6,8成神经细胞和神经胶质细胞和星形胶质细胞的钙波3受体通道表达的自发活动模式。由于在SVZ细胞是未成熟或有胶质属性，它们不火动作电位11,12，也就是说毫秒表示成熟的网络中的活动的电压电势的变化是不适用的在这个区域。因此，捕捉速度较慢的钙事件（以秒计）不仅是生物学意义的，但也许是最相关的...

<table border="1"><tbody><tr><td> 溶质 </td><td> 公司 </td><td> 目录编号 </td><td> 夹层（MM） </td></tr><tr><td>蔗糖</td><td>西格玛</td><td> S0389 </td><td>解剖：147毫米 学联：0毫</td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S9888 </td><td>解剖：42毫米 学联：126毫米</td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P3911 </td><td>夹层：2.5毫米 学联：2.5毫米</td></tr><tr><td>氯化镁2 .6 H 2 O </td><td>西格玛</td><td> M9272 </td><td>夹层：4.33毫米 学联：1毫米</td></tr><tr><td>的NaH 2 PO 4·H 2 O </td><td>西格玛</td><td> S8282 </td><td>夹层：1.25毫米 学联：1.25毫</td></tr><tr><td>葡萄糖</td><td>西格玛</td><td> G8270 </td><td>狄氏ction：10mM的 学联：10毫米</td></tr><tr><td> 碳酸氢钠 </td><td>西格玛</td><td> S6014 </td><td>解剖：26毫米 学联：26毫米</td></tr><tr><td>氯化钙2 .2 H 2 O </td><td>西格玛</td><td> C3306 </td><td>解剖：1.33毫米 学联：2mM的</td></tr></tbody></table> 表1。夹层的解决方案和学联的化学品清单和食谱。 <table border="1"><tbody><tr><td></td><td></td><td></td><td> [头] </td></tr><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Vibratome </td><td>徕卡</td><td> VT 1000S </td><td></td></tr><tr><td>超能胶</td><td> Surehold或3M </td><td> 3G超级胶或3M兽医债券的Surehold </td><td></td></tr><tr><td>解剖工具</td><td> Roboz或特德·佩拉</td><td></td><td></td></tr><tr><td>氟钙离子敏感的染料</td><td> Invitrogen公司</td><td> F14201 </td><td></td></tr><tr><td>俄勒冈绿BAPTA-AM钙离子敏感的染料</td><td> Invitrogen公司</td><td> O6807 </td><td></td></tr><tr><td>的Pluronic F-127的20％的DMSO溶液</td><td> Invitrogen公司</td><td> P3000MP </td><td></td></tr><tr><td>立式激光共聚焦显微镜</td><td>奥林巴斯</td><td> FV300或FV1000 </td><td></td></tr><tr><td>浸水目标</td><td>奥林巴斯</td><td> LUMPlanFl 40×W / IR（NA 0.80）; LUMPlanFl 60×W / I（NA 0.90） </td><td></td></tr><tr><td>微操纵器</td><td>萨特</td><td> MPC-200/MPC-325/MPC-385 </td><td></td></tr><tr><td>压力控制器&lt;/ TD&gt; <td>派克汉尼汾</td><td> Picospritzer </td><td> &lt;3 PSI过程中的应用</td></tr><tr><td>用移液管拉马</td><td>萨特或Narshige </td><td>萨特P-97或Narshige PP-830 </td><td></td></tr><tr><td>玻璃吸液管</td><td>萨特</td><td> BF150-110-10 </td><td> ID：1.10，OD：1.50 </td></tr><tr><td>蠕动泵</td><td>哈佛设备</td><td>型号720 </td><td>流速：1毫升/分钟</td></tr><tr><td>商会浴</td><td>华纳仪器</td><td> RC-26 GLP </td><td>低调允许的客观间隙</td></tr><tr><td>管道</td><td>聚乙烯</td><td></td><td></td></tr><tr><td>温度控制器</td><td>华纳仪器</td><td> TC-324B/344B </td><td></td></tr></tbody></table> 表2。材料/设备清单。

preparation of acute subventricular zone slices for calcium imaging

脑室下区（SVZ）是在出生后的大脑的神经区域之一。 SVZ密密麻麻的细胞，包括神经祖细胞与星形胶质细胞的功能（SVZ星形胶质细胞），神经母细胞和中间祖细胞。出生在SVZ的神经母细胞切向迁移到嗅球，它们分化成的interneurons很大的距离。细胞间粘附分子和扩散信号的信号通过发挥着重要的作用，在控制神经发生。许多这些信号触发内和细胞间的信息发送的胞内钙离子活性。钙活性因此反射的活性的胞外信号，是一个最佳的方式来理解SVZ的细胞的功能之间的细胞间信号。 其他许多地区和类型的细胞，包括成熟的星形胶质细胞和神经元钙活动进行了研究。然而，传统的方法来加载，D细胞与钙指示剂染料（ 即浴加载）的效率不高在加载所有SVZ的细胞类型。事实上，细胞密度在SVZ排除内部的组织的染料扩散。此外，制备矢状切片，将更好地保留SVZ的细胞，特别是流延髓尾轴上的成神经细胞迁移的三维配置的。 在这里，我们描述的方法来准备矢状部分包含SVZ，装载的SV​​Z细胞与钙离子指示剂染料，收购活性钙与时间推移电影。我们使用荧光凌晨4点染料加载SVZ星形胶质细胞内组织施加压力。使用扫描共聚焦显微镜，让一个精确的分辨率为区分单个细胞钙活动的记录。我们的方法是适用于包括成人海马颗粒下层和胚胎神经源性区域的其他神经性区。此外，其他类型的染料可以使用所描述的方法被应用。

Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

一个方法来加载脑室下区（SVZ）细胞中的钙离子指示剂染料记录活性钙。产后SVZ包括神经祖细胞和神经母细胞的细胞排列紧密。而不是使用清洗负载注入染料内的组织可以更好的染料扩散的压力。

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Research

JoVE Journal

Neuroscience

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Neonatal Subventricular Zone Electroporation

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...