钙成像的急性下区片的制备
Summary
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。
Abstract
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SVZ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。
Protocol
1。制备的解决方案，夹层，Vibratome的解，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。 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。 准备一盘冰浴中。在冰浴中放置一个夹层?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Solute | Company | Catalog Number | Dissection (mM) |
| Sucrose | Sigma | S0389 | Dissection: 147 mM ACSF: 0 mM |
| NaCl | Sigma | S9888 | Dissection: 42 mM ACSF: 126 mM |
| KCl | Sigma | P3911 | Dissection: 2.5 mM ACSF: 2.5 mM |
| MgCl2.6H2O | Sigma | M9272 | Dissection: 4.33 mM ACSF: 1 mM |
| NaH2PO4.H2O | Sigma | S8282 | Dissection: 1.25 mM ACSF: 1.25 mM |
| Glucose | Sigma | G8270 | Dissection: 10 mM ACSF: 10 mM |
| NaHCO3 | Sigma | S6014 | Dissection: 26 mM ACSF: 26 mM |
| CaCl2.2H2O | Sigma | C3306 | Dissection: 1.33 mM ACSF: 2 mM |
Table 1. Chemical list and recipes of dissection solution and ACSF.
| [header] | |||
| Name | Company | Catalogue Number | Comments |
| Vibratome | Leica | VT 1000S | |
| Super Glue | Surehold or 3M | Surehold 3G Super Glue or 3M Vet-Bond | |
| Dissection tools | Roboz or Ted Pella | ||
| Fluo-4 AM calcium-sensitive dye | Invitrogen | F14201 | |
| Oregon Green BAPTA-1 AM calcium-sensitive dye | Invitrogen | O6807 | |
| Pluronic F-127 20% solution in DMSO | Invitrogen | P3000MP | |
| Upright confocal microscope | Olympus | FV300 or FV1000 | |
| Water-immersion objectives | Olympus | LUMPlanFl 40 x W/IR (NA 0.80); LUMPlanFl 60 x W/I (NA 0.90) | |
| Micromanipulators | Sutter | MPC-200/MPC-325/MPC-385 | |
| Pressure controller | Parker Hannifin | Picospritzer | <3 PSI during application |
| Pipette puller | Sutter or Narshige | Sutter P-97 or Narshige PP-830 | |
| Glass pipettes | Sutter | BF150-110-10 | I.D.:1.10, O.D.: 1.50 |
| Peristaltic pump | Harvard Apparatus | Model 720 | flow rate: 1 ml/min |
| Chamber bath | Warner Instruments | RC-26 GLP | Low profile allows for objective clearance |
| Tubing | Tygon | ||
| Temperature Controller | Warner Instruments | TC-324B/344B |
Table 2. Materials/equipment list.
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Tags
Acute Subventricular ZoneSlicesCalcium ImagingNeurogenic ZoneSVZ AstrocytesNeuroblastsIntermediate Progenitor CellsInterneuronsAdhesion MoleculesDiffusible SignalsIntercellular Calcium ActivityFunctional Intercellular SignalingCalcium Indicator DyeBath LoadingCellular DensitySagittal SlicesThree-dimensional ArrangementNeuroblast Migration
Cite This Article
Lacar, B., Young, S. Z., Platel, J., Bordey, A. Preparation of Acute Subventricular Zone Slices for Calcium Imaging. J. Vis. Exp. (67), e4071, doi:10.3791/4071 (2012).