成像细胞内Ca<sup> 2+</sup>从成年小鼠使用基因编码的钙指标纹状体星形胶质细胞信号
Summary
The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.
Abstract
Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.
Introduction
星形胶质细胞是脑的无处不在的和丰富的神经胶质细胞。它是公认的星形胶质细胞起到重要的支持和内环境稳定的作用，包括钾离子浓度在细胞外空间缓冲，摄取神经递质以及提供营养素。然而，最近的研究表明，它们也显示的[Ca 2+] i的信号，自发，并且增加了神经元的活动1。星形胶质细胞的存在的[Ca 2+] i的信令已被越来越多地认为是触发它们与神经元的通信，并因此被解释为“ 钙离子兴奋”星形胶质细胞内的形式。在过去的二十年里，现有数据表明两个设置在其中的星形胶质细胞和神经元可能通信，也许以双向方式。首先，星形胶质细胞经常与我当神经递质和激活增加的[Ca 2+]回应从神经元释放2神经调节。第二，内[Ca 2+]的星形胶质细胞中的我的增加导致从该反过来可影响神经元和血管的星形胶质细胞的信号分子的释放。有证据表明，由星形胶质细胞释放的分子，导致经由星形细胞向神经细胞信号传导的改变突触，电路和最终行为3-5的功能。然而，这仍然是一个快速发展的研究领域，它已经认为更好，详细了解星形胶质细胞的[Ca 2+] i的需要，以解决目前的一些不确定因素6。
在过去的工作中，已证明该批量加载的有机的Ca 2+指示剂染料为星形胶质细胞不能可靠地检测的[Ca 2+]在培养和原位 7-10整个星形胶质细胞内的I信号。这些研究结果已经被美国和其他6,11,12讨论。该emergin克图象是内[Ca 2+] i的星形胶质细胞过程（ 例如，分支和小枝），这是在主站点与神经元和血管内的相互作用的信号，很少被详细探讨。最近，利用基因编码的钙指标（GECIs），如胞浆GCaMP3，GCaMP5G和GCaMP6和质膜拴版本（ 例如 ，lck的-GCaMP3）已批准的[Ca 2+] i的信号的研究星形胶质细胞等的小隔间薄工艺，近质膜和整个领土7,8内。然而，GECIs有超过有机钙指示剂染料有一个缺点，那就是要求遗传方式有选择地提供编码基因的星形胶质细胞在体内数周的时间来完成的GECIs要适当地表达出来。表达在体内通常实现使用转基因小鼠，基因敲除小鼠或基于病毒递送的应用程序蟑螂。在本朱庇特的文章，我们报告来输送GECIs使用腺相关病毒纹状体星形胶质细胞的方法和程序。我们专注于CYTO-GCaMP3作为一个例子，但是相同的基本过程适用于任何其他GECI或荧光蛋白的基于报告。
Protocol
Representative Results
Discussion
本文描述的方法，使我们能够表达CYTO-GCaMP3纹状体星形胶质细胞在体内原位后续的[Ca 2+] i的成像。这种方法比使用转基因或基因敲除小鼠，其中包括有针对性的蛋白质，快速稳健的表达和实施实验和解剖特异性的灵活性优势。用AAV2 / 5 GCaMP3的表达被认为是特定的和健壮。的GFAP GfaABC 1（D）的启动子与血清型5的腺相关病毒的结合是至关重要的，实现了特异性。此处所?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
大部分的工作和所涉及的人员由美国国立卫生研究院拨款NS060677，部分由美国国立卫生研究院授予MH099559和MH104069（以BSK）的支持。有些工作也是由CHDI基金会的支持。
Materials
| Syringe Pump | Harvard Apparatus | 704506 | |
| Glass Capillaries | World Precision Instruments | 1B100-4 | |
| Micropipette puller | Narishige | PC-10 | |
| Micropipette grinder | Narishige | EG-40 | |
| pZac2.1 GfaABC1D.cyto-GCaMP3 | Addgene | 44331 | a plasmid sent to UPenn Vector Core for virus packaging |
| I mL syringe | BD | 309628 | |
| syringe needle | BD | 305109 | |
| AAV2/5 virus | UPenn vector core | NA | |
| Sudan red IV | Sigma-Aldrich | 67386 | |
| Mineral oil | CVS Pharmacy | 152355 | |
| Cryostat | Leica | CM3050 S | |
| Stereotaxic instrument | David Kopf Instruments | 900LS | |
| High Speed Rotary Micromotor Kit | FOREDOM | K.1070 | |
| Paraformaldehyde | Santa cruz biotechnology | sc-281692 | |
| Super Glue | Krazy®Glue | KG925 | |
| Microslicer | Ted Pella | DTK-Zero 1 | |
| Confocal microscopes | Olympus | FV300 and FV1000 | |
| Normal goat serum | Vector | S-1000 | |
| chicken anti-GFP | Abcam | ab13970 | |
| mouse anti-s100β | Sigma-Aldrich | S2532 | |
| mouse anti-NeuN | Millipore | MAB377 | |
| mouse anti-glutamine synthetase | Millipore | MAB302 | |
| goat anti-mouse-Alexa546 | Invitrogen | A11003 | |
| goat anti-chicken-Alexa488 | Invitrogen | A11039 | |
| Microscope Slides | Fisher Scientific | 12-550-15 | |
| Cover Glass | Fisher Scientific | 12-548-5J | |
| Mounting Medium | Vector | H-1000 |
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