Name: imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators
Uploaded: 2014-11-19T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

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MH10406​​9（以BSK）的支持。有些工作也是由CHDI基金会的支持。

所有的动物都是协议符合卫生指南的美国国家研究院​​实验动物的护理和使用，并经实验动物管理和使用委员会在加州大学洛杉矶分校。 1.1）编写微管和AAV2 / 5加载病毒<ol><li>用细硼硅玻璃为微量注射的病毒。使用两步骤拉方案具有垂直拉出拉微量。斜角移液器在40℃通过使用移液管磨机的角度。这是理想的利用吸移管将具有20的前端直径为 - 40微米，6的柄长度 - 7mm左右。高压釜的吸管和手术器械。钻头是通过用70％乙醇擦拭消毒。 </li><li>存储该病毒的AAV2 / 5-GfaABC 1 D.cyto-GCaMP3.SV40（1.5×10 13 GC /毫升）中，在-80℃下在10微升等分试样。刚好手术前，取出等分试样进行冷冻机的和在冰上储存直至用于填充第Ë微量。 </li><li>填充用病毒注射泵微量。紧紧一个管件，的一端连接，通过一个适当尺寸的管道压力接头，以在玻璃注射器用钢筋柱塞。通过一个适当尺寸的可移动针压缩接头连接的高压灭菌微量到管件的另一端。 </li><li>前加载的病毒载体，通过填充系统与矿物油着色苏丹红IV（〜1毫克/ 50毫升）的1毫升注射器针头（27G的除去从所述泵送系统包括管道，注射器和微量气泡）。 </li><li>将一块干净的封口膜在玻璃微吸管，并免除5 - 10微升的病毒载体上的封口膜。 </li><li>吸病毒载体通过使注射器的柱塞向后以0.5微升/分钟，并标记上的玻璃移液管将载体和油之间的边界。 </li></ol> 1.2）麻醉，剪发，HEAD固定，准备开颅<ol><li>把鼠标（P49-63，C57BL / 6）放入盛有N 2 O和O 2和5％异氟醚室。由损耗有目的的运动和减缓呼吸速率​​（ - 90 /分钟〜60）评估麻醉深度。剪下的头发在头上当鼠标麻醉。 </li><li>适合鼠标在立体框架，其头部被钝器耳棒和鼻子放入麻醉和通风系统安全。保持持续的异氟醚在2 - 3％。应用人工泪液软膏双眼，因为老鼠失去了在麻醉状态下的眨眼反射。 </li><li>通过皮下注射来缓解疼痛管理0.05毫升丁丙诺啡（0.1毫克/毫升）的。 </li><li>用10％聚维酮碘和70％乙醇3次清洁手术区域，这两种溶液之间交替并开始与聚维酮碘。开始从正面和擦拭朝向背面以除去任何已切断毛发。 </li><li>做一个皮肤切口上顶OF的眼睛之间开始和耳朵之间结束了头。 </li></ol> 1.3）开颅手术和显微注射<ol><li>用棉签刷去除骨膜，然后用干的牙科棉球头骨的表面。使注射部位周围的标记，并钻周围用小钢毛刺搭载了高速钻头的标记的边缘。 </li><li>除去骨和清洁表面用生理盐水。 </li><li>将吸管插入左背外侧具有以下坐标（毫米）纹状体:前后0.8，内侧，外侧:2.0，背腹的软脑膜表面:-2.4 13。需要注意的是这些坐标表示的显微注射针的尖端位于正上方的背外侧纹状体（ 图1A，B）中 ，这意味着它只能略微这个核内贯通。避免损坏血管。如果吸移管恰好是正确的血管上，稍微调整通过坐标〜0.1 - 0.2毫米。 </li><li>开始注射用喷射率设定为0.2微升/分钟，并且通常注入1〜1.5微升病毒。 </li><li>留在原地的移液管注入10分钟后，再抽出吸管缓慢。 </li><li>通过闭合手术切口连续缝合外的尼龙缝合完成。 </li></ol> 1.4）恢复<ol><li>把鼠标在干净的笼子上的一个加热垫24小时。不返回鼠标已经动过手术，以其他动物的公司，直到完全康复。不要让无人看管的鼠标，直到它已经恢复了足够的意识，保持胸骨斜卧。 </li><li>添加苄啶Sulfamethoxiazole（每500毫升的水5毫升，含有40mg的甲氧苄啶和200毫克sulfamethoxiazole）在水中一周。手术后给予丁丙诺啡，每天2次，3天。 </li><li>维持小鼠在12小时明暗周期，进料并每日浇水，一次被加权每天一次至3天手术后。在这3天，所有鼠标具有大于10％的体重损失被认为是受到损害，并会被安乐死，而不是进行实验。 </li><li>另外，每周两次更换鼠笼，并至少一次，每天监测总体健康状况。 3周立体显微注射后 - 通常情况下，鼠标在2使用。 </li></ol> 2.急性脑切片准备共聚焦钙成像 2.1）准备切割和记录解决方案。 <ol><li>准备切割溶液包含（mM ​​计）:194蔗糖，30的NaCl，4.5的KCl，10个D-葡萄糖，1的MgCl 2，1.2的NaH 2 PO 4和26的NaHCO 3中，用95％O 2和5％CO 2饱和的。 </li><li>准备记录溶液含有（mM计）:124氯化钠，氯化钾4.5，1的MgCl 2，10 D-葡萄糖，2的CaCl 2，1.2的NaH 2 PO 4的</子&gt;，和26 碳酸氢钠 ; pH为7.3 - 7.4 290 - 295毫渗透摩尔浓度，用95％ 的 O 2和5％CO 2饱和的。充满录音解决方案的脑切片拿着烧杯中，并保持在34℃。 </li></ol> 2.2）切片<ol><li>经过两到三周立体显微注射，深入，晚期麻醉鼠标，然后斩首了。 </li><li>提取大脑，并使用叶片以除去未注射的右半球，和左1安装到使用超级胶水的vibratome托盘。填充的vibratome托盘用冰冷的切割缓冲液中，并保持在95％O 2和5％CO 2饱和的。 </li><li>切冠状或矢状纹状体脑片在300微米的厚度。通常情况下，4 - 5冠状或6 - 7矢状纹状体片可以收集。 </li><li>转移的切片的切片保持烧杯加热至34℃，并且将它们存储在室温下用于随后recordi之前保持它们有30分钟NG。 </li><li>的[Ca 2+] i的成像前孵育在含氧记录缓冲器的脑切片在室温下放置至少30分钟。 </li></ol> 3.免疫组织化学（IHC） <ol><li>病毒注射后两周内，灌注小鼠transcardially用PBS随后在PBS中的10％福尔马林中。 </li><li>除去，后固定过夜，并cryoprotect大脑中缓冲的30％蔗糖，至少2天。 </li><li>准备用冷冻切片机切片机40微米的部分。 </li><li>冲洗切片3次，用PBS在10分钟的时间间隔。 </li><li>孵育切片1小时，在PBS中，用10％正常山羊血清和0.5％的Triton X-100。 </li><li>在制备在PBS中，用0.5％的Triton X-100的第一抗体过夜孵育切片在4℃下。分别用第一抗体如下:鸡抗GFP 1:1000，小鼠抗S100β1:400，小鼠抗神经元核抗原1:600，小鼠抗谷氨酰胺合成酶1:300。 </li><li>冲洗切片3次，用PBS以10米在时间间隔。 </li><li>孵育制备在PBS中，用10％正常山羊血清进行2小时，在室温下的二级抗体的部分。所述第二抗体分别为:山羊抗小鼠Alexa546 1:500，山羊抗鸡Alexa488 1:500。 </li><li>安装在载玻片上，干燥的部分并应用抗褪色封固剂的数滴，然后慢慢盖上玻璃盖玻片的部分。存放载玻片在4℃下。 </li><li>拍摄图像上的一个共焦显微镜用40X油浸镜头具有1.3的数值孔径。 </li></ol> 4.共焦的[Ca 2+] i的成像注:内[Ca 2+] i的成像使用共聚焦显微镜用40X水浸物镜透镜用0.8的数值孔径已完成。 <ol><li> 2毫升每 - 在1至5小时的切片，切片位置在记录室中，用含氧记录缓冲器superfuse在室温下用1流量后分钟。然后放置在片的顶部的铂竖琴用尼龙绳的实验过程中最小化的运动。 </li><li>可视化和找到下一个10倍的水浸泡的目的背侧纹状体与明场致发光。 </li><li>切换到40X水浸物镜透镜，并打开氩激光器的488纳米线。通过改变焦平面，发现细胞位于大约20微米的薄片表面的下方，显示基础荧光的细胞体，树枝和小枝。值得注意的是，受损细胞通常会显示高于平均荧光水平，这应该被避免。 </li><li>使用"帧扫描"模式，开始扫描。通常，激光强度调节到0.5 -最大输出的5％是足够的，以图像的[Ca 2+] i的与CYTO-GCaMP3。对于监控的[Ca 2+] i的动态星形胶质细胞的全境，"帧扫描"，每帧或更快的1秒的扫描率recommended，但这需要取决于实验中的问题来决定。 </li><li>如果有必要，图像的[Ca 2+] i的过程中，使用了2个数字放大- 3倍，监视1 - 2星形胶质细胞在整个视场。 </li><li>为了避免拍摄过程中饱和像素，使用"高住低训&#39;的查找模式，伪细胞。始终以"试点记录"约5分钟开始，如果显示为红色，饱和度指标进行测试。如果是这样，降低激光的输出功率，或降低PMT /增益/偏移值，并执行另一个导频记录到测试中的像素值处于非饱和的范围内。典型地，用于成像的星形胶质细胞的组合参数的[Ca 2+] i的纹状体如下:激光输出功率0.5 - 5％（10毫瓦），光电倍增管550 - 650伏，增益2.0 - 3.5X，和偏移0 - 5％。 </li></ol>

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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?...

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。

<table><tbody><tr><td>Syringe Pump</td><td>Harvard Apparatus</td><td>704506</td><td></td></tr><tr><td>Glass Capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Micropipette grinder</td><td>Narishige</td><td>EG-40</td><td></td></tr><tr><td>pZac2.1 GfaABC&lt;sub&gt;1&lt;/sub&gt;D.cyto-GCaMP3</td><td>Addgene</td><td>44331</td><td>a plasmid sent to UPenn Vector Core for virus packaging</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>syringe needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>AAV2/5 virus</td><td>UPenn vector core</td><td>NA</td><td></td></tr><tr><td>Sudan red IV</td><td>Sigma-Aldrich</td><td>67386</td><td></td></tr><tr><td>Mineral oil</td><td>CVS Pharmacy</td><td>152355</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050 S</td><td></td></tr><tr><td>Stereotaxic instrument</td><td>David Kopf Instruments</td><td>900LS</td><td></td></tr><tr><td>High Speed Rotary Micromotor Kit</td><td>FOREDOM</td><td>K.1070</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Santa cruz biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>Super Glue</td><td>Krazy&amp;reg;Glue</td><td>KG925</td><td></td></tr><tr><td>Microslicer</td><td>Ted Pella</td><td>DTK-Zero 1</td><td></td></tr><tr><td>Confocal microscopes</td><td>Olympus</td><td>FV300 and FV1000</td><td></td></tr><tr><td>Normal goat serum</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>chicken anti-GFP</td><td>Abcam</td><td>ab13970</td><td></td></tr><tr><td>mouse anti-s100&amp;beta;</td><td>Sigma-Aldrich</td><td>S2532</td><td></td></tr><tr><td>mouse anti-NeuN</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>mouse anti-glutamine synthetase</td><td>Millipore</td><td>MAB302</td><td></td></tr><tr><td>goat anti-mouse-Alexa546</td><td>Invitrogen</td><td>A11003</td><td></td></tr><tr><td>goat anti-chicken-Alexa488</td><td>Invitrogen</td><td>A11039</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Cover Glass</td><td>Fisher Scientific</td><td>12-548-5J</td><td></td></tr><tr><td>Mounting Medium</td><td>Vector</td><td>H-1000</td><td></td></tr></tbody></table>

imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators

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.

为CYTO-GCaMP3的纹状体中的星形胶质细胞特异性表达，我们使用腺相关病毒（AAV）的5血清型，并且GFAP GfaABC 1（D）的启动子（ 图1A），已被先前显示驱动健壮GCaMP3和报告基因在海马和皮层星形胶质细胞8,14表达。两个星期后，病毒注射到小鼠纹状体中，小鼠（〜10周龄）中的灌注和免疫组化物上薄的大脑切片进行评估纹状体中（ 图1B）CYTO-GCaMP3表达。我们发现C...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

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.

成像细胞内Ca<sup&gt; 2+</sup&gt;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

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Research

JoVE Journal

Neuroscience

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

14.8K 次观看.  University of California Los Angeles. 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. 

视频: Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

神经科学

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

体内 使用大颅窗的小鼠宽场和双光子钙成像

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

小鼠齿状回细胞神经元活动的实时成像 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

作者聚焦:用微型显微镜研究齿状回状颗粒细胞的神经活动

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

小鼠海马齿状回管颗粒细胞的体内钙成像 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

成像细胞内Ca

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近质膜和全球星形胶质细胞内钙动力学测量

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

在体内小鼠劣质橄榄中的钙成像

活体转基因小鼠脑围产层钙和血液学成像

使用遗传编码的Ca^{2 +} 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca^{2 +} 摄取进行成像

体内使用大颅窗的小鼠宽场和双光子钙成像

体内完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

小鼠海马齿状回管颗粒细胞的体内钙成像

成像细胞内Ca

摘要

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此视频中的章节

近质膜和全球星形胶质细胞内钙动力学测量

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

在体内 小鼠劣质橄榄中的钙成像

活体转基因小鼠脑围产层钙和血液学成像

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

体内 使用大颅窗的小鼠宽场和双光子钙成像

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

小鼠海马齿状回管颗粒细胞的体内钙成像

在体内小鼠劣质橄榄中的钙成像

使用遗传编码的Ca^{2 +} 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca^{2 +} 摄取进行成像

体内使用大颅窗的小鼠宽场和双光子钙成像

体内完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像