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 border="0" cellpadding="0" cellspacing="0" >
	<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 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I 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&#174;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&#946;</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

14.7K 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

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

科研

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

成像膜电位与基因编码荧光电压传感器的两种类型

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

从成人和老化小鼠的河马切片中尽量减少低氧

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

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.