我们感谢博士学Stella和海伦VUONG向钙成像协议的贡献。我们感谢K.医生表拍戏面试场景。我们感谢阿琳平野博士，她对稿件的意见。该研发项目是由作者在医学大卫格芬医学院在加州大学洛杉矶分校进行的，是可能由被授予和管理由美国陆军医学研究与装备司令部（USAMRMC）的合同协议，远程医疗与先进技术研究中心（TATRC），在德特里克堡，下合同编号MD:W81XWH-10-2-0077。支持这些研究也来自美国国立卫生研究院EY04067和VA价值评议（NB）。 NCB是弗吉尼亚州职业研究科学家。

所有实验均进行了按照由人类善待实验动物和加州大学洛杉矶分校（UCLA）的动物研究委员会，美国公共卫生服务政策颁布实验动物福利的准则。雄性和雌性的成年Sprague-Dawley大鼠的3-5周龄之间（查尔斯河实验室，威尔明顿，马萨诸塞州）被使用。 1，动物和组织准备Wholemount视网膜和免疫组化协议<ol><li>准备以下的材料和工具:解剖显微镜，镊子2用很细的技巧，剪刀，纤维素滤纸，塑料吸管和一个显微镜载玻片。 </li><li>深深地麻醉用1-3％的异氟醚后斩首了断头台动物安乐死在一个封闭的腔室中的老鼠。一对虹膜剪刀和地点在包含外液的培养皿中取出的眼睛。冬眠，艾姆斯我dium或生理的解决方案建议。 </li><li> [可选步骤，如果视网膜神经节细胞具有荧光4回填需要。在步骤2.3-2.4描述进行荧光4标签。 </li><li>使用解剖显微镜（光强度:1.6×10 8光子/毫米2⋅sec），切断的眼球前部取出角膜。取出晶状体和玻璃体从视网膜内表面用钳子。要删除的玻璃体，通过举办巩膜坚定一个钳子稳定的眼球。轻轻剥离朝向与其他钳子的视网膜中心的玻璃基底。的制备在这个阶段被称为眼罩，其用于冷冻切片。关于冰冻切片的更多细节可以参考文献[14,15]中找到。 </li><li>取下眼罩视网膜。然后，提出四切让视网膜平躺着。轻轻地安装在与感光层一个显微镜载玻片视网膜。添加一滴地解决了视网膜，把C​​E在视网膜上llulose滤纸。一旦视网膜附着在滤纸上，放置在视网膜后面的溶液。如果需要的话，压平，用刷子或镊子视网膜。 </li><li>将wholemounted视网膜到4％PFA 10-15分钟进行固定。 </li><li>为30分钟，在0.1M磷酸缓冲液（PB），pH值7.4洗wholemounted视网膜三次。方框，用5％正常山羊血清或驴血清视网膜在0.1 PB含0.3％的Triton-X 100和0.1％NaN 3的在4℃下过夜。我们推荐的500微升最终体积的所有步骤，以节省封闭液和抗体。 </li><li>第二天，孵育视网膜wholemounts与第一抗体5-7天，在4℃。最佳稀释度必须由用户来确定。 </li><li>在PB中的0.1M洗视网膜3×30分钟，并在其针对第一抗体（浓度为1:1000）的种类对应的荧光团标记的二抗孵育过夜，在4℃。 </li><li>之后每30分钟用PB最后三洗，安装在安装介质中的视网膜。轴封采用指甲油长时间存放盖玻片。商店的样品在4℃下并避光。 </li></ol> 2，贴标神经节细胞轴突与大鼠Wholemount视网膜与钙指示剂染料<ol><li>制备的1000ml哺乳动物林格氏含有（以mM计）125氯化钠，氯化钾3，2的CaCl 2，1.25和NaH 2 PO 4，1的MgCl 2，25的NaHCO 3和10葡萄糖通入95％O 2/5％CO &#173; 2。 </li><li>在步骤1.2-1.4描述进行清扫。 </li><li>要使用注射器装载视网膜神经节细胞（RGC的）和其轴突与钙指示剂染料，注入0.5μL荧光4 pentapotassium盐（40毫米的股票在H 2 O）进入视神经断端约1mm后的眼球。为了测量动态增加神经节细胞内[Ca 2 + </SUP&gt;] i的高亲和力，如荧光4与345纳米的杀敌的指标，是最佳的。的Fluo-4提供具有非常高的量子效率造成的&gt; 100倍的排放量增加的时候结合到钙的额外好处。 </li><li>将眼罩在哺乳动物林格入95％O2 / 5％CO2 &#173; 2为1小时，在室温下在黑暗中。 </li></ol> 3，钙成像协议的视网膜神经节细胞和轴突。 <ol><li>按照步骤1.1-1.4描述取下眼，晶状体和玻璃体。隔离眼罩视网膜，分成象限。安装一个象限神经节细胞的一面朝上载玻片上，用竖琴片网格，以稳定它。保持其他部分暗适应在哺乳动物林格入95％O2 / 5％CO2 &#173; 2在冰上以供以后使用。 </li><li> Superfuse哺乳动物林格氏液在记录室和保持该溶液用95％O 2/5％CO 2的连续鼓泡。 </li><li>图像在显微镜下的组织。行为实验在室温（〜23℃）或温热的阶段，试样下加热该水浴或施加暖空气在试样的表面达到生理温度。 注意:需要注意的是许多细胞功能是由温度，这对于维持细胞内稳态16关键作用调节是很重要的。然而，引进变暖措施可提高漂白，由热膨胀引起17的介质和焦点偏移的蒸发速率。利用室内温度降低荧光4 18细胞内隔离。 </li><li>在不存在药物进行的两个成对的高K +的脉冲的控制。视网膜神经节细胞去极化和轴突研资局由每个脉冲期间提高K +Ø从3毫米到60毫米，33秒钟即可激活VGCCs同一个八信道的重力驱动的表面灌流系统。减少钠离子通过57毫米，以保持isosmolarity在高架K +的解决方案。 </li><li>评估不同VGCCs的与使用一般或特定的钙通道阻滞剂钙信号的贡献。例如，一种非特异性钙离子通道阻断剂，钴施用以下中的钙信号的减少，提供的VGCCs的高K + -evoked钙信号的贡献的半定量测量。 </li><li>通过将感兴趣区域（ROI）围绕感兴趣的视网膜神经节细胞的区域（ 图2）获得的荧光强度值。 </li><li>正常化在由所述第二高K +峰由第一高K +峰产生的变化所产生的荧光强度的变化。然后，对于具体的钙通道阻滞剂的测试，只有在第二高的K +脉冲对用药和规范针对此峰第一峰值。以测量在高K +峰的药物的影响，由该第一对分割所述第二K +峰的幅度。分析应该对这些值就被执行以从在不存在药物的测定配对高K +的峰来自他们的匹配对照。 </li><li>获得5秒的间隔图像。以避免光致漂白，保持激光激发尽可能低。提供激励的氩激光488nm的线，而光电倍增管通过505纳米LP滤波器收集荧光发射。 </li></ol>

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作者没有披露。

在这篇文章中，我们描述了两种不同的方法:1）免疫组化染色显示荧光4定位于神经节细胞的视网膜wholemounts，和2）钙成像分析钙动力学的视网膜神经节细胞及其轴突。 使用wholemounts免疫组已被用于揭示蛋白质的定位在啮齿动物视网膜20-23。不过，也有一些限制。染色的质量在很大程度上依赖于该抗体的识别其相应的抗原和其穿透视网膜组织对感兴趣的特定层的能力的?...

视网膜神经节细胞（RGC的）表达L，N，P / Q-和T-型VGCC通过整个细胞的这些成分的药理学阻断测定Ca通道电流1,2。 VGCC是涉及递质释放，基因转录，细胞调节和突触可塑性3,4,5该跨膜多聚体蛋白。官能VGCCs是由至少三种不同类的亚基组成:大，跨膜α1孔形成在建立通道的生物物理和药理性质，主要是细胞外的辅助α2δ亚基和胞内β亚基的亚基。后两种形式的异配合不同的α 亚单位和改变门控动力学的渠道质膜6和贩运。 在最近的几十年中，许多技术已被用于研究蛋白质expre裂变，如免疫组织化学，酶联免疫吸附测定法，western分析和流式细胞术。这些技术需要使用特定的抗体对于感兴趣的给定的蛋白质的检测，并提供本地化和特异性蛋白质的分布在不同组织中的有力工具。用于检测和定量特定蛋白质的mRNA表达水平的技术，例如Northern印迹分析，RT-PCR，实时定量RT-PCR， 原位杂交，cDNA芯片和核糖核酸酶保护测定法提供了另一种方法，当抗体是不容易特定的蛋白质可用，或者表达水平低7。然而，一个限制，以使用这样的分子生物学技术是基因序列的所需鉴定。 在视网膜本地化蛋白，免疫组织化学可wholemount视网膜进行。由于视网膜神经节细胞的可及性，wholemount准备提供给特定蛋白质的本土化研究，以研资局胞体和轴突一个很好的平台。 除了其定位，VGCCs在视网膜神经节细胞的一些功能特性可以通过使用钙成像技术来证明。我们描述了钙成像协议选择性标记视网膜神经节细胞与钙指示剂染料测量细胞内钙离子动态。不同VGCCs来在不同的细胞区室中的钙信号的贡献可以分离与使用亚型特异性钙通道阻滞剂。 也许这里描述的钙成像技术的最有利的方面之一是，从多个视网膜神经节细胞和其轴突同时和独立地记录的能力。虽然许多生理技术，如全细胞膜片钳记录，提供膜电流的高时间分辨率的录音，躯体或采取这些轴突源记录ê电流不能歧视和录音只能从每次的单个神经元的进行。多电极阵列（MEAs）中能够从许多细胞同时记录的尖峰，但既不能检测或识别的激活，例如，不同的钙离子通道的亚型。 MEAS从细胞位于接近该给定的电极8和记录从细胞中产生大的尖峰9优先记录。光学成像方法提供了一种替代的策略，使细胞的整个种群可与从单细胞微电极和膜片钳记录和MEA记录中获得的信息结合起来的同时和独立记录。虽然这里所描述的钙成像技术被用于研究视网膜神经节细胞的钙动力学，膜片钳和多边环境也可使用在平行于进一步阐明离子电流和视网膜神经节细胞的动作电位发放。 <pCLASS ="jove_content"&gt;由于流离失所无长突细胞组成的神经元群在小鼠视网膜10的神经节细胞层的约60％，我们的目标是利用装载技术，选择性地标记与视网膜神经节细胞的合成钙指示剂染料wholemount准备。虽然合成钙指示剂染料为细胞内钙离子动力学研究一个很好的平台，它的广泛使用已经阻碍无法给定的网络中有效地加载神经元的特定人群。如批量加载11和电穿孔8,12许多技术已被执行的加载单元的整个人群中，然而，这种技术不特定的细胞类型之间进行区分。遗传编码的钙指标提供以选择性标记的细胞的特定群体的能力，但是，这样的方法要求的转基因动物13的产生。我们的技术描述了其实​​现方法具d，来选择经由视神经断端注射钙指示剂染料的wholemount准备标记视网膜神经节细胞。 综上所述，本文中介绍的结构和生理技术提供研究视网膜神经节细胞和其轴突的定位和VGCCs的钙信号的作用的平台。

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			<td height="21" style="height:21px;width:216px;">Straight scissors</td>
			<td style="width:112px;">WPI</td>
			<td style="width:119px;">14124-G</td>
			<td style="width:229px;">dissecting tools</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Straight Forceps</td>
			<td style="width:112px;">WPI</td>
			<td style="width:119px;">501985</td>
			<td>dissecting tools</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">curved iris scissors</td>
			<td style="width:112px;">WPI</td>
			<td>504487</td>
			<td>dissecting tools</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellulose filter paper</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:119px;">HABP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hibernate A</td>
			<td>Invitrogen</td>
			<td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=A1247501">A12475-01</a></td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield</td>
			<td>Vector</td>
			<td>H-1000&#160;</td>
			<td><a href="https://www.google.com/url?sa=t&amp;rct=j&amp;q=&amp;esrc=s&amp;source=web&amp;cd=1&amp;cad=rja&amp;sqi=2&amp;ved=0CC0QFjAA&amp;url=http%3A%2F%2Fwww.vectorlabs.com%2Fcatalog.aspx%3FcatID%3D279&amp;ei=VAHbUb_fBceeiQLK2IHIDw&amp;usg=AFQjCNHijFTTB8p19FMPQ5hg_qTawXU9mA&amp;sig2=FJCfB9fzKR_TmjxoGvhLcg&amp;bvm=bv.48705608,d.cGE">Mounting Media for Fluorescence</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 pentapotassium</td>
			<td>Invitrogen</td>
			<td>F-14200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td>Abbott Laboratories</td>
			<td>05260-05</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope slide</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">22-178-277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss LSM 5 Pascal microscope</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axioplan 40x (NA 0.8) objective lens</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:112px;">Molecular Probes</td>
			<td style="width:119px;">A-11034</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RBPMS</td>
			<td style="width:112px;">ProSci, Poway, CA</td>
			<td style="width:119px;"></td>
			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
		</tr>
	</tbody>
</table>

immunohistochemical and calcium imaging methods in wholemount rat retina

在本文中，我们描述了工具，试剂，并且需要的实际步骤:1）成功制备wholemount视网膜的免疫组化和，2）钙成像的电压门控性钙通道（VGCC）在视网膜介导的钙信号研究神经节细胞。钙成像方法，我们描述关于位移的无长突细胞在神经节细胞层的非特异性加载规避问题。

用特异性抗体免疫标记，为研究在视网膜和钙成像技术感兴趣的特定蛋白质的定位平台允许的VGCCs在视网膜神经节细胞和它们的轴突的钙动力学的贡献的研究。 通过用抗体对神经节细胞蛋白RBPMS（RNA具有多个剪接结合蛋白），该选择性标记的RGC 19，我们能够显示，荧光4标记限定为神经节细胞（ 图1）。残端注射不会导致所有的神经节细胞的如可预期的，从...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

免疫组化协议，用于研究在视网膜特定蛋白质的定位。钙成像技术被用来研究视网膜神经节细胞及其轴突钙的动态。

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

immunohistochemical-calcium-imaging-methods-wholemount-rat

Research

JoVE Journal

Neuroscience

14.4K 次观看.  University of California, Los Angeles. 免疫组化协议，用于研究在视网膜特定蛋白质的定位。钙成像技术被用来研究视网膜神经节细胞及其轴突钙的动态。 

视频: Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

科研

神经科学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute 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+ ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

脑毛细管中培养细胞细胞钙测量和钙成像研究

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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 ...