支持内授予EY014850。

1。 Sylgard盖菜，试验室，和刀片的制备<ol><li> 35毫米猎鹰的Petri Sylgard弹性体涂层的菜是一个孤立的视网膜上获得单的感光细胞需要适当切碎。根据供应商的说明和少量浇到每道菜，其底部覆盖一层弹性体准备。更换的菜后，涂层覆盖，并将其储存。在几天的时间弹性体变硬的菜都准备好了。 </li><li>隔离光感受器需要坚持的实验室的底部，让他们固定在成像实验的过程中。这是通过涂层与聚- L -赖氨酸或聚- L -鸟氨酸分庭的底部。加入200微升0.01％，其中每室之一的解决方案，并分庭，用纸巾覆盖，防止灰尘。解决方案已经晾干后，用蒸馏水和存储在一个封闭的盒子商会。使用2个星期内。 </li><li>要在一个实验结束时的清洁商会，他们用100％的乙醇删除任何油从油浸镜头和细胞碎片。要去除细胞碎片，用棉签涂药，仔细擦洗室底部。之后，用蒸馏水冲洗，并让钱伯斯干重涂前。 </li><li>切成小块，用金属切割机（8从一个刀片）双刃刀片。 </li></ol> 2。准备的解决方案<ol><li>该解决方案的组成取决于品种。对于两栖类动物，林格（mmol / L的）：110氯化钠，氯化钾2.5，1.6 氯化钙 ，氯化镁2，1 5 HEPES，pH值= 7.55 。应调整pH值用NaOH的最终值。对于哺乳动物，林格（mmol / L的）：130氯化钠，氯化钾，0.5，2 MgCl 2的氯化钙 ，25 hemisodium - HEPES，pH值= 7.40。林格氏液可在室温下保存几个月良好的密封。 </li><li>联合葡萄糖溶液，1 mol / L的，保存在-20 ° C，以避免细菌的生长。 </li><li>在一天的实验，添加到终浓度为5 mmol / L葡萄糖林格氏液在这一天结束，丢弃的林格氏液，含有葡萄糖，因为它可能会增长细菌。 </li></ol> 3。对视网膜的分离<ol><li>对于适当的视网膜切除重要的是，动物牺牲前至少2-3小时的暗适应。动物应在一个合适的通风容器在暗室暗适应。 </li><li>填补中途林格氏液2张35毫米的培养皿中。 </li><li>昏暗的红灯下牺牲的动物和消除眼睛。随后，所有的程序都使用解剖显微镜或一个视频监控摄像头的红外光下进行。 </li><li>删除任何剩余的组织，从眼睛的外表面。剪切和删除的前部，然后转移到一个充满林格氏培养皿眼罩。 </li><li>取出玻璃体和视网膜仔细分离眼罩的其余部分按捏或切割任何附件。轻轻抬起视网膜和它完全分开的眼罩。 </li><li>视网膜传送到第二个培养皿中，用塑料移液管。保持菜中含有轻紧框视网膜。 </li></ol> 4。单一的感光细胞的分离<ol><li>所有的程序下进行红外光。视网膜切小块，用塑料吸管转移到Sylgard盖菜。应包含视网膜片的解决方案的体积约250μL。 </li><li>抓斗一小片刀片与刀座 - 叶片的边缘应在约45 °角向持有人。 Sylgard层上的一块平展的视网膜和使用刀片砍一块视网膜精细，同时保持它坚持Sylgard层。 </li><li>转移200μL含有细胞的实验室的解决方案 - Sylgard覆盖菜留下任何视网膜的剩余部分。保持在光紧中分离出的细胞室。 </li><li>等待10分钟解决的细胞，然后加入2-3毫升林格氏。在这个阶段，可以分离出的细胞装入一个特定的荧光染料（例如FURA - 2）根据染料的加载协议。 </li><li>这些细胞现在可以采取在显微镜阶段的实验。 </li></ol> 5。荧光成像<ol><li>传输室epifluorescence显微镜阶段。灌注调整解决方案，温度传感器，红外线照射等。 </li><li>拉上窗帘，开始实验。打开的红外光显微镜内笼，重点实验室的底部，和细胞的移动舞台。 </li></ol> 6。代表性的结果S： 图图 1显示了健康的隔离杆，与本议定书获得蝾螈（Ambystoma tigrinum）视网膜锥光感受器的形态。蝾螈的细胞已被广泛用于单细胞荧光成像技术的研究，因为其庞大的规模和其生存的能力，从视网膜分离后的几个小时。此外，从蝾螈视网膜中，可以定期获得杆和锥光感受器。 细胞健康的重要标准之一是存在一个完整的椭圆体（ 图 1），部分细胞线粒体集中。当细胞下的DAPI光学看，这种浓度的线粒体，给人强烈的荧光信号（ 图2）由于NADH的存在。缺乏一个完整的椭球是一个受损细胞的标志，一般不宜用于实验。图。 3显示了一个损坏的蝾螈棒感光，肿胞体和凝结核。这些细胞显示荧光下的DAPI光学低得多，但观看下FITC标记的光学强FAD的信号显示在椭球区域（氧化黄素核苷酸和flavoproteins的）。另一种孤立的光感受器的健康标准是自己的能力产生刺激他们的后外节光全反式视黄醇（维生素A ）。维生素A的生成需要大量的NADPH的，这取决于一个完整的代谢机械。图4和图5显示形成的维生素在一个完整的青蛙和鼠标杆光感受器外节。 <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="图1"/> 图1。健康的单杆和锥光感受器细胞中分离出从虎蝾螈视网膜。 Phototransduction外段和椭球与线粒体密集的包装。棒负责昏暗的灯光下视力，明亮的灯光视觉锥。 <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="图2"/> 图2。荧光生活娃娃鱼杆和锥，这是暗适应细胞，显示在各自的椭球的强烈NADH的荧光，并没有明显的维生素A在其外层细分市场的荧光。荧光图像捕捉可见光，暗适应时间后的第一次接触。 <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="图3"/> 图3。受损的蝾螈杆感光肿胞体和凝结核损害的指标。被氧化的细胞，是最小的NADH的信号（DAPI光学），但更强大的FAD的信号（FITC标记光学）。 <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="图4"/> 图4。 NADH和视黄醇的青蛙棒 。这是一个健康的青蛙棒感光，在椭球区域呈现出强大的NADH的荧光。光线照射前有外段的最小荧光。光线照射后，由于维生素A的形成是有显着增加外段荧光<img src="/files/ftp_upload/2789/2789fig5.jpg" alt="图5"/> 图5。鼠标杆与视黄醇 ，这是一个健康的鼠标杆感光，由于光线照射后形成维生素A的鼠标杆光感受器椭球区域不表现出强烈的荧光信号显着的外段荧光。

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如果未取得健康孤立的细胞，但问题在于与隔离或视网膜的健康，或者其斩。通常情况下，取出后，眼睛的玻璃体前，视网膜随时升空色素上皮细胞。如果没有，尝试从外围眼罩开始剥离它关闭。如果它仍然是难以分开，一个可能性是，动物还没有足够的时间内，暗适应，或太亮了红灯。确保适当的暗适应的动物，和昏暗的红灯。在视网膜上的杆光感受器的存在，可以很容易地通过检查孤立的视?...

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>黑暗的房间（100-150英尺2） </td><td></td><td></td><td></td></tr><tr><td> 19个红灯</td><td></td><td></td><td>网上商店</td></tr><tr><td>红外光源和红外图像的观众</td><td> FJW光学系统，公司</td><td></td><td></td></tr><tr><td>解剖显微镜19 </td><td></td><td></td><td>配备红外观众</td></tr><tr><td>封闭在一个光紧笼19 Epifluorescence显微镜</td><td></td><td></td><td></td></tr><tr><td>剖析工具（剪刀，镊子，刀片持有） </td><td> Roboz或精细的科学工具</td><td></td><td></td></tr><tr><td> Sylgard弹性体</td><td>埃塞克斯郡（北卡罗莱纳州夏洛特） </td><td> Sylgard 184弹性体套件</td><td></td></tr><tr><td>聚- L -鸟氨酸（0.01％） </td><td> Sigma - Aldrich公司</td><td> P4957 </td><td></td></tr><tr><td>聚- L -赖氨酸（0.1％） </td><td> Sigma - Aldrich公司</td><td> P8920 </td><td>稀释至0.01％ </td></tr><tr><td>实验商会</td><td>华纳仪器（哈姆登，CT） </td><td> D3512P </td><td></td></tr><tr><td>培养皿，塑料移液器</td><td> Fisher Scientific则</td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

在脊椎动物视网膜，phototransduction，光转换为电信号，进行了由杆和锥感光细胞1-4。棒感光器负责在昏暗的光线，在明亮的光线锥的视野。 Phototransduction的感光细胞外，一个专门的车厢，其中包含一个视觉色素，主光探测器的高浓度段的地方。视色素是一个生色，11 - 顺视网膜附着的蛋白质，视蛋白组成。视觉色素吸收光子异构从11生色团- 独联体国家全反式 。这种光致异构化带来了一个启动一个级联反应，最终在膜电位的变化，为电信号的传导光刺激的视觉色素的构象变化。光刺激的细胞恢复涉及的失活，由光激活的中间体和膜电位的重建。的Ca 2 +调节几个phototransduction涉及的酶的活性，减少光刺激后，其浓度。在这样的Ca 2 +在细胞光刺激，其背景光的适应复苏起着重要作用。 恢复过程的另一个重要组成部分，已在光检测摧毁其11的光致异构化的视觉色素的再生-独联体生色全反式5-7。这种再生开始的全反式视网膜的光敏色素的释放，留下的APO -蛋白视蛋白。被释放的全反式视网膜利用NADPH的全反式视黄醇，视蛋白反应迅速减少，结合新鲜的11 -顺视网膜带来改革的视觉色素外段。全反式视黄醇，然后转移到邻近的细胞外段和专门载体Interphotoreceptor维甲酸结合蛋白（IRBP）。 单一的感光细胞的荧光成像可用于他们的生理和细胞生物学研究。的Ca 2 +敏感的荧光染料，可用于详细研究外段的Ca 2 +的变化和反应之间的相互作用 ，以光 8-12以及内心的Ca 2段中的作用的Ca 2 +存储+动态平衡13,14 。荧光染料，也可用于测量镁2 +浓度，pH值，以及水和膜车厢 16示踪剂。最后，全反式视黄醇（维生素A）的内在荧光可以用来监测其形成的动力学和17日至19日在单一的感光细胞去除。

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

一个方法是为单身的生活，从不同的脊椎动物物种的荧光成像的感光细胞的准备。该方法可用于图像的内源性荧光基团，如NADH或维生素A，的荧光，或外源性补充钙敏感的荧光染料，<sup&gt; 2 +</sup&gt;或其他因素。

生活荧光成像的隔离脊椎动物的感光细胞的制备

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

preparation-living-isolated-vertebrate-photoreceptor-cells-for

Research

JoVE Journal

Neuroscience

12.2K 次观看.  Medical University of South Carolina. 一个方法是为单身的生活，从不同的脊椎动物物种的荧光成像的感光细胞的准备。该方法可用于图像的内源性荧光基团，如NADH或维生素A，的荧光，或外源性补充钙敏感的荧光染料， 2 +或其他因素。

视频: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

毫秒氢/氘交换质谱法用于生理条件下α-突触核蛋白结构动力学的研究

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...