This work was supported by the Deutsche Forschungsgemeinschaft DFG FOR701 and FE464/11-1 to A.F. The authors want to acknowledge the excellent technical assistance of Andrea Nerz.

所有程序都根据用于由德国联邦共和国发出动物实验的准则进行。 注意:由于视网膜组织将在此过程中的延长部分是无氧，所有步骤应尽可能快地进行。小鼠应该暗适应解剖前至少3小时。以避免视网膜，制备的光适应和切片应在昏暗的红光下进行。 1.媒体和其他化学品的制备<ol><li> 准备艾姆斯介质6。解剖后，沉浸视网膜组织埃姆斯"在任何时候都网上平台。 <ol><li>溶解2.2克艾姆斯培养基和298毫克的HEPES在250ml蒸馏水中。 HEPES浓度为5毫米。轻轻搅拌，直到所有的颗粒溶解。 </li><li>气泡艾姆斯介质在室温下用卡波金（95％氧气，5％的车盂兰盆氧化钛）为至少5分钟。供给卡波金用玻璃巴斯德吸管到溶液中。经由适当的管道的巴斯德吸管连接至气体调节器的出口。 </li><li>添加475毫克钠 - 碳酸氢盐溶液中。碳酸氢盐的起泡后加入防止碳酸钙的沉淀。 </li></ol></li><li> 制备低凝胶温度琼脂糖。琼脂糖需要vibratome切片期间视网膜组织包埋。 <ol><li>溶解于9ml埃姆斯"媒介180毫克琼脂糖和1ml蒸馏水。使用微波，直至所有固体溶解缓慢加热该琼脂糖溶液。热火在5 - 10秒为增量，以避免溢出。溶液应澄清透明。 </li><li>商店在水浴琼脂糖溶解在37℃的过程的持续时间。 </li></ol></li></ol> 2.设置Vibratome 注:所有切片已用进行在材料表vibratome上市。给定的设置指这类只vibratome的。有关神经组织vibratome切片进一步详情，请参阅参考资料7。 <ol><li>将vibratome喷油器刀片插入刀座。 </li><li>测量用"vibrocheck"装置中的叶片的垂直偏转。使用的刀片保持器的调节螺钉来倾斜叶片，直到它平行于切割平面。叶片的精确定位是必要的，以防止部分表面上百叶影响，并产生一个平坦部分，它包含在其表面上活细胞。 </li><li>设置vibratome以下设置:振幅，1.00;速度，0.20;厚度，180 - 200微米。 </li></ol> 3.鼠标视网膜解剖<ol><li>由约1ml异氟烷气化在气密容器麻醉成年C57BL / 6小鼠。当麻醉完成后，由颈椎脱位安乐死的动物。该鼠标不应早于3个月，否则片的质量将变差。 </li><li>通过放置一个弯钳眼睛下方取出眼球。轻轻抬起眼球会露出视神经。切用弹簧剪刀视神经和眼球转移到含2ml埃姆斯&#39;培养基在35毫米培养皿中。 </li><li>开展立体显微镜的阶段的所有后续步骤。调整放大倍数值使得下面的过程（步骤3.4 - 3.9）最佳的视觉控制。 </li><li>穿刺角膜在过渡到与尖镊子巩膜或20G的针。在此步骤中，稳眼球与另一个钳。要小心，不要损坏穿刺装置视网膜。 </li><li>将弹簧剪刀到在步骤3.3所做的小孔，减少角膜周围一圈。此外，稳健的眼球用钳子切割时。保持切口非常接近表面，以便不至cUT斯达康到视网膜。 </li><li>小心取出角膜，晶状体和玻璃体钳。玻璃体包括在眼睛后部仅一薄透明层。但是，它是用于vibratome切片的玻璃体完全除去必要的。 </li><li>在陪替氏培养皿，在某种程度上一个看起来通过孔向下到视网膜上的位置眼罩。开口的周缘应仅由巩膜组织。如果该圆形切口（步骤3.4）已进行了太靠近眼罩的赤道，视网膜组织也将存在接近切口。千万要注意了，不要破坏步骤3.8视网膜。 </li><li>抓住用钳子巩膜，仔细撕裂组织。不要在此步骤破裂视网膜至关重要。如果需要的话，通过用钳子稳住组织的巩膜部分，并仔细剥离与其他镊子视网膜脱离从锯齿缘视网膜。保持镊子关闭;从来没有办理再蒂娜与自组织钳很柔软，这样会马上给。 注:在这一步骤中，在视网膜和眼罩的其余部分将只在视神经头相连。 </li><li>切断附着位点在视神经乳头与弹簧剪刀。视网膜现在应该自由浮动在艾姆斯中单件的组织。 </li><li>通过举办视网膜随着钳的边缘，它操纵到位的使用弯曲手术刀片约23毫米6小矩形 - 切视网膜成4。避免夹持矩形内视网膜组织。取从视网膜，而不是远周边的中央部分件。 </li><li>转移视网膜件具有大直径的塑料巴斯德吸管35毫米的塑料陪替氏培养皿，其底部的已被取代的具有网格尺寸小于1毫米的网格。固定烧杯或锥形瓶中的上三分之一内培养皿并在埃姆斯&#39;中蒲式耳淹没视网膜件bbled与卡波。 </li></ol> 4.嵌入视网膜饮片琼脂糖<ol><li>小心地转移2 - 3视网膜片放入35毫米的塑料培养皿连同一些艾姆斯媒介。它是最方便使用的大直径的塑料巴斯德吸管转让在该步骤中，视网膜片的确切取向并不重要。 </li><li>用玻璃巴斯德吸管除去尽可能剩余艾姆斯培养基。一旦流体已被删除与步骤4.3进行。不要让视网膜组织干的。 </li><li>立即覆盖视网膜片用2ml琼脂糖溶解（37℃）。定在步骤3.10中提到的尺寸，片不会在琼脂糖内卷曲。 </li><li> 内的液体琼脂糖，轻轻地推视网膜件培养皿的底部。神经节细胞层应朝下。使用立体显微镜的精确位置的判断。为定位，处理视网膜PI欧洲经委会用小铲。视网膜定位为快速，精确地因为琼脂糖会很快凝固。 <ol><li>安排平行于玻璃盘底部视网膜件;否则部分将切或斜。单件不应该接触对方，但也不宜相距甚远也。尝试把视网膜碎片上同一水平更多或更少。 </li></ol></li><li>把玻璃培养皿嵌入视网膜片放入冰箱（4 - 6℃）至少2分钟。这将迅速变硬，以便进一步处理的琼脂糖。一旦琼脂糖聚合下一步骤继续进行。 </li></ol> 5.安装视网膜饮片<ol><li>小心取出含有从培养皿视网膜个琼脂糖块。用刮刀或类似设备，以解除从玻璃琼脂糖块。由于粘合力，这可以证明是相当困难的。但是，避免折断琼脂糖块。进行在室温下这和所有后续步骤。 </li><li>放置在玻璃盖玻片琼脂糖块，并用手术刀刀片调整其大小。留下1 - 在视网膜件每一边琼脂糖2毫米。块的最终尺寸应约为1066毫米（10毫米指块，6mm至其边缘的长度的高度）。 </li><li>用胶水瞬间胶粘剂琼脂糖块到vibratome的试样架。视网膜件应该位于10毫米高的块的顶部和取向平行于它的表面，而底部固定在支架上。 </li><li>通过倾倒先前鼓泡艾姆斯中等入vibratome的缓冲盘，立即覆盖块。 </li></ol> 6.准备水平Vibratome部分<ol><li>开始切片。首先，设置手动切片的厚度直至视网膜组织的片段出现在该切片。然后将设置更改为在步骤2.3。由于小鼠视网膜仅约200微米厚，就会有不超过2 - 每琼脂糖块3片。 </li><li>从用玻璃棒缓冲纸盘中取出切片，并将它们在35毫米培养皿转移至2ml艾姆斯培养基。 </li><li>商店切片立即在37℃（5％二氧化碳/ 55％O 2）的培养箱。周围部分中的琼脂糖会留在培养箱固体和可用于处理的部分，而不会损坏视网膜。相比氧化艾姆斯在RT中的各自气氛中，在37℃下储存时，会获得更好的片质量。部分可以立即使用或可被存储到4小时中具有良好的生理效果的孵化器。 </li><li>嵌入在琼脂糖和重复第4下一视网膜片 - 6。 </li></ol>

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The authors have nothing to disclose.

在对比垂直切片，如水平细胞和无长突细胞径向定向神经元的树突形态保存完好。相比于整个视网膜的准备工作，这些细胞类型是微电极方便且可由两个电生理和成像技术进行研究。在这里，我们专注于视网膜外层;然而，类似的方法已经报道尤其用于内视网膜10的无长突细胞。 水平切片准备的属性有功能性研究的几个问题。锥感光细胞和突触后水平细胞之?...

视觉信息被编码为感光体活化的动态空间 - 时间模式，及光感受器和二阶神经元之间的色带突触确定的视觉信息的下行传输。哺乳动物视网膜的垂直切片制备是高度有价值的工具来研究在垂直通路的信号处理， 即感光细胞和双极细胞之间，和之间双极细胞和某些类型的无长突细胞1，2，3，4。 然而，垂直切片总是导致许多视网膜神经元的树突领域的严重截断，导致突触联系的相当大的损失。特别是在外侧视网膜，水平细胞由于它们的横向扩展的扩展树突字段和轴突终端系统5的影响。在垂直因而切片制备，数百锥感光体终端到水平细胞的树突的突触输入被降低到几稀疏联系人。这可能足以研究单个突触的性质，但是它确实绝非表示感光体色带突触的同步激活基础的信号处理能力。 因此，我们开发了一个横向切片准备，这让几乎所有的锥感光的突触输入水平细胞树突的完整和功能。平行于视网膜表面切片运行的平面上，通过水平细胞和无长突细胞之间的内核层理想地切割。因此，外视网膜的水平片段创建含有上突触前部位，用内外段以及突触终端，水平细胞和双极细胞上的突触后现场光感受器。这种准备是非常适合ŤØ通过其树突领域内的所有锥感光细胞研究协调突触的投入水平细胞树突。它因此可能证明是有用的，以便更好地理解在感光色带突触，这是视觉信息的传送从感光体活化的基质成突触后响应关键的运作。

<table><tbody><tr><td>Ames&#039; Medium</td><td>Sigma</td><td>A1420</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Carbogen</td><td>Air Liquide</td><td></td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9045</td><td></td></tr><tr><td>35 mm Petri dish (plastic)</td><td>Nunc Thermofisher</td><td>150318</td><td></td></tr><tr><td>Glass Petri dish</td><td>Chemoline</td><td>50-1470-40</td><td></td></tr><tr><td>Isoflurane</td><td>Dispensary university hospital</td><td></td><td></td></tr><tr><td>Vibratome injector blade</td><td>Biosystems</td><td>39053250</td><td></td></tr><tr><td>Vibratome</td><td>Leica Microsystems</td><td>VT1200</td><td></td></tr><tr><td>Vibrocheck</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Microwave</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>20 G needles</td><td></td><td></td><td></td></tr><tr><td>Spring scissors</td><td></td><td></td><td></td></tr><tr><td>Curved scalpel blades</td><td></td><td></td><td></td></tr><tr><td>Stereo microscope</td><td></td><td></td><td></td></tr><tr><td>Plastic Pasteur pipette</td><td></td><td></td><td></td></tr><tr><td>Glass Pasteur pipette</td><td></td><td></td><td></td></tr></tbody></table>

preparation of horizontal slices of adult mouse retina for electrophysiological studies

垂直切片准备工作已经非常成熟，研究电路和信号传输在成年哺乳动物的视网膜。这些制剂切片的平面垂直于视网膜表面，使其成为理想的像光感受器和双极细胞径向定向神经元的研究。然而，水平细胞，宽视场的无长突细胞和神经节细胞的大树突乔木大多截短，留下显着降低突触活动在这些细胞中。而神经节细胞和无长突移位细胞可以在视网膜的一个整体安装的制剂进行研究，水平细胞和位于内核层无长突细胞是只很差在整个视网膜组织电极访问。 为了实现最方便的访问和突触的完整性，我们开发了小鼠视网膜的水平切片准备，并就读于光感受器和水平之间的突触信号传递细胞。横切片允许（1）容易和明确为电极定位水平细胞体的视觉识别，和（2）保存在扩展水平细胞树突的字段，作为完整且功能锥体突触输入到水平细胞树突的先决条件。 从水平切片水平细胞表现出补品突触活动在黑暗中，他们回答了介绍光闪烁以减少内向电流和减弱突触的活动。免疫细胞化学证据表明，水平细胞的树突领域内几乎所有的锥体建立与周边树突突触。因此，水平片段制备是公适合研究水平延伸视网膜神经元的跨越选定突触生理特性以及传感信号传输和整合。

<html>在小鼠视网膜的水平片段，水平细胞体可以很容易地根据它们与几个主要树突从多边形细胞体（ 图1A）的新兴独特的形态识别。位于切片的表面附近水平细胞机构是对膜片钳电极例行访问。期间记录，将细胞填充有荧光染料，揭示其复杂树突分支，这非常类似于完整小鼠视网膜8（ 图1B）的轴突承载水平细胞的形态。用荧光?...

Watch this Scientific Journal Video about Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies at JoVE.com

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

我们开发了成年哺乳动物视网膜的水平切片准备具有平行切片于视网膜表面的平面。非径向导向的视网膜神经元树突领域并没有截断，使信号处理与膜片钳和成像技术研究。

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

preparation-horizontal-slices-adult-mouse-retina-for

Research

JoVE Journal

Neuroscience

10.0K 次观看.  Friedrich-Alexander-University (FAU) Erlangen-Nürnberg. 我们开发了成年哺乳动物视网膜的水平切片准备具有平行切片于视网膜表面的平面。非径向导向的视网膜神经元树突领域并没有截断，使信号处理与膜片钳和成像技术研究。 

视频: Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

视网膜水平切片制备

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

科研

生物学

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

神经科学

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

研究小鼠分裂视网膜中的双极和水平神经元细胞

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

作者聚焦：使用 Split Retina 技术增强访问和加速实验 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.