这项工作是由美国国立卫生研究院资助EY019312和EY021126（VJK），EY002687到眼科及视觉科学系在华盛顿大学的支持，以及防盲研究。

所有的实验方案符合指南实验动物的护理和使用，被批准的机构动物研究委员会在华盛顿大学。 1.设置灌注和标本持有人<ol><li>制备用于视网膜灌注新鲜的实验当天溶液。使用蒸馏水和去离子水。使用以下三种解决方案之一。 <ol><li>制备碳酸氢盐的含艾姆斯溶液（1L）:1瓶艾姆斯的"媒介和1.9克碳酸氢钠的， </li><li>制备洛克溶液（以mM计）:112.5氯化钠，3.6氯化钾，2.4的MgCl 2，1.2氯化钙2，10.0 HEPES，20.0 碳酸氢钠 ，3.0钠琥珀酸盐，0.5钠谷氨酸，0.02 EDTA和10.0葡萄糖，0.1％MEM维生素和氨基酸酸</li><li>制备HEPES缓冲林格氏溶液（以mM计）:133.3氯化钠，3.3氯化钾，2.0的MgCl 2，1.0氯化钙2，10.0葡萄糖，0.01 EDTA，12.0 HEPES，pH值调节至7.5以〜5.8毫升的1M氢氧化钠，加0.72克/升的Leibovitz培养基的L-15。使用20 - 50μMDL-AP4和50 - 100μM氯化钡2隔离带任何媒体灌注感光响应。 </li></ol></li><li>制备用于电极28（以mM计）溶液:140.0氯化钠，3.6氯化钾，2.4的MgCl 2，1.2氯化钙2，3 HEPES，0.01 EDTA和调节pH值至7.4 - 7.5用NaOH。电极溶液可以储存在RT几个月。 </li><li>准备和测试样品支架。 <ol><li>胶在试样保持器的底部的圆顶顶部黑/灰滤纸（ 见图1A）。小心地双组分5分钟环氧树脂胶围绕圆顶的平坦顶部的边缘。如果有必要，这样做解剖显微镜下。 </li><li>等到胶是几乎干燥（约4分钟），并通过使用一个扁平项按下圆顶的滤纸。胶滤纸至少一天实验前。该滤纸可多次使用，但在一个月的录音后应更换。用70％乙醇安装更换纸张之前，小心地从任何残胶清洁的圆顶。 </li><li>与电极液试图避 ​​免任何气泡和螺杆封闭带螺纹适配器插入电极通道粒料电极（ 见图1A和B）填充电极通道。连接的顶部和底部件的试样保持器的四个螺钉和填充灌注线与电极液。 </li><li>测量用万用表（ 图1B），每个电极对的导线之间的电阻和电压。电阻应该低于100Ωk和低于10毫伏的电压，如果信道是无气泡和电极是在良好的条件。 </li></ol></li><li>倾400 - 700毫升灌注介质中的玻璃瓶中。分开在视网膜和s的解剖中使用另外300ml的在冰箱撕毁了它。设置在热交换器区块灌注管，并放置在加热板上将预热块（参见图1D）。 </li><li>封闭瓶上的帽具有连接到CO 2 / O 2的气体（如果艾姆斯或洛克的使用）和灌注管（参见图1D）。预热瓶媒体37℃，并将其放置在加热板上或强光刺激单元的水浴设为37顶- 39℃下17。 <ol><li>总理灌注线用灌注填充媒体向他们发起重力驱动的流动。 <ol><li>在LKC系统，将瓶上的刺激器单元17的顶部，以提供一个显著重力流动，然后由流速调节器，而不会受到在实验过程中，在瓶子中灌注溶液的降低水平进行调整。 </li><li>在Ocuscience系统，放置在p法拉第笼内部erfusion瓶以减少噪声和放置从所述试样保持长期灌注输出线（见步骤2.8）大大低于试样保持器（和灌注瓶）的水平，以使溶液的重力驱动。这些屏蔽输出线和屏蔽连接到功放的地面，以防止电磁噪声的ERG信号的耦合。 </li></ol></li></ol></li><li>通过使用流速调节器5毫升/分钟 - 调节灌注到3。连接5％CO 2/95％O 2的气体从适当的调节器的气缸，并调整流量以确保介质在瓶子的稳定鼓泡通过空气石。 </li></ol> 2.样品制备<ol><li>组装清洁和锋利清扫工具包括直刀刃microscissors，一个或两个45°镊子，刀片和一块长方形的滤纸。 </li><li>倒入约200毫升冷perfu的在一个大的陪替氏培养皿锡永溶液中，使所述试样保持器（包括圆顶）的整个底部部分可以浸入到该溶液中。安装在圆顶的视网膜时，这一步就变得很重要（见步骤2.6）。虽然一些解决方案被设计成与卡波金（5％CO 2/95％O 2）是饱和的，这是不适合的夹层目的必不可少，并没有在这里描述的实验中进行。 </li><li>对于一个典型的实验中，让动物们在12/12小时黑暗/光照周期和暗适应他们6 - 录音前12小时。安乐死的动物通过CO 2吸入，随后在昏暗的红灯颈椎脱位，并做下暗红色或红外光所有的下列程序（ 如使用红色过滤器显微镜光源的前面）。 <ol><li>拉的眼睛通过使用镊子，把他们的报道。将一只眼睛在一个时间上的一小片纸（ 例如，一些正规的过滤纸），并作为点燃大约在锯齿缘的水平，同时保持与镊子眼睛（这是解决方案之外在这里完成）。 </li></ol></li><li>沿着锯齿缘切（或接近眼睛的赤道）配microscissors并去除角膜和晶状体。将眼罩在冷介质在大陪替氏培养皿，并用另一只眼睛重复相同的过程。 </li><li>通过保持视网膜和巩膜之间的剪刀，以保持视网膜作为完整尽可能削减从眼罩朝着视神经顶部的小切口。握从切口的两侧通过使用两个镊子和拉镊子远离彼此分离的视网膜巩膜。 <ol><li>切视神经和尝试隔离视网膜与身体接触的远端表面的最小量。视网膜色素上皮将自动从视网膜中的解剖过程大多分离。以避免视网膜的机械干扰比来执行更重要的是夹层很快，一般视网膜可以培养在溶液至少30分钟而对响应特性显著影响。 </li></ol></li><li>安装在试样保持器（ 图1C）的圆顶的视网膜。沉浸在培养皿与解剖视网膜的试样保持器的底部。滑动视网膜，感光体一侧（隔离视网膜的凸面）向上，在圆顶上并提起试样保持器，以使视网膜附着在滤纸上。重复上述步骤为其他视网膜。 </li><li>仔细擦干保持架板，以防止视网膜之间的电串扰，以及噪声和信号分流。试样保持器具有围绕底部圆顶O型环，以防止夹持器的底部和顶部部分之间溶液溢出以及帮助个体视网膜的光感受器和神经节细胞侧的电隔离。用四个螺丝安装支架的顶部部分（参见图1B），并通过用注射器和针填充灌注通道灌注溶液。 </li><li>传送旁边的热交换器区块的试样保持器，并连接在输入和输出灌注线连接到试样保持器（ 图1D）。连接电极以ERG的放大器（顶部电极连接到接地/减销中的放大器），并通过使用一个适配器附着在试样保持器的刺激器/控制单元或滑动加热垫成取决于所述刺激器单元在其上的体内 ERG系统被使用。 </li></ol> 3.录音<ol><li>通过使用在体内系统的软件配置放大器和刺激设置。采集频率设置为1至10千赫和低通值滤波，以300赫兹。不要使用高通滤波。使用60赫兹（或在欧洲50赫兹）如果必要的话陷波滤波器。 </li><li>没有光刺激和机智记录基线ħ暗淡刺激（ 例如 ，绿光的-35 dB或0.3 MCD平方米-2）来测试电极和视网膜样品之间良好的电连接。等待10 - 20分钟在开始收集数据之前，使视网膜温度和功能达到一个稳定的状态。 <ol><li>根据任何具体的实验选择的刺激参数，并开始数据收集。 例如，用绿光从-40到0 dB或从0.1上升到1000 MCD SM -2为暗反应的家庭。使用约2到3个对数单位亮闪烁明视录音哪里棒必须由背景光被抑制（3 - 30 CDM -2）或探针闪光灯（约1.3镉平方米-2 = 1分贝参见图3）。 </li><li>但是，请记住确切的光强值可能会一定程度上取决于你的系统校准和实验条件。作为一个经验法则，规模强度下降了5 - 10倍相比， 在体内 </eM&gt;使用绿灯时17录音。黑色覆盖有孔上面的视网膜（包含在商用适配器系统）可用于以减少光散射在样品支架和由商业体内系统的Ganzfeld球体同时促进视网膜的均匀和相等的刺激。 注:对于记录从暗适应视网膜中，在典型的实验漂白颜料只有微不足道的分数使用的测试闪烁的强度以使缺少RPE驱动再生的是不是一个问题。此外，先前已显示，锥体颜料再生仍然可以通过米勒细胞视觉周期20发生在隔离视网膜。 </li></ol></li><li>作为实验持续典型地从30分钟到几个小时，监测基线漂移，噪声水平和响应稳定性试验期间的变化在这些可能表示的技术问题。 注:噪音的增加或减少D响应幅度可以指示气泡在灌注或电极通道，过低或过高的温度，溶液泄漏/溢出或视网膜的位移。 </li></ol> 4.清洗<ol><li>从灌注线取下样品支架，打开它，并从滤纸冲洗的视网膜。取出电极，并用蒸馏水（不要使用乙醇）冲洗。清洁试样保持器（包括灌注通道）与乙醇和/或蒸馏水。同花顺灌注管小心地用&gt; 70％的乙醇。 </li><li>冲洗瓶灌注用蒸馏水（不要使用清洁剂）。乙醇也可以用来清洗瓶子。 </li></ol>

<ol>
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Sci. 47, 2583-2588 (2006).</li><li>Walter, P., Luke, C., Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+and+light+responses+in+superfused+bovine+retina.">Antibiotics and light responses in superfused bovine retina.</a> Cell. Mol. Neurobiol. 19, 87-92 (1999).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+profile+of+alkylphosphocholines+in+the+model+of+the+isolated+perfused+vertebrate+retina.">The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.</a> Graefes Arch. Clin. Exp. Ophthalmol. 248, 511-518 (2010).</li><li>Januschowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+toxicity+testing+of+VEGF+Trap-Eye+in+an+isolated+perfused+vertebrate+retina+organ+culture+model.">Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.</a> Acta Ophthalmol. 92, e305-e311 (2014).</li><li>Kolesnikov, A. V., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transretinal+ERG+recordings+from+mouse+retina:+rod+and+cone+photoresponses.">Transretinal ERG recordings from mouse retina: rod and cone photoresponses.</a> J Vis Exp. , (2012).</li><li>Koskelainen, A., Hemila, S., Donner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+sensitivities+of+short-+and+long-wavelength+sensitive+cone+mechanisms+in+the+frog+retina.">Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.</a> Acta Physiol. Scand. 152, 115-124 (1994).</li><li>Lyubarsky, A. L., Falsini, B., Pennesi, M. E., Valentini, P., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-+and+midwave-sensitive+cone-driven+retinal+responses+of+the+mouse:+a+possible+phenotype+for+coexpression+of+cone+photopigments.">UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.</a> J. Neurosci. 19, 442-455 (1999).</li><li>Lyubarsky, A. L., Daniele, L. L., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+candelas+to+photoisomerizations+in+the+mouse+eye+by+rhodopsin+bleaching+in+situ+and+the+light-rearing+dependence+of+the+major+components+of+the+mouse+ERG.">From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.</a> Vision Res. 44, 3235-3251 (2004).</li><li>Azevedo, A. W., Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+protocols+alter+phototransduction:+the+implications+for+retinal+processing+at+visual+threshold.">Experimental protocols alter phototransduction: the implications for retinal processing at visual threshold.</a> J. Neurosci. 31, 3670-3682 (2011).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+I.+Structural+analysis+using+light+and+electron+microscopy.">Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.</a> J. Comp. Neurol. 188, 245-262 (1979).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+vertebrate+photoreceptors.">Adaptation in vertebrate photoreceptors.</a> Physiol. Rev. 81, 117-151 (2001).</li><li>Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-driven+translocation+of+signaling+proteins+in+vertebrate+photoreceptors.">Light-driven translocation of signaling proteins in vertebrate photoreceptors.</a> Trends Cell Biol. 16, 560-568 (2006).</li><li>Schneeweis, D. M., Schnapf, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photovoltage+of+macaque+cone+photoreceptors:+adaptation,+noise,+and+kinetics.">The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics.</a> J. Neurosci. 19, 1203-1216 (1999).</li><li>Heikkinen, H., Vinberg, F., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+background+lights+enhance+dark-adapted+cone+ERG+flash+responses+in+the+intact+mouse+retina:+a+possible+role+for+gap+junctional+decoupling.">Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.</a> J. Neurophysiol. 105, 2309-2318 (2011).</li><li>Gouras, P., MacKay, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+in+amplitude+of+the+human+cone+electroretinogram+with+light+adaptation.">Growth in amplitude of the human cone electroretinogram with light adaptation.</a> Invest. Ophthalmol. Vis. Sci. 30, 625-630 (1989).</li><li>Peachey, N. S., Goto, Y., al-Ubaidi, M. R., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+mouse+cone-mediated+electroretinogram+during+light+adaptation.">Properties of the mouse cone-mediated electroretinogram during light adaptation.</a> Neurosci. Lett. 162, 9-11 (1993).</li></ol>

圣路易斯华盛顿大学与Xenotec，Inc.的许可协议，并可以从出售体外适配器收到版税。

<html>我们在这里表明，用于通过使用在体内的ERG系统组件与离体的ERG适配器一起同时获得高品质的离体的ERG记录从两个分离的小鼠视网膜的关键步骤。在此研究中，我们灌注既视网膜从具有相同的溶液（或者艾姆斯，洛克的或林格氏）的动物，但它也可以将灌注每个视网膜与不同的溶液如用于药物测试的目的。为了获得高质量的数据，最重要的步骤是从电磁噪声，小心?...

电图（ERG）是一种公认​​的技术，可以用来记录由光引发的视网膜的电活动。 ERG的信号由引起径向电流的电压变化（沿感光细胞和双极细胞的轴）中流动的视网膜的电阻外空间，主要产生的。第一ERG信号是从的鱼眼1表面记录在1865年霍姆格伦。艾因特霍芬和乔利1908除以2的ERG响应于光的发作分为三个不同的波，称为A，B，和c波，现在已知以反映感光体的主要的活性，ON双极细胞，和色素上皮细胞，分别3-8。 ERG可以从麻醉动物或人类（ 体内 ）的眼睛被记录时，从分离的眼制剂9，穿过分离完整视网膜（ 离体 ）3,10-15或跨越特定视网膜层与微电极（本地ERG）4,16。在这些中， 在体内 ERG是目前最广泛使用的方法，以评估视网膜的功能。它是一种非侵入性的技术，可用于诊断目的或跟随在动物或患者的视网膜疾病的进展。然而， 在体内 ERG录音制作一个复杂的信号，层峦叠成分，往往是由生理性眼外噪音（ 如呼吸和心脏活动）污染。 本地ERG可用于记录整个视网膜的特定层的信号，但它是最侵入性和具有最低信噪比（SNR），为相对于其他的ERG记录的配置。本地ERG也是技术要求高，需要昂贵的设备（ 如，显微镜和显微操作）。 Transretinal ERG从完整的，孤立的视网膜（ 体外 ERG）提供了在体内和局部ERG方法之间的妥协，允许稳定HIG^ h SNR录音来自动物或人17完整的视网膜。最近，这种方法已成功地用于研究哺乳动物，灵长类动物和人类视网膜18-20杆和视锥感光功能。另外，由于不存在在体外视网膜色素上皮，视网膜电图信号的正C波分量被除去，一个突出的负慢PIII成分显露在体外的录音。缓慢PIII组件已经显示从米勒胶质细胞的活性，以发起在视网膜21-23。因此，也可以采用离体的ERG的方法来研究Müller细胞在完整视网膜。几项研究也表明， 体外 ERG记录可以用来测量药剂浓度周围视网膜24和 ​​测试药物25-27的安全性和有效性。 在体内系统中的多个商业可用和许多实验室不一定具有广泛的背景电使用。相比之下， 体外设备还没有着落直到最近17并因此只有极少数的实验室目前正在这个强大的技术优势。这将是有益的，使提供给更多的实验室体外 ERG录音，以推进我们对视网膜的生理和病理知识，并开发新的疗法致盲疾病。我们在这里展示了一个简单而经济实惠的体外 ERG装置17，并显示如何可以组合使用几种市售体内的ERG系统来记录棒状和锥形介导的信号（a-和b-波）和功能Müller细胞（慢PIII）从完整的野生型小鼠视网膜。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>In vivo ERG system</td>
			<td>OcuScience</td>
			<td>HMsERG</td>
			<td><a href="http://www.ocuscience.us/id77.html">www.ocuscience.us/id77.html</a></td>
		</tr>
		<tr>
			<td>In vivo ERG system</td>
			<td>LKC Technologies</td>
			<td>UTAS-E 3000</td>
			<td><a href="http://www.lkc.com/products/UTAS/bigshot.html">www.lkc.com/products/UTAS/bigshot.html</a></td>
		</tr>
		<tr>
			<td>Ex vivo adapter</td>
			<td>OcuScience</td>
			<td>Ex VIVO ERG adapter</td>
			<td><a href="http://www.ocuscience.us/id107.html">www.ocuscience.us/id107.html</a></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>North Central Instruments</td>
			<td>Leica M80</td>
			<td><a href="http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;idCategory=27">May use any brand</a></td>
		</tr>
		<tr>
			<td>IR emitter</td>
			<td>Opto Diode Corp.</td>
			<td>OD-50L</td>
			<td><a href="http://www.optodiode.com/">www.optodiode.com</a></td>
		</tr>
		<tr>
			<td>Prowler Night Vision Scopes</td>
			<td>B.E. Meyers Electro Optics</td>
			<td>D4300-I</td>
			<td>Military grade product.</td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Rosco Laboratories</td>
			<td>Roscolux #27 Medium Red</td>
			<td>May be used instead of IR system</td>
		</tr>
		<tr>
			<td>Red head light</td>
			<td>OcuScience</td>
			<td>ERGX011</td>
			<td><a href="http://www.ocuscience.us/catalog/i29.html">www.ocuscience.us/catalog/i29.html</a></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WPI, Inc.</td>
			<td>500086</td>
			<td><a href="http://www.wpiinc.com/">www.wpiinc.com/</a></td>
		</tr>
		<tr>
			<td>Dumont tweezers #5</td>
			<td>WPI, Inc.</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com</a></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metler Toledo</td>
			<td>AB54-S/FACT</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>pH meter and electrode</td>
			<td>Beckman Coulter</td>
			<td>pHI 350</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60129</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63020</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>B0750</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris Bioscience</td>
			<td>101</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Succinic acid disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>224731</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2834</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Leibovitz culture medium L-15</td>
			<td>Sigma-Aldrich</td>
			<td>L4386</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>MEM vitamins</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M5550</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>UN3156</td>
			<td>5% CO2</td>
		</tr>
	</tbody>
</table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

我们记录从暗适应的野生型闪光灯反应（WT）的C57BL / 6小鼠的视网膜通过以下通过使用不同的标准灌注液（ 图2）如上所述和在图1中所示的实验方案。的响应波形和动力学以及视杆细胞的灵敏度出现在埃姆斯&#39;和洛克的媒体（ 图2A和B）类似。另一方面，在HEPES缓冲的Ringer溶液（无碳酸氢盐或5％的CO 2/95％O 2）的反应振幅均显著小。我们还...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.1K 次观看.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

视频: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...