Name: patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina
Uploaded: 2016-10-13T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.

伦理批准 - 涉及受试动物的程序是按照规则和研究动物卫生准则的国家机构的规定进行的，所批准的机构动物护理和使用贝勒医学院的委员会。 1.外部和内部解决方案<ol><li>在视网膜剥离，并在随后电生理记录外液使用哺乳动物的林格氏液。制备哺乳动物林格氏上记录的一天从10倍原液（没有钙）溶液中，添加氯化钙2滴carbogenation的15分钟（95％O 2和5％CO 2）之后。最终1倍溶液含有（以mM计）:120氯化钠，5氯化钾，25的NaHCO 3，0.8 的 Na 2 HPO 4，0.1的NaH 2 PO 4，2的CaCl 2，1用MgSO 4和10 D-葡萄糖。 </li><li>使用两个内部解决方案，以表征SAC振荡。测试制备的溶液在长时间全细胞膜片钳记录对成年小鼠视网膜神经节细胞的有用性，并存储于500μl等份的验证批次在-20℃直到使用。 <ol><li>用于记录膜电位振荡，使用包含（以mM计）基于钾内部溶液:125 K-葡糖酸盐，8氯化钠，4的ATP的Mg，0.5立GTP，5 EGTA，10 HEPES和0.2％生物胞素（重量/ v）中。调节pH值至7.3 KOH。 </li><li>用于记录兴奋性和抑制性突触后电流，使用包含（以mM计）基于铯内部溶液:100铯甲磺酸盐，8氯化钠，4的ATP的Mg，0.5立GTP，5 EGTA，10 HEPES和0.2％生物胞素（重量/体积）。调节pH值至7.3用CsOH。 </li></ol></li></ol> 2.准备录制的日子<ol><li>制备具有开放孔的硝化纤维素膜，人工与来自钝为16G注射器针头制成的定制冲床冲膜和压平两个干净的玻璃板之间的薄膜。对于whole-安装视网膜，使中央孔直径为0.2mm以及由4较大的孔是1-1.2毫米直径围绕着它。 </li><li>使装载内部溶液到电极定制回填长丝，熔融10微升枪头在中间，直到它冷却变硬轻轻延长熔融部分。只是在使用前，用干净的刀片修剪细丝，直到它比补丁移液器稍长。 </li><li>在从硼硅玻璃管的可编程拉出器具有内部长丝拉补丁移液器。制造上实验当天的微量，并立即使用。 注:对于录音的SAC，我们用下面的车夫设置:热量:484;拉:0;速度:25;延迟时间:1;压力:400和462的硼硅酸盐管的外径和内径的斜坡值分别为1.65和1.0mm。移液器的电阻10-14MΩ。 </li><li>一个HR组织收获前，解冻被萨基内部解决方案的管纳克它为&gt; 30分钟的旋涡。 </li></ol> 3.视网膜解剖<ol><li>麻醉4％的异氟醚的动物中氧气直到动物失去反应到脚趾捏。颈椎脱位牺牲的动物。 </li><li>在1-2毫米从直指出虹膜剪视神经头离开切断视神经两个眼球。 </li><li>滚上干净的纸巾眼球，以清除血液，然后在carbogenated哺乳动物的林格氏液沉浸其中。按机器上使用23政针缘的孔，并伴随着微剪刀切缘平分眼球。删除使用细镊子角膜和晶状体。 <ol><li>对于视网膜整体安装，轻轻揭下用细镊子的色素上皮细胞的整个视网膜，并从向视神经乳头边缘4 1.5-2毫米正交削减。 </li></ol></li><li>沉浸穿孔硝酸纤维素膜，放入盘中，轻轻拖动与视网膜在它保利协鑫的一面朝上。制备整个安装视网膜当视神经乳头放入中心孔。与视网膜膜内转印到另一个干净的菜。轻轻压平视网膜用细刷油漆，看起来像一个马耳他十字打下了孔四边。 <ol><li>如果该视网膜的一小部分是在一个时间进行检查，切来自步骤3.3制备成3-4件用刀片与色素上皮保持连接每个眼罩。避光未使用的部分在carbogenated外部解决方案，12小时内使用。 </li></ol></li><li>吸干用干燥的滤纸的硝酸纤维素膜，并删除与镊子玻璃体和内界膜和漆刷。 </li><li>确保所有的视网膜边缘装配转印到记录室在底部密封的玻璃盖片之前完全附着到膜上。用真空脂膜固定到盖玻片和rehydratË与外部溶液视网膜。要小心，不要捕获任何气泡的组件下方。 </li><li>该室设置到一个直立式显微镜的阶段。以每分钟〜3毫升灌注速度用温水（34-35℃）carbogenated外液室。 </li><li>检查10倍物镜先下视网膜，然后用60倍的水浸泡镜头下看到微分干涉对比（DIC）和/或落射荧光GCL和INL神经元。为了形象化tdTomato表达的SAC，使用白光LED组合光源的波长554激发过滤器和581 nm的发射滤光片。 </li></ol> 4.全细胞膜片钳记录从平面贴装视网膜<ol><li>过滤通过针筒式滤器插入定制回填灯丝的内部溶液。 </li><li>插入灯丝成新鲜的拉微量和直到溶液覆盖银电极线为&gt;分配尖端附近的内部溶液为5mm。紧固微量到与抽吸杆的电极保持器，通过该电极内部的压力可以通过推动或拉动管10连接毫升的塑料注射器的柱塞进行调整。 </li><li>找到目标下的吸管，并把它归结为视网膜以上〜100微米。根据目前的跟随模式（I = 0），使用DC偏移为零立起的直流电压信号。通过吸移管注入固定振幅的方波电流测量电流钳（Ⅰ 钳 ）模式下的移液管电阻，而它是在浴中和通过转动r存取旋钮中和的差。使用对r存取旋钮读取由欧姆定律来计算吸液管电阻。 </li><li>慢慢把10微米视网膜上方的电极〜。施加正压到电极。观看枪头附近的反射变化，因为它接近视网膜。很快，但轻轻用力吸液管进入保利协鑫，降低了积极pressu立即重新。 </li><li>移动吸管向神经元标记。避免接触其他神经元，血管和Muller细胞endfeet。如果需要防止电极堵塞施加更大的正压力。 </li><li>直到一个凹坑是可见的标记的神经元的中线附近枪头定位。释放正压和允许质膜反弹到枪头。 </li><li>应用20〜120 pA的负电流，以移液管，以帮助一个千兆欧姆密封的形成。施加轻柔抽吸如果必要质膜拉入吸管。等待后密封形成5分钟以破裂细胞膜。这使得泻内部解决方案，通过灌流清除。用温和的吸力破裂膜。 </li><li>膜破裂后，而在电流钳模式，开启桥平衡，并使用r存取旋钮进行调整。 注:对于像国资委一小格，只需要稍作调整，如果有的话。 Alternatively，记录兴奋性和通过保持在氯化物的逆转电位的细胞（约-75毫伏）和谷氨酸（约0毫伏）分别在电压钳模式（V 钳位 ）抑制性突触后电流。检查并在必要时以适应偶尔的视网膜和/或显微漂移调整吸管的位置。 </li><li>到之前记录膜电位或电流变化，期间和药物治疗后，制备突触阻滞剂（ 如，CNQX，AP5，印防己毒素，筒箭毒碱等 ）和信道调制器（ 例如，氟吡汀，多巴胺，甲氯芬那酸等）新鲜上实验从冻结的股票一天。用稀释carbogenated林格氏液的药物。通过灌注以间歇方式应用它们。 </li><li>录制完成后，轻柔地从索玛吸管。从该室转移装配到一个干净的菜。分离和视网膜重新连接到另一个扁平硝酸纤维素膜上打孔不HOLES以固定期间保证视网膜平整度。 </li><li>通过在4％低聚甲醛浸渍在RT 30分钟固定的视网膜。取下硝酸纤维素膜在视网膜和用1×PBS广泛冲洗。由O / N染色用染料缀合的链霉在1×PBS稀释用0.4％的Triton X-100在4℃下14显示记录细胞的形态。在安装中防褪色视网膜和观察使用扫描共聚焦显微镜记录细胞。 注:涉及多聚甲醛，这是易挥发和有毒程序，应该在安全室草案进行。 </li></ol>

<ol>
	<li>Choi, H., 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=Intrinsic+bursting+of+AII+amacrine+cells+underlies+oscillations+in+the+rd1+mouse+retina.">Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.</a> J Neurophysiol. 112 (6), 1491-1504 (2014).</li><li>Gregg, R. G., 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=Nyctalopin+expression+in+retinal+bipolar+cells+restores+visual+function+in+a+mouse+model+of+complete+X-linked+congenital+stationary+night+blindness.">Nyctalopin expression in retinal bipolar cells restores visual function in a mouse model of complete X-linked congenital stationary night blindness.</a> J Neurophysiol. 98 (5), 3023-3033 (2007).</li><li>Hellmer, C. B., Ichinose, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+light-evoked+postsynaptic+responses+in+neurons+in+dark-adapted,+mouse+retinal+slice+preparations+using+patch+clamp+techniques.">Recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques.</a> J Vis Exp. (96), (2015).</li><li>Tu, H. Y., Bang, A., McQuiston, A. R., Chiao, C. C., Chen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+dendritic+branching+in+direction+selective+retinal+ganglion+cells+in+nob1+mice.">Increased dendritic branching in direction selective retinal ganglion cells in nob1 mice.</a> Invest Ophthalmol Vis Sci. 55 (13), (2014).</li><li>Tu, H. Y., Chen, Y. J., Chiao, C. C., McQuiston, A. R., Chen, C. K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+membrane+potential+fluctuations+of+cholinergic+amacrine+cells+in+mice+lacking+ERG+b-waves.">Rhythmic membrane potential fluctuations of cholinergic amacrine cells in mice lacking ERG b-waves.</a> Invest Ophthalmol Vis Sci. 56 (7), (2015).</li><li>Demas, J., 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=Failure+to+maintain+eye-specific+segregation+in+nob,+a+mutant+with+abnormally+patterned+retinal+activity.">Failure to maintain eye-specific segregation in nob, a mutant with abnormally patterned retinal activity.</a> Neuron. 50 (2), 247-259 (2006).</li><li>Tu, H. Y., Chen, Y. J., McQuiston, A. R., Chiao, C. C., Chen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Retinal+Oscillation+Mechanism+in+an+Autosomal+Dominant+Photoreceptor+Degeneration+Mouse+Model.">A Novel Retinal Oscillation Mechanism in an Autosomal Dominant Photoreceptor Degeneration Mouse Model.</a> Front Cell Neurosci. 9, 513 (2015).</li><li>Borowska, J., Trenholm, S., Awatramani, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intrinsic+neural+oscillator+in+the+degenerating+mouse+retina.">An intrinsic neural oscillator in the degenerating mouse retina.</a> J Neurosci. 31 (13), 5000-5012 (2011).</li><li>Margolis, D. J., Detwiler, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+origin+of+spontaneous+ganglion+cell+spike+activity+in+animal+models+of+retinitis+pigmentosa.">Cellular origin of spontaneous ganglion cell spike activity in animal models of retinitis pigmentosa.</a> J Ophthalmol. , (2011).</li><li>Stasheff, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+sustained+spontaneous+hyperactivity+and+temporary+preservation+of+OFF+responses+in+ganglion+cells+of+the+retinal+degeneration+(rd1)+mouse.">Emergence of sustained spontaneous hyperactivity and temporary preservation of OFF responses in ganglion cells of the retinal degeneration (rd1) mouse.</a> J Neurophysiol. 99 (3), 1408-1421 (2008).</li><li>Trenholm, S., Awatramani, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+spontaneous+activity+in+the+degenerating+retina.">Origins of spontaneous activity in the degenerating retina.</a> Front Cell Neurosci. 9, 277 (2015).</li><li>Trenholm, S., 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=Intrinsic+oscillatory+activity+arising+within+the+electrically+coupled+AII+amacrine-ON+cone+bipolar+cell+network+is+driven+by+voltage-gated+Na++channels.">Intrinsic oscillatory activity arising within the electrically coupled AII amacrine-ON cone bipolar cell network is driven by voltage-gated Na+ channels.</a> J Physiol. 590 (Pt 10), 2501-2517 (2012).</li><li>Chen, C. K. J., 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=Cell-autonomous+changes+in+displaced+cholinergic+amacrine+cells+lacking+Gbeta5.">Cell-autonomous changes in displaced cholinergic amacrine cells lacking Gbeta5.</a> Invest Ophthalmol Vis Sci. 56 (7), (2015).</li><li>O'Brien, B. J., Isayama, T., Richardson, R., Berson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+physiological+properties+of+cat+retinal+ganglion+cells.">Intrinsic physiological properties of cat retinal ganglion cells.</a> J Physiol. 538 (Pt 3), 787-802 (2002).</li><li>Lee, S., 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=An+Unconventional+Glutamatergic+Circuit+in+the+Retina+Formed+by+vGluT3+Amacrine+Cells.">An Unconventional Glutamatergic Circuit in the Retina Formed by vGluT3 Amacrine Cells.</a> Neuron. 84 (4), 708-715 (2014).</li><li>Hoggarth, A., 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=Specific+wiring+of+distinct+amacrine+cells+in+the+directionally+selective+retinal+circuit+permits+independent+coding+of+direction+and+size.">Specific wiring of distinct amacrine cells in the directionally selective retinal circuit permits independent coding of direction and size.</a> Neuron. 86 (1), 276-291 (2015).</li><li>Margolis, D. J., Gartland, A. J., Singer, J. H., Detwiler, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+oscillations+drive+correlated+spiking+of+ON+and+OFF+ganglion+cells+in+the+rd1+mouse+model+of+retinal+degeneration.">Network oscillations drive correlated spiking of ON and OFF ganglion cells in the rd1 mouse model of retinal degeneration.</a> PLoS One. 9 (1), e86253 (2014).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+retinal+preparation+to+record+light+response+from+genetically+labeled+retinal+ganglion+cells.">An isolated retinal preparation to record light response from genetically labeled retinal ganglion cells.</a> J Vis Exp. (47), (2011).</li><li>Ivanova, E., Yee, C. W., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+phosphorylation+of+Cx36+gap+junctions+in+the+AII+amacrine+cells+of+RD+retina.">Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina.</a> Front Cell Neurosci. 9, 390 (2015).</li><li>Enoki, R., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+horizontally+sliced+preparation+of+the+retina.">A method of horizontally sliced preparation of the retina.</a> Methods Mol Biol. 935, 201-205 (2013).</li><li>Akrouh, A., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+function+of+three+VIP-expressing+amacrine+cell+types+in+the+mouse+retina.">Morphology and function of three VIP-expressing amacrine cell types in the mouse retina.</a> J Neurophysiol. 114 (4), 2431-2438 (2015).</li><li>Wei, W., Elstrott, J., Feller, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+targeted+recording+of+GFP-expressing+neurons+for+light+responses+and+live-cell+imaging+in+the+mouse+retina.">Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.</a> Nat Protoc. 5 (7), 1347-1352 (2010).</li></ol>

The authors have nothing to disclose.

许多实验室已经从保利协鑫神经元记录台安装准备15-18，但我们的程序允许从INL神经元的记录。在此，我们强调的几个步骤是成功的日常记录的关键。 视网膜的新鲜度和平整度是有记录吸管穿透力很重要。在这方面，在视网膜的冲裁硝酸纤维素膜的牢固附着是非常重要的，并通过的溶液中，随后及时补液（步骤3.4-3.6）瞬态吸收实现的。在此短时间内，通常小于30秒，?...

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in Rho&#916;CTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.

<table><tbody><tr><td>Fixed-stage fluorescent microscope with DIC</td><td>Olympus</td><td>BX51-WI</td><td></td></tr><tr><td>Micromanipulators</td><td>Sutter</td><td>MP-225</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>A-M System</td><td>AM2400</td><td></td></tr><tr><td>AD converter</td><td>National Instrument</td><td>NI-USB-6221</td><td></td></tr><tr><td>Heater controller</td><td>Warner Instrument</td><td>TC-324B</td><td></td></tr><tr><td>Inline heater</td><td>Warner Instrument</td><td>SC-20</td><td></td></tr><tr><td>Peristaltic pump</td><td>Rainin</td><td>Dynamax</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instrument</td><td>P-1000</td><td></td></tr><tr><td>Glass tube with filament</td><td>King Precision Glass</td><td>Customized</td><td></td></tr><tr><td>Stimulator</td><td>A.M.P.I.</td><td>Master-8</td><td></td></tr><tr><td>Biocytin</td><td>Sigma</td><td>B4261</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S6191</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>BP328-1</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S0876</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S5011</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C5670</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M1880</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G6152</td><td></td></tr><tr><td>K-gluconate</td><td>Sigma</td><td>G4500</td><td></td></tr><tr><td>ATP-Mg</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>Li-GTP</td><td>Sigma</td><td>G5884</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E0396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Cs-methanesulfonate</td><td>Sigma</td><td>C1426</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Syringer filter</td><td>Nalgene</td><td>171</td><td></td></tr><tr><td>1 ml syring</td><td>Rainin</td><td>17013002</td><td></td></tr><tr><td>10 &amp;mu;l pipette tip</td><td>Genesee Scientific</td><td>24-130RL</td><td></td></tr><tr><td>Streptavidin-488</td><td>ThermoFisher</td><td>S-11223</td><td></td></tr><tr><td>10x PBS</td><td>Lonza</td><td>17-517Q</td><td></td></tr></tbody></table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

哺乳动物视网膜是多种神经元类型构成的分层组织。为了理解如何视觉信号的错综复杂突触网络内进行处理，电生理记录经常用于研究单个神经元之间的连接。我们已经优化了遗传标记的神经元在这两个GCL（神经节细胞层）和INL鼠标视网膜的膜片钳记录（内核层）一台安装准备。在平面支架记录INL神经元是有利的过度分片，因为纵向和横向连接在前者配置保护，这样，具有大的横向分量的视网膜电路进行研究。我们已经用这种方法来比较视网膜镜像神经元结成伙伴的反应，如胆碱能爆无长突细胞（评估中心）。

ON-和从deafferentated小鼠视网膜关型的SAC的代表性记录示于图1。在这两个GCL和INL胆碱能细胞可通过tdTomato荧光可靠地识别和DIC下针对全细胞膜片钳记录（ 图1A）以揭示其膜电位（顶部的痕迹），并推动它（底部的痕迹， 图1B）突触电流的振荡。抑制和兴奋性突触电流通过保持在分别为0毫伏和-75毫伏，单元，电压钳的条件（ 图1B，<...

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

此协议演示了如何从一台安装在准备视网膜神经元进行全细胞膜片钳记录。

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Research

JoVE Journal

Neuroscience

8.2K 次观看.  Baylor College of Medicine. 此协议演示了如何从一台安装在准备视网膜神经元进行全细胞膜片钳记录。 

视频: Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

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 ...

科研

神经科学

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 ...

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 ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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. ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

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 ...

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.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

摘要

探索更多视频

此视频中的章节

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

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

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

在完整的视网膜电生理特性表达GFP的细胞群

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

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

成年小鼠视网膜电生理研究水平切片的制备

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂