Name: implementing dynamic clamp with synaptic and artificial conductances in mouse retinal ganglion cells
Uploaded: 2013-05-16T13:00:00
Description: Watch this Scientific Journal Video about Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells at JoVE.com

这项工作是支持的澳大利亚研究理事会（ARC DP0988227）和生物医学研究计划资助的生物医学科学学科，悉尼大学。设备膜片钳放大器是由EPC 8的启动资金从悉尼大学生物医学科学学科。从Rebecca L.库珀从悉尼大学生物医学科学学科的基础和启动基金的资金购买设备InstruTech产品立8 +8的数据采集系统。我们想感谢匿名审稿人，他们有见地的建议和意见。

1。一般设置和组织准备<ol><li>按住鼠标在黑暗中30分钟（旨在减少其压力水平）。在等待时，准备外液1升。首先Milli-Q水半升碳酸氢钠1.9克溶解。通过鼓入95％O 2和5％CO 2，它的pH值保持在7.4。五分钟后，溶解8.8克艾姆斯中等Milli-Q水100毫升，加入碳酸氢钠溶液，Milli-Q水1升，拌匀。保持本方案的carboxygenated实验的其余部分。 </li><li>艾姆斯中carboxygenated 250毫升，并成立了流体灌注系?...

<ol>
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F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends in Neuroscience. 27, 218-224 (2004).</li><li>Williams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+compartmentalization+and+functional+impact+of+conductance+in+pyramidal+neurons.">Spatial compartmentalization and functional impact of conductance in pyramidal neurons.</a> Nature Neuroscience. 7 (9), 961-967 (2004).</li><li>Perkins, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-attached+voltage-clamp+and+current-clamp+recording+and+stimulation+techniques+in+brain+slices.">Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices.</a> Journal of Neuroscience Methods. 154 (1-2), 1-18 (2006).</li><li>Feng, S. S., Jaeger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+SK+calcium-dependent+potassium+currents+in+regulating+the+activity+of+deep+cerebellar+nucleus+neurons:+a+dynamic+clamp+study.">The role of SK calcium-dependent potassium currents in regulating the activity of deep cerebellar nucleus neurons: a dynamic clamp study.</a> Cerebellum. 7 (4), 542-546 (2008).</li><li>Butera, R., Lin, R., Destexhe, A., Bal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+factors+for+improving+dynamic+clamp+performance.+In:+Dynamic-Clamp+From+Principles+to+Applications+Series.">Key factors for improving dynamic clamp performance. In: Dynamic-Clamp From Principles to Applications Series.</a> Dynamic-Clamp From Principles to Applications Series: Springer Series in Computational Neuroscience. 1, (2009).</li><li>Marco, S. D. i., Nguyen, V. A., Bisti, S., Protti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+functional+reorganization+of+retinal+circuits+induced+by+early+long-term+visual+deprivation.">Permanent functional reorganization of retinal circuits induced by early long-term visual deprivation.</a> The Journal of Neuroscience. 29 (43), 13691-13701 (2009).</li><li>Zhao, Y., Inayat, S., Dikin, D. A., Singer, J. H., Ruoff, R. S., Troy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+technique:+review+of+the+current+state+of+the+art+and+potential+contributions+from+nanoengineering.">Patch clamp technique: review of the current state of the art and potential contributions from nanoengineering.</a> Proceedings of the Institution of Mechanical Engineers, Part N: Journal of Nanoengineering and Nanosystems. 222 (1), 1-11 (2009).</li><li>Lobb, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+NMDA+receptor+conductance+in+rat+midbrain+dopaminergic+neurons+using+the+dynamic+clamp+technique.">Application of a NMDA receptor conductance in rat midbrain dopaminergic neurons using the dynamic clamp technique.</a> J. Vis. Exp. (46), e2275 (2010).</li><li>Briggman, K. L., Euler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+electroporation+and+population+calcium+imaging+in+the+adult+mammalian+retina.">Bulk electroporation and population calcium imaging in the adult mammalian retina.</a> Journal of Neurophysiology. 105 (5), 2601-2609 (2011).</li><li>Middleton, T. P., Protti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+spontaneous+synaptic+activity+in+retinal+ganglion+cells.">Cannabinoids modulate spontaneous synaptic activity in retinal ganglion cells.</a> Visual Neuroscience. 28 (5), 393-402 (2011).</li><li>Pottek, M., Knop, G. 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这里，我们表明的使用，动态钳位评估的兴奋和抑制视网膜神经节细胞的输出的比值和相对定时的影响。动态钳利用计算机模拟引入生理记录或人工活体神经元的突触传导到。这种方法提供了一个互动的工具，通过它可以修改和电导注入神经元计算其影响神经元的反应。能够得到电导波形的视觉刺激实验中，用于激活光感受器的控制条件和药理学试剂的存在下在特定的细胞或电路隔离的贡献。注?...

在视网膜后面的眼睛里，是一个近乎透明的神经组织。许多研究使用视网膜的模型，以探讨视觉处理和突触信号机制的第一步。由于视网膜网络中的整个安装准备解剖后保持不变的，它代表了一个理想的系统研究突触的相互作用，作为其生理反应非常相似， 在体内条件。因此，使用一个孤立的视网膜神经元的特性，可以研究采用膜片钳技术（技术的评论，看到6,9,13）。的识别的确切贡献的具体电路和神经递质的神经节细胞的反应，但是，通常阻碍药理学试剂作用于各种网站。 视网膜神经元的生理反应轻，自然的刺激，可以记录充满了细胞内液与玻璃吸管。使用补丁CL可以记录膜电位的波动（电流钳）或电流（电压钳）放大器技术，神经元对光刺激的反应。通过在不同的电压和实施后验电导分析膜电位，它是未能隔离抑制和兴奋性突触输入5,12。这种类型的实验，可以进行正常沐浴介质中，并在不同的药理剂的存在下，以隔离的贡献不同的神经递质和受体的神经元的反应。从许多实验室的研究特征在于丰富的尖峰输出和兴奋性和抑制输入刺激属性，例如大小，对比度，空间和时间的频率，方向，方位和其他刺激变量的依赖。虽然这些实验方法提供尖峰输出和突触输入功能刺激物业之间的关系的信息，解释特定的细胞类型和细胞的兴奋性突触输入的贡献并不简单。这是由于这样的事实，通常兴奋性和抑制输入随刺激属性，因此，它是无法评估的确切影响，这些输入的变化对神经元扣球。 另一种方法来规避这些限制是开展动态钳记录，这也让个别突触输入输出扣球的贡献的关键评价。动态钳技术允许电流直接注入到单元格，在一个给定的时间依赖于电流注入量的记录，可在那个时候1,2,3（综述见7,14）的膜电位。它是一个修改后的电流钳设置在一个实时记录和下方的单元格的装置，其特征在于，包括专门的硬件，软件之间的相互作用，快速反馈和一台计算机来实现。电流注入到细胞的量的计算。因此，这种方法的优点是可以刺激细胞与电导波形的不同组合，其响应将模仿激活的受体介导的突触输入。例如，注射兴奋性和抑制的一个小点，响应注射兴奋性的一个小点的电导电导响应相比，只提供抑制对细胞响应的影响的信息。同样地，其他的组合生理记录的电导可以共同注入揭示了如何依赖于刺激兴奋性和/或抑制电导的变化会影响尖峰输出。 在我们的研究中，动态钳技术用于演示的相对幅度和时序上的发射性能的视网膜神经节细胞的突触输入的影响。各种电导在控制的条件下或在药理学试剂的存在下得到的生理实验作为输入。此外，人工电导基于阿尔法功能也被用于为了研究如何集成神经元突触输入。因此，这是一个多功能的技术，可以使产生的各种类型的电导生理，药理或计算被注入到相同的神经节细胞，所以这些输入的响应的比较可以。

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			<td>Reagent</td>
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			<td>Isoflurane Inhalation Anaesthetic</td>
			<td>Pharmachem</td>
			<td>&nbsp;</td>
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			<td>Ames Medium with L-Glutamate (Powder)</td>
			<td>Sigma-Aldrich</td>
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			<td>Potassium Gluconate, Anhydrous</td>
			<td>Sigma-Aldrich</td>
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			<td>HEPES Sodium salt</td>
			<td>Sigma-Aldrich</td>
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			<td>Magnesium chloride solution (4.9 mol/l)</td>
			<td>Sigma-Aldrich</td>
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			<td>Adenosine 5'-triphosphate (ATP) disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
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			<td>Guanosine 5'-triphosphate sodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
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			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid</td>
			<td>Sigma-Aldrich</td>
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			<td>Paraformaldehyde Powder, 95%</td>
			<td>Sigma-Aldrich</td>
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			<td>Anti-Lucifer Yellow, Rabbit IgG Fraction (3 mg/ml)</td>
			<td>Invitrogen</td>
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			<td>Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L) 2 mg/ml</td>
			<td>Invitrogen</td>
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			<td>Fluorescent Preserving Media</td>
			<td>BioFX Laboratories Inc.</td>
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			<td>Capillary Glass Tubing with flame polished ends (OD = 1.50 mm, ID = 0.86 mm, Length = 15 cm)</td>
			<td>Warner Instruments</td>
			<td>64-0794</td>
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			<td>Single Stage Glass Microelectrode Puller</td>
			<td>Narishinge Japan</td>
			<td>Model PP-830</td>
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			<td>Minipuls 2</td>
			<td>Gilson</td>
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			<td>Millex-GV 0.22 &mu;m Filter Unit</td>
			<td>Millipore Corporation</td>
			<td>SLGV004SL</td>
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			<td>Luer Lock Reusable Hypodermic Needle: 30 G</td>
			<td>Smith &amp; Nephew (Australia)</td>
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			<td>Single Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>Model SH-27B</td>
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			<td>Dual Automatic Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
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			<td>Olympus Stereomicroscope SZ61</td>
			<td>Olympus Corporation</td>
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			<td>Olympus Microscope BX50WI: with 40X objective</td>
			<td>Olympus Corporation</td>
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			<td>0-30 V 2.5 A DC Power Supply</td>
			<td>Dick Smith Electronics</td>
			<td>Q1770</td>
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			<td>Digital Microscopic Camera ProgResMF cool</td>
			<td>Jenoptik</td>
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			<td>Micromanipulator MP-225</td>
			<td>Sutter Instrument Company</td>
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			<td>Patch Clamp Amplifier EPC 8</td>
			<td>HEKA Elektronik</td>
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			<td>InstruTECH LIH 8+8 Data Acquisition System</td>
			<td>HEKA Elektronik</td>
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			<td>Computer: DELL</td>
			<td>Dell Corporation</td>
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implementing dynamic clamp with synaptic and artificial conductances in mouse retinal ganglion cells

神经节细胞输出的视网膜神经元，他们的活动体现了集成多个突触输入所产生的特定的神经回路。膜片钳技术，在电压钳和电流钳配置，常用来研究神经元的生理特性和表征其突触输入。虽然这些技术的应用是非常翔实，他们带来种种限制。例如，它是难以量化的兴奋性和抑制输入交互操作的精确确定响应输出。为了解决这个问题，我们使用了修改后的电流钳技术，动态钳，也称为电导钳1，2，3，检查的影响兴奋性和抑制性神经元兴奋性突触输入。这种技术需要电流注入到单元格，是依赖于在那个时候，其膜电位的实时反馈。 injecteD电流计算从预定的兴奋性和抑制性突触传导，其反转的潜力和细胞的瞬时膜电位。实验程序的详细信息，说明在这个视频文章膜片钳细胞的动力钳技术，实现了全细胞的结构和就业。这里，我们表明小鼠视网膜神经节细胞的反应的生理实验在控制的条件下或在药物的存在下得到的各种导波形。此外，我们使用人工使用alpha调查反应的细胞的功能产生的兴奋和抑制电导。

不同来源的神经节细胞的反应的抑制性输入的贡献表现通过应用各种电导波形。这些波形不同的亮度得到的刺激，在正常条件下和在TTX，电压门控Na +通道阻断剂的存在下，仅在一个子集，抑制视网膜中间块动作电位的产生。 图2A示出了有代表性的反应注射兴奋和抑制电导波形记录在刺激反应，在正常情况下在灰色背景上一个小黑点。当电导有一个黑色斑点的大小不同的波形?...

Watch this Scientific Journal Video about Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells at JoVE.com

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

此视频文章说明设置，程序，修补细胞组织，以及如何实现动态钳记录整个安装鼠标视网膜神经节细胞的。这种技术允许调查的兴奋性和抑制性突触输入精确的贡献，他们的相对幅度和时机神经扣球。

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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 ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

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

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. ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...