Name: wide-field single-photon optical recording in brain slices using voltage-sensitive dye
Uploaded: 2019-06-20T14:00:00
Description: Watch this Scientific Journal Video about Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye at JoVE.com

TT 获得 JSPS KAKENHI 赠款 (JP16H06532, JP16K21743、JP16H06524、JP16K0038 和 JP15K00413)来自 MEXT,卫生、劳动和福利部(MHLW-kagaku-ippan-H27 [15570760] 和 H30 [18062156])。我们要感谢编辑(www.editage.jp)的英语语言编辑。

所有动物实验均按照德岛邦里大学动物护理和使用委员会批准的规程进行。以下切片制备协议几乎是一个标准程序27,但修改是VSD染色和记录的协议。
1. 实验前的准备
<ol><li>准备股票A(表1),股票B(表2)和股票C(表3)溶液,并存放在冰箱中。</li>
	<li>准备1L的人工脑脊液(ACSF)(表4,见步骤3),并保存在冰箱中。</li>
	<li>?...

<ol>
	<li>Tominaga, Y., Taketoshi, M., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overall+Assay+of+Neuronal+Signal+Propagation+Pattern+With+Long-Term+Potentiation+(LTP)+in+Hippocampal+Slices+From+the+CA1+Area+With+Fast+Voltage-Sensitive+Dye+Imaging.">Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging.</a> Frontiers in Cellular Neuroscience. 12, 389 (2018).</li><li>Tominaga, T., Kajiwara, R., Tominaga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSD+Imaging+Method+of+Ex+Vivo+Brain+Preparation.">VSD Imaging Method of Ex Vivo Brain Preparation.</a> Journal of Neuroscience and Neuroengineering. 2 (3), 211-219 (2013).</li><li>Homma, R., 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=Wide-field+and+two-photon+imaging+of+brain+activity+with+voltage-+and+calcium-sensitive+dyes.">Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes.</a> Methods Mol Biol. 364 (1529), 2453-2467 (2009).</li><li>Grinvald, A., Hildesheim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSDI:+a+new+era+in+functional+imaging+of+cortical+dynamics.">VSDI: a new era in functional imaging of cortical dynamics.</a> Nature Reviews Neuroscience. 5 (11), 874-885 (2004).</li><li>Tasaki, I., Watanabe, A., Sandlin, R., Carnay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+fluorescence,+turbidity,+and+birefringence+associated+with+nerve+excitation.">Changes in fluorescence, turbidity, and birefringence associated with nerve excitation.</a> Proceedings of the National Academy of Sciences. 61 (3), 883-888 (1968).</li><li>Cohen, L., Keynes, R., Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Scattering+and+Birefringence+Changes+during+Nerve+Activity.">Light Scattering and Birefringence Changes during Nerve Activity.</a> Nature. 218 (5140), 438-441 (1968).</li><li>Hill, D., Keynes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opacity+changes+in+stimulated+nerve.">Opacity changes in stimulated nerve.</a> The Journal of Physiology. 108 (3), 278-281 (1949).</li><li>Waggoner, A., Salzberg, B., Davila, H., Cohen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Large+Change+in+Axon+Fluorescence+that+Provides+a+Promising+Method+for+Measuring+Membrane+Potential.">A Large Change in Axon Fluorescence that Provides a Promising Method for Measuring Membrane Potential.</a> Nature New Biology. 241 (109), 159 (1973).</li><li>Salzberg, B., Davila, H., Cohen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Recording+of+Impulses+in+Individual+Neurones+of+an+Invertebrate+Central+Nervous+System.">Optical Recording of Impulses in Individual Neurones of an Invertebrate Central Nervous System.</a> Nature. 246 (5434), (1973).</li><li>Cohen, L., lzberg, B., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Monitoring+Neuron+Activity.">Optical Methods for Monitoring Neuron Activity.</a> Annual Review of Neuroscience. 1 (1), 171-182 (1978).</li><li>Ross, W. 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切片生理学对于收集正确的信号至关重要。在本议定书中使用环膜过滤系统可确保切片在整个程序2、16、17中保持健康且不变形。其他系统可用于在记录期间保持切片生理学,但切片不应在任何时候变形,因为成像需要切片的每个部分都保持健康。环形膜过滤系统也更适合染色,因为这有助于我们最大限度地减少所需的染色溶液的?...

宽场单光子电压敏感染料(VSD)成像的散装染色脑切片制剂已成为一个有用的定量工具,以评估神经回路1,2,3,4的动态.在分析了膜激发5、6、7、VSD成像引起的光学特性变化后,科恩等人于20世纪70年代初首次描述了VSD成像。 9;当染料直接探测膜电位变化(即神经元的主要信号)时,它是实时监测大脑功能的合适方法。
最早的VSD具有理解大脑系统的理想特征,如快速时间常数,以跟踪神经元膜电位事件的快速动力学,以及线性随膜电位变化9,10,11,12,13,14,15.与其他成像实验类似,这种技术需要广泛的特定调谐,如照相机、光学、软件和切片生理学,以达到预期的结果。由于这些技术缺陷,对于大多数不专门从事这种技术的实验室来说,在初步努力期间的预期效益并不一定实现。
技术难度的主要原因是VSD在切片制剂的批量染色时对膜潜在变化的灵敏度较低。光学信号的大小(即荧光的分量变化)通常在生理条件下是控制(F0)信号的10-4-10-3。神经元膜电位变化的时间尺度约为毫秒到几百毫秒。为了测量神经元膜电位的变化,用于录制的摄像机应该能够获取高速(10 kHz 到 100 Hz)的图像。VSD 的低灵敏度和跟随神经信号所需的速度要求在高速的摄像机上收集大量光,并且具有高信噪比 (S/N)2、16。
记录系统的光学元件也是确保收集足够光线和改善S/N的关键元件。光学器件实现的放大倍率通常过低,例如 1 倍到 10 倍,以可视化局部功能神经回路。例如,为了可视化海马电路的动态,大约5的放大倍率是合适的。这种低放大倍率具有低荧光效率;因此,先进的光学设备将有利于这种记录。
此外,切片生理学也是必不可少的。由于成像分析要求切片完好无损,因此需要仔细处理切片。此外,为维持切片的可存活性而采取的措施,亦是重要的18。
本文介绍了切片制备、VSD 染色和测量的协议。本文还概述了对VSD、成像器件和光学器件的改进,以及对实验系统的其他改进,这些改进使这种方法能够用作一种简单、强大和定量的测定,用于可视化大脑功能的修饰19,20,21,22,23,24,25。该技术还可用于海马切片1的CA1区域的长期强权。此外,该技术在光记录单个神经细胞26的膜电位方面也很有用。

<table border="0" cellpadding="0" cellspacing="0" style="width:940px;" width="942">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">High speed image acquisition system</td>
			<td style="width:195px;">Brainvision co. Ltd.</td>
			<td style="width:353px;">MiCAM - Ultima</td>
			<td style="width:171px;">Imaging system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High speed image acquisition system</td>
			<td>Brainvision co. Ltd.</td>
			<td>MiCAM 02</td>
			<td>Imaging system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Macroscepe for wide field imaging</td>
			<td>Brainvision co. Ltd.</td>
			<td>THT macroscope</td>
			<td>macroscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High powere LED illumination system with photo-diodode stablilizer</td>
			<td>Brainvision co. Ltd.</td>
			<td style="width:353px;">LEX-2G</td>
			<td style="width:171px;">LED illumination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Image acquisition software</td>
			<td>Brainvision co. Ltd.</td>
			<td>BV-ana</td>
			<td>image acquisition software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multifunctional electric stimulator</td>
			<td>Brainvision co. Ltd.</td>
			<td>ESTM-8</td>
			<td>Stimulus isolator+AD/DA converter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Slicer</td>
			<td>Leica</td>
			<td>VT-1200S</td>
			<td>slicer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Slicer</td>
			<td>Leica</td>
			<td>VT-1000</td>
			<td>slicer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blade for slicer</td>
			<td>Feather Safety Razor Co., Ltd.</td>
			<td>#99027</td>
			<td>carbon steel razor blade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Membrane filter for slice support</td>
			<td>Merk Millipore Ltd., MA, USA</td>
			<td>Omnipore, JHWP01300, 0.45 &#181;m pores,</td>
			<td>membrane filter/ 0.45 13</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Numerical analysis software</td>
			<td>Wavemetrics Inc., OR, USA</td>
			<td>IgorPro</td>
			<td>analysing software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stimulation isolator</td>
			<td>WPI Inc.</td>
			<td>A395</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AD/DA converter</td>
			<td>Instrutech</td>
			<td>ITC-18</td>
			<td>AD/DA converter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Voltage sensitive dye Di-4-ANEPPS</td>
			<td>Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA</td>
			<td>catalog number: D-1199</td>
			<td>VSD: Di-4-ANEPPS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">poloxamer</td>
			<td>Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA</td>
			<td>Pluronic F-127 P30000MP</td>
			<td>poloxamer / Pluronic F-127 (20% solution in DMSO)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">polyethoxylated castor oil</td>
			<td>Sigma-Aldrich</td>
			<td>Cremophor EL C5135</td>
			<td>polyethoxylated castor oil</td>
		</tr>
	</tbody>
</table>

wide-field single-photon optical recording in brain slices using voltage-sensitive dye

宽场单光子电压敏感染料(VSD)成像脑切片制剂是评估神经回路功能连接的有用工具。由于光信号的分量变化,很难将这种方法用作定量测定。本文介绍了特殊的光学和切片处理系统,使该技术稳定可靠。本文详细介绍了VSD染色海马切片的切片处理、染色和记录。该系统长期保持生理条件,具有良好的染色,并防止切片在记录过程中的机械运动。此外,它还能用少量的染料染色切片。光学器件在低放大倍率下实现高数值光圈,从而能够以10 kHz的最大帧速率记录VSD信号,具有100像素x100像素的空间分辨率。由于帧速率和空间分辨率高,该技术允许应用后记录滤波器,提供充足的信噪比,以评估神经回路的变化。

图5显示了在小鼠海马切片的CA1区域对舍弗附带物进行电刺激时具有代表性的光信号。图 5A中的连续图像显示应用任何空间和时间滤波器之前的光信号,而图 5B显示应用 5 x 5 x 5 立方滤波器(高斯内核卷积、5 x 5 空间卷积和 5 到时间维度)两次。由于帧速率高(0.1 ms/frame)和高空间分辨率(100 像素 x 100 像素),滤波器...

Watch this Scientific Journal Video about Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye at JoVE.com

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

介绍了一种使用电压敏感染料的脑切片可重复、稳定的光学记录方法。本文介绍了电压敏感染料染色和记录光信号使用传统的海马切片制剂。

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

带电压敏感染料的光学记录方法被认为是一种理想的技术，可以直观地显示大脑是如何工作的。这种方法使我们能够记录神经心理动态稳定可靠地超过12小时，给我们一个心理活动的看法。我们正在努力扩展应用，以检测化学品在开发的所有阶段引起的电路变化，例如对母亲的宝贵酸应用。这项技术已经很成熟了。请尝试按照此处介绍的方法复制研究。要开始此过程，请将被斩首的头浸入冰冷的 ACSF 中，放在不锈钢手术托盘中。在一分钟内提取大脑，并放在一个烧杯中含有冷藏ACSF五分钟。接下来，用手术刀将中线的大脑部分分开，将两个半球放在4%的阿加块上。然后，将两个半球的大脑块放在 4% 的阿加块上。用滤纸擦拭块中多余的 ACSF。随后，将超级胶水应用到振动台。将胶块放在其上，然后用滤纸擦拭过多的粘合剂。轻轻地从脑粘滞块顶部轻轻涂抹少量冰冷ACSF，以帮助凝固多余的超级胶水，并防止胶水覆盖大脑和干扰切片。之后，将平台固定到振动器托盘并倒注修改后的 ACSF。将振动器设置为慢速，并且刀片频率达到其最大设置。然后开始切片。按顺序将切片放在切片室的一角，以便可以轻松区分切片的顺序。通常可以从一个半球获得三到五个切片。接下来，用30针切断脑干部分。使用小倾斜的油漆刷，将切片放在用有机玻璃环持有的膜过滤器上。将环放在潮湿的回收室中，并固定盖，以保持内部压力高。将切片放在膜过滤器上时，调整切片在环内的方向和位置，以确保其居中良好且具有一致的方向。将试样在28摄氏度下保存30分钟，然后在室温下至少保存10至30分钟。要染色切片，请轻轻将 100 微升染色溶液涂抹在每片上，并在 20 分钟内孵育室温。接下来，在容器中准备 50 到 100 毫升的 ACSF。将带试样环放在内以冲洗染色溶液。然后，将冲洗过的切片转移到另一个孵化室。在实验前等待一个多小时的恢复。要开始此过程，请打开放大器、计算机和摄像机系统并打开软件。将ACSF倒入50毫升的管子中，用碳原泡。使用近光泵循环 ACSF，将流量调整为每分钟约 1 毫升。然后，调整吸液器的高度，使实验室内的液位始终保持不变。将接地电极放在腔室中。随后，用少量ACSF填充玻璃电极，并放在电极支架中。将支架连接到操纵器的杆上。使用放大器，确保电极电阻约为 1 兆兆兆赫。在记录会话中，将一片从湿润室转移到实验室。将环的边缘牢固地压入硅胶 O 环中。小心不要破坏膜或实验室的底部。在显微镜下，将刺激电极的尖端和场位记录电极放在切片上。通过提供刺激来检查引起的反应。确认视场覆盖了光学记录系统中的右神经回路。接下来，将激发光强度调整到相机最大容量的 70 到 80%。使用荧光光源，使用采集系统调整焦点。然后，在采集后开始记录和分析图像采集软件中的数据。此图显示了小鼠海马切片CA1区域沙弗线子醇电刺激时具有代表性的光信号。这些连续图像显示光学信号之前，任何空间和时间过滤器应用，而这些图像显示相同的数据后，应用五五五立方滤波器两次。由于帧速率高、空间分辨率高，滤波器的应用没有改变信号，而是滤光了在单个试验中以像素为单位记录的时间过程中也可以观察到的噪声。以下是小鼠和大鼠之间海马切片CA1区域的典型反应的比较。在小鼠海马切片24毫秒后，在CA1远端观察到小的超极化响应，但更大规模的超极化响应超过了大鼠海马切片中的去极化响应。一旦切片被染色，他们应该持续超过12小时，你可以执行其他电生理录音。

Research

JoVE Journal

Neuroscience

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

科研

神经科学

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

区域选择性基因枪瞄准的器官脑片用改进的基因枪

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Whole-cell Patch-clamp Recordings in Brain Slices

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...