这项工作得到NIH NINDS授予R01 NS047557的支持。

<ol>
	<li>Sykova, E., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+in+brain+extracellular+space.">Diffusion in brain extracellular space.</a> Physiol Rev. 88 (4), 1277-1340 (2008).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+related+transport+mechanisms+in+brain+tissue.">Diffusion and related transport mechanisms in brain tissue.</a> Rep Prog Phys. 64 (7), 815-884 (2001).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-selective+microelectrodes+and+diffusion+measurements+as+tools+to+explore+the+brain+cell+microenvironment.">Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment.</a> J Neurosci Methods. 48 (3), 199-213 (1993).</li><li>Nicholson, C., Phillips, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+diffusion+modified+by+tortuosity+and+volume+fraction+in+the+extracellular+microenvironment+of+the+rat+cerebellum.">Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.</a> J Physiol. 321, 225-257 (1981).</li><li>Nicholson, C., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+space+structure+revealed+by+diffusion+analysis.">Extracellular space structure revealed by diffusion analysis.</a> Trends Neurosci. 21 (5), 207-215 (1998).</li><li>Xie, L. L., 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=Sleep+drives+metabolite+clearance+from+the+adult+brain.">Sleep drives metabolite clearance from the adult brain.</a> Science. 342 (6156), 373-377 (2013).</li><li>Hrabetova, S., Nicholson, C., Michael, A. C., Borland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+of+brain+extracellular+space+explored+with+ion-selective+microelectrodes,+integrative+optical+imaging+and+related+techniques.">Biophysical properties of brain extracellular space explored with ion-selective microelectrodes, integrative optical imaging and related techniques.</a> Electrochemical Methods for Neuroscience Neuroscience. , 167-204 (2007).</li><li>Rice, M. E., Okada, Y. C., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+and+heterogeneous+diffusion+in+the+turtle+cerebellum:+implications+for+volume+transmission.">Anisotropic and heterogeneous diffusion in the turtle cerebellum: implications for volume transmission.</a> J Neurophysiol. 70 (5), 2035-2044 (1993).</li><li>Vargova, L., 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=Diffusion+parameters+of+the+extracellular+space+in+human+gliomas.">Diffusion parameters of the extracellular space in human gliomas.</a> Glia. 42 (1), 77-88 (2003).</li><li>Haack, N., Durry, S., Kafitz, K. W., Chesler, M., Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-barreled+and+concentric+microelectrodes+for+measurement+of+extracellular+ion+signals+in+brain+tissue.">Double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue.</a> J Vis Exp. (103), (2015).</li><li>Xiao, F., Hrabetova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+extracellular+space+of+aquaporin-4-deficient+mice+does+not+enhance+diffusion+of+Alexa+Fluor+488+or+dextran+polymers.">Enlarged extracellular space of aquaporin-4-deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers.</a> Neuroscience. 161 (1), 39-45 (2009).</li><li>Sherpa, A. D., Pvan de Nes, ., Xiao, F., Weedon, J., Hrabetova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliotoxin-induced+swelling+of+astrocytes+hinders+diffusion+in+brain+extracellular+space+via+formation+of+dead-space+microdomains.">Gliotoxin-induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead-space microdomains.</a> Glia. 62 (7), 1053-1065 (2014).</li><li>Kume-Kick, 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=Independence+of+extracellular+tortuosity+and+volume+fraction+during+osmotic+challenge+in+rat+neocortex.">Independence of extracellular tortuosity and volume fraction during osmotic challenge in rat neocortex.</a> J Physiol. 542 (Pt 2), 515-527 (2002).</li><li>Saghyan, A., Lewis, D. P., Hrabe, J., Hrabetova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+diffusion+in+laminar+brain+structures+exemplified+by+hippocampus.">Extracellular diffusion in laminar brain structures exemplified by hippocampus.</a> J Neurosci Methods. 205 (1), 110-118 (2012).</li><li>Fedirko, N., Svichar, N., Chesler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+use+of+high-speed,+concentric+H+-+and+Ca2+-selective+microelectrodes+suitable+for+in+vitro+extracellular+recording.">Fabrication and use of high-speed, concentric H+- and Ca2+-selective microelectrodes suitable for in vitro extracellular recording.</a> J Neurophys. 96 (2), 919-924 (2006).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+from+an+injected+volume+of+a+substance+in+brain+tissue+with+arbitrary+volume+fraction+and+tortuosity.">Diffusion from an injected volume of a substance in brain tissue with arbitrary volume fraction and tortuosity.</a> Brain Res. 333 (2), 325-329 (1985).</li><li>Nicholson, C., Tao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindered+diffusion+of+high+molecular+weight+compounds+in+brain+extracellular+microenvironment+measured+with+integrative+optical+imaging.">Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging.</a> Biophys J. 65 (6), 2277-2290 (1993).</li><li>Thorne, R. G., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+diffusion+analysis+with+quantum+dots+and+dextrans+predicts+the+width+of+brain+extracellular+space.">In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space.</a> Proc Natl Acad Sci U S A. 103 (14), 5567-5572 (2006).</li><li>Wolak, D. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+macromolecules+in+the+brain:+implications+for+drug+delivery.">Diffusion of macromolecules in the brain: implications for drug delivery.</a> Mol Pharm. 10 (5), 1492-1504 (2013).</li></ol>

作者宣称他们没有竞争的经济利益。

<img alt="图10" class="xfigimg" src="/files/ftp_upload/55755/55755fig10.jpg" /> 图10:展示常见技术问题的非理想数据。 （ a ）离子电渗微电极的常见技术问题图:功能性离子电渗微电极的TMA正常释放与三个来源的技术问题的比较。 [高放大倍数，a1]理想的离子电渗源中的电流通过TMA释放和氯化物摄取而均匀地进行。 [高倍率，A2]一种离子电渗微电极具有低N t个释放更少TMA?...

<html>细胞外空间（ECS）是所有脑细胞外部相互通路的网络，包含间质液和细胞外基质（ 图1a和图1b ）。脑细胞功能所需的许多物质（包括营养素，激素和神经递质）的分布通过ECS扩散发生。在该空间中的物理参数，包括体积，几何形状和细胞外基质，变化可以显着地通过ECS和局部离子浓度洗澡脑细胞，从而对脑细胞功能1,2产生深远的影响影响扩散。 实时离子电渗疗法（RTI）被用于确定一个大脑区域的两个结构特点:体积分数和弯曲度3，4，"xref"&gt; 5。体积分数（ α ）是代表基础体积中ECS（ V ECS ）相对于总组织体积（ V 组织 ）占据的组织体积的比例; <img alt="方程" src="/files/ftp_upload/55755/55755eq1.jpg" />与没有障碍物的介质相比，Tortuosity（ λ ）是通过脑区域扩散时物质遇到的相对障碍; <img alt="方程" src="/files/ftp_upload/55755/55755eq2.jpg" />其中D * （cm 2 s -1 ）是物质在脑中的有效扩散系数， D （cm 2 s -1 ）是物质在游离介质如稀释琼脂糖凝胶中的自由扩散系数。 今天是R最常用的探针物质TI方法是小阳离子四甲基铵（TMA）。 TMA的分子量为74g / mol，在溶液中完全解离，并具有一个正电荷。这种离子的RTI研究已经证明α <img alt="方程" src="/files/ftp_upload/55755/55755eq3.jpg" /> 0.2和λ <img alt="方程" src="/files/ftp_upload/55755/55755eq3.jpg" /> 1.6 1,2。这意味着ECS大约是总体积的20％，并且小的惰性分子的扩散在ECS中比在没有障碍物3的介质中大约慢2.5倍。然而， α和λ随着脑年龄，区域和状态以及病理状况而变化1 。这些参数的改变与大脑发育，衰老，睡眠，癫痫以及脑中许多其他基本过程和疾病有关1， 6 。虽然其他技术测量α和λ ，RTI可以实时测量生物组织的局部区域。因此，RTI在急性和可逆性挑战中已成为调查α和λ变化的不可缺少的工具。 该理论支撑器R最初由尼克尔森和Phillips验证，并且该技术已经被自那时起4,7广泛使用。使用RTI的实验开始于通过离子电渗法从源微电极释放到脉冲TMA的稀释琼脂糖凝胶。一旦弹出，离子从点源自由扩散，从潜在的无限数量的随机路径中选择（ 图1d ）。使用大约定位的离子选择性微电极（ISM）随时间测量离子的变化浓度100米远（ 图1c ）。 TMA浓度的变化被绘制并适合于允许计算D和离子电渗微电极（在方案中讨论的参数）的输送数的曲线。利用这些值，在感兴趣的脑区域中重复该过程以获得D *并计算α和λ 。离子电渗微电极的控制，TMA浓度曲线的数据收集，绘图和拟合以及实验参数的计算都通常由专门为此目的而设计的程序Wanda和Walter完成（软件及其手册是可根据要求自由提供）。 本评论的"议定书"部分描述了在啮齿动物脑切片中设计和执行RTI所需的基本程序。该技术也被用于无杆ENT模型，包括人脑切片和体内脑制剂1，4，6，8，9。 "代表结果"部分提供理想和非理想的结果，以突出数据解释的细微差别。最后，讨论部分简要介绍了故障排除技术，RTI的限制，用于研究ECS的替代技术以及RTI的未来应用。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55755/55755fig1.jpg" /> 图1:ECS扩散图。 （ a ）ECS图:在典型的脑部分显示ECS的大小和位置。黄色标记了灰色脑细胞过程之间的ECS。 ECS的体积大约是总组织体积的20％（ 即，体积分数= 0。2）生理条件下。 （ b ）ECS的放大图:重点介绍有助于曲折的物理参数，包括脑细胞几何（灰色）和细胞外基质（图示为多色糖胺聚糖和蛋白多糖的网格）。 （ c ）从点源扩散的3D图:证明惰性分子从离子电渗源到ISM的净运动。排除扩散阻挡层和细胞摄取，分子在所有方向向外扩散，产生球形浓度前沿。 ISM量化从离子电渗源释放的惰性分子的局部浓度。 （ d ）大脑ECS中扩散的计算机模拟:[左侧]蒙特卡罗模拟的设置;绿色球体代表脑细胞过程，红色十字表示点源。这个设置模拟图1a所示的脑组织。 [中图] 3和6个分子在通过大脑的细胞外空间扩散时进行随机运动，如图2所示。 [远]从点源释放的许多分子的随机游走。来自点源的所有分子的净运动是向外的， 如图1c所示 。累积随机散点概述了单元格之间的空格（ 即 ECS;参见参考文献5进行进一步说明）。 <a href="http://ecsource.jove.com/files/ftp_upload/55755/55755fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

<table><tbody><tr><td>A/D and D/A converter</td><td>National Instruments Corporation</td><td>NI USB-6221 DAQ</td><td>The NI USB-6221 is still sold as a &#039;Legacy&#039; device by NI. They recommend using NI USB-6341 X Series DAQs for new installations, however we have not tested the newer units. We describe the use of the NI USB-6221 with MATLAB and Windows 7 (32-bit). Alternatives: the much older PCI-MIO-16E-4 A/D converter (Used under Windows XP or older OS only) with BNC-2090 BNC connector panel and SH68-68-EP cable. As noted in the Wanda Manual, an experimental MATLAB program to use Axon Binary Files is available.</td></tr><tr><td>agarose</td><td>Lonza</td><td>NuSieve GTG Agarose #50081</td><td>to prepare dilute agarose gel for RTI measurements</td></tr><tr><td>amplifier for ISM</td><td>Dagan</td><td>Model IX2-700 Dual Intracellular Preamplifier</td><td>ion and reference voltage amplifier with N=0.1 (for reference barrel) and N=0.001 (for ion barrel) headstages</td></tr><tr><td>biological compound miscroscope (with 4x and 10x objective)</td><td></td><td></td><td>for chipping the microelectrode tips and inspecting microelectrodes; various suppliers, &lt;em&gt;e.g.&lt;/em&gt; AmScope</td></tr><tr><td>borosilicate theta capillary glass tubing</td><td>Harvard Apparatus</td><td>Warner Instruments model TG200-4; order #64-0811</td><td>double-barreled glass tubing for ion-selective microelectrodes and iontophoretic microelectrodes; O.D. 2.0 mm, I.D. 1.4 mm, septum 0.2 mm, length 10 cm</td></tr><tr><td>brush</td><td>Winsor &amp;amp; Newton</td><td>University Series 233, size 0</td><td>round shoft handle brush, available from Amazon</td></tr><tr><td>bunsen burner</td><td>Fisher</td><td></td><td></td></tr><tr><td>camera for visualizing micropipettes</td><td>Olympus</td><td>OLY-150</td><td>requires monitor, IR filter on substage illuminator is optional</td></tr><tr><td>chart recorder</td><td></td><td></td><td>to record continuously voltages on ion-selective microelectrode during calibration in tetramethylammonium standards and during RTI experiment; &lt;em&gt;e.g.&lt;/em&gt; Kipp &amp;amp; Zonen type BD112 dual-cannel chart recorded, available refurbished</td></tr><tr><td>chlorotrimethylsilane, puriss., &gt; 99%</td><td>Sigma-Aldrich</td><td>catalog # 92360</td><td>for silanization; CAUTION: flammable, acute toxicity (oral, dermal, inhalation), skin corrosion, eye damage, reacts violently with water, see Sigma-Aldrich Safety Information for full description</td></tr><tr><td>Commercial Software</td><td>The MathWorks</td><td>MATLAB, Data acquisition toolbox</td><td>for data acquisition and analysis using Wanda and Walter programs. Note that an academic license is available.</td></tr><tr><td>eye protective goggles</td><td>Fisher</td><td></td><td></td></tr><tr><td>fixed-stage compound microscope</td><td>Olympus</td><td>BX51WI</td><td>can use other compound microscopes with fixed stages</td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>#11251-10</td><td>to chip glass capillary; Dumond #5, preferably used and no longer needed for fine work</td></tr><tr><td>fume hood</td><td></td><td></td><td>for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; various supliers, &lt;em&gt;e.g.&lt;/em&gt; Captair with approriate filter sold by Erlab</td></tr><tr><td>glass microscope slide</td><td>Fisher</td><td>#12-550A</td><td>to chip microelectrode tips</td></tr><tr><td>heater/stirrer</td><td>Fisher</td><td>Corning PC-420D</td><td>to prepare dilute agarose gel and stir solutions</td></tr><tr><td>iontophoretic unit</td><td>Dagan</td><td>ION-100 and PS-100</td><td>ION-100 is a single channel iontophoresis unit +/- 130 V compliance; PS-100 is an external power supply; alternatives: &lt;em&gt;e.g.&lt;/em&gt; Axoprobe-1A made by Axon Instruments (now Molecular Devices), out of production, check for availability of refurbished units (eBay and other sites)</td></tr><tr><td>liquid ion exchanger (LIX) for tetramethylammonium</td><td>World Precision Instruments</td><td>IE190 Potassium Ion Exchanger</td><td>Note: this is equivalent to the original Corning potassium exchanger 477317 based on tetraphenlyborate - do not confuse with neutral carrier potassium exchanger originating from the laboartory of Dr. Simon, ETH, Zurich, which does not sense tetramethylammonium, and is sold by Fluka. You can also make liquid ion exchanger for tetramethylammonium yourself: 3% by weight potassium tetrakis = (p-chlorophenyl) borate dissolved in 2,3-dimethylnitrobenzene. Buy chemicals from Fluka (now part of Sigma). See Oehme and Simon (1976) Anal. Chim. Acta 86: 21-25; CAUTION: The toxicological properties of this liquid ion exchanger have not been fully determined. Ingestion or contact with the human body may be harmful. Exercise due care! Liquid ion exchangers should be stored in a cool place out of direct sunlight.</td></tr><tr><td>microelectrode holder</td><td>WPI</td><td>M3301EH</td><td>to hold ion-selective microeletrode prefabricate for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; WPI sells two versions of this holder, clear M3301EH and black M3301EH. In our experience, the clear M3301EH appears to be sturdier then the black M3301EH.</td></tr><tr><td>micromanipulator</td><td>Narishige</td><td>MM-3</td><td>to position ion-selective microelectrode prefabricate during silanization and filling the tip of ion-selective barrel with liquid ion exchanger; can be substituted with any three-axis micromanipulator in good working condition</td></tr><tr><td>micropipette puller</td><td>Sutter Instruments</td><td>Model P-97</td><td>to pull double-barreled glass tubing; other pullers can be used as long as they can accommodate large diameter double-barreled glass tubing</td></tr><tr><td>microprobe thermometer</td><td>Physiotemp</td><td>Model BAT-12R</td><td>fine probe of this thermometer is placed close to recording site</td></tr><tr><td>needle</td><td>BD</td><td>Syringes and Needles # 305122 (25 gauge)</td><td>for silanization; BD PrecisionGlide needles 25 G x 5/8 in (0.5 mm x 16 mm)</td></tr><tr><td>objective 5X dry</td><td>Olympus</td><td>MPlan N</td><td></td></tr><tr><td>objective 10X water immersion</td><td>Olympus</td><td>UMPlan FL N</td><td>10X objective is water immersion, numerical aperture is 0.3, working distance is 3.3 mm</td></tr><tr><td>plastic containers (with lids)</td><td>Fisher</td><td>#14-375-148</td><td>to store tetramethylammonium standard solutions and microelectrodes</td></tr><tr><td>platform and x-y translation stage for fixed-stage microscope</td><td>EXFO</td><td>Gibraltar Burleigh</td><td>platform holds slice chamber, micromanipulators and accesorries, x-y translational stage moves microscope without compromising recording stability</td></tr><tr><td>porous minicup</td><td></td><td></td><td>for RTI measurements in a dilute agarose gel; homemade</td></tr><tr><td>reusable adhesive</td><td>Bostik</td><td>Blu-Tack</td><td>for securing microelectrodes to holding vessel and other uses; various suppliers, available from Amazon</td></tr><tr><td>robotic micromanipulator with precise x,y,z positioning</td><td>Sutter Instruments</td><td>MP-285</td><td>two mircomanipulators are needed to hold separately ion-selective microelectrode and iontophoretic microelectrode. Also possible to glue micropipettes in a spaced array (see text).</td></tr><tr><td>signal conditioning unit with low-pass filter</td><td>Axon Instruments</td><td>CyberAmp 320 or 380</td><td>no longer available from the manufacturer but may be available from E-Bay; alternatives: &lt;em&gt;e.g.&lt;/em&gt; FLA-01 Filter/Amplifier from Cygnus Technology. This is a single channel instrument with a minimum cutoff at 10 Hz using a multipole Bessel filter but the company may be willing to modify it for a lower cutoff frequency (2 Hz) if needed.</td></tr><tr><td>silver wire</td><td>A-M Systems</td><td>#7830</td><td>diameter 0.015&quot;, bare (no coating)</td></tr><tr><td>slice chamber</td><td>Harvard Apparatus</td><td>Warner Model RC-27L</td><td>this is submersion slice chamber; do not use interface slice chamber</td></tr><tr><td>stereomicroscope</td><td></td><td></td><td>for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; horizontally mounted; various suppliers</td></tr><tr><td>syringe, 10 mL</td><td>BD</td><td>Syringes and Needles #309604</td><td>to backfill microelectrodes and for silanization; BD Luer-Lok tip</td></tr><tr><td>syringe filter 0.22 &amp;micro;m pore</td><td>Whatman</td><td>#6780-1302</td><td>to filter backfill solutions; available from Fisher</td></tr><tr><td>syringe needle, 28 gauge, 97mm</td><td>World Precision Instruments</td><td>MicroFil MF28G-5</td><td>to backfill microelectrodes</td></tr><tr><td>Teflon (=PTFE) tubing</td><td>Component Supply</td><td>STT-28 PTFE tube light wall (28 gauge)</td><td>for silanization of ion-selective barrel; fits on BD PrecisionGlide needles 25 G x 5/8 in. Note: Teflon is essential, PVC tubing would melt by hot wax.</td></tr><tr><td>temperature control system</td><td>Harvard Apparatus</td><td>Warner Models TC-344B and SH-27A</td><td>TC-344B is a dual automatic temperature controller, SH-27A is an in-line heater; controller and heater work with Warner slice chambers</td></tr><tr><td>tetramethyammonium (TMA) chloride</td><td>Sigma-Aldrich</td><td>T-3411</td><td>5 M solution; CAUTION: acute toxicity (oral, dermal, inhalation), carcinogenicity, hazardous to the aquatic environment, see Sigma-Aldrich Safety Information for full description</td></tr><tr><td>vibrating blade microtome</td><td>Leica</td><td>VT1000S</td><td>to cut brain slices</td></tr><tr><td>xylenes</td><td>Fisher</td><td>X5-1</td><td>for silanization; CAUTION: flammable, acute toxicity (oral, dermal, inhalation), skin corrosion, eye damage, carcinogenicity, see Fisher Safety Information for full description</td></tr></tbody></table>

real-time iontophoresis with tetramethylammonium to quantify volume fraction and tortuosity of brain extracellular space

该评论描述了执行实时离子电渗（RTI）方法的基本概念和方案，探索和定量活体大脑的细胞外空间（ECS）的金标准。 ECS围绕所有脑细胞并且包含间质液和细胞外基质。大脑活动所需的许多物质的运输，包括神经递质，激素和营养物质，都是通过ECS扩散发生的。这种空间的体积和几何形状的变化发生在正常的脑部过程，如睡眠和病理状况，如缺血。然而，大脑ECS的结构和调节，特别是在患病状态，仍然在很大程度上未被探索。 RTI方法测量活体大脑的两个物理参数:体积分数和曲折度。体积分数是ECS占据的组织体积的比例。柔软度是衡量物质在通过脑部扩散时遇到的相对阻碍与没有障碍物的介质相比。在RTI中，惰性分子从源微电极脉冲到脑ECS中。当分子从该源扩散时，使用离子选择性微电极定位大约100微米，离子的浓度随时间变化。从所得扩散曲线可以计算体积分数和曲折度。这种技术已被用于来自多个物种（包括人类）的脑切片和体内研究ECS的急性和慢性变化。与其他方法不同，RTI可以用于实时检测脑ECS的可逆和不可逆变化。

RTI技术的实用性在设计用于测量α和低渗摩洛尔挑战期间变化的实验中得到证明（ 图8和图9 ）。先前已经表明，降低了ECS的渗透压通过洗涤上低渗ACSF将产生α的降低，并增加了λ13。 在该实验中，在两个对照条件下和在低渗ACSF的洗涤期间对大?...

Watch this Scientific Journal Video about Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space at JoVE.com

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

该方案描述了实时离子电渗疗法，一种测量活体大脑细胞外空间（ECS）物理参数的方法。释放到ECS中的惰性分子的扩散用于计算ECS体积分数和曲折度。它是研究脑ECS急性可逆变化的理想选择。

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

real-time-iontophoresis-with-tetramethylammonium-to-quantify-volume

Research

JoVE Journal

Neuroscience

11.9K 次观看.  University of Virginia. 该方案描述了实时离子电渗疗法，一种测量活体大脑细胞外空间（ECS）物理参数的方法。释放到ECS中的惰性分子的扩散用于计算ECS体积分数和曲折度。它是研究脑ECS急性可逆变化的理想选择。 

视频: Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

科研

神经科学

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脑组织中的双管和同心微电极用于测量细胞外离子的信号

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

使用路西法黄电磷学可视化星形形态

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

绘制脑血流和电场图:小鼠的电极放置

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

作者聚焦:基于刺激的方法改善阿尔茨海默病模型中的脑血流 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

摘要

探索更多视频

此视频中的章节

谷氨酸和GABA快速微离子导入：一个有用的工具，调查突触整合

方法以使对新生儿鼠害的腹头骨开颅手术

脑组织中的双管和同心微电极用于测量细胞外离子的信号

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

一种基于微 ct 的小动物脑损伤和定位电极表征方法

使用路西法黄电磷学可视化星形形态

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

摘要

探索更多视频

此视频中的章节

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

方法以使对新生儿鼠害的腹头骨开颅手术

脑组织中的双管和同心微电极用于测量细胞外离子的信号

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

一种基于微 ct 的小动物脑损伤和定位电极表征方法

使用路西法黄电磷学可视化星形形态

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

谷氨酸和GABA快速微离子导入：一个有用的工具，调查突触整合