Name: double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue
Uploaded: 2015-09-05T14:00:00.000+00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

作者感谢C.罗德利哥专家技术援助。我们感谢S.·克勒（高级影像中心，海涅大学杜塞尔多夫），用于视频制作的帮助。研究作者的实验室已经由德国研究协会（DFG:罗2327 / 8-1到CRR）时，海涅大学杜塞尔多夫（以NH）和美国国立卫生研究院授予R01NS032123（以MC）。

<ol>
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The authors declare no competing interests.

液舰载，离子选择性电极已被成功地使用了几十年，对许多离子，高度特异性的传感器可用22-26。当在脊椎动物脑制剂外空间（ECS）中使用，必须牢记，然而，这是一个相当侵入性技术:当ECS的宽度大约只有20-50纳米，直径离子选择性微电极约1微米（双管电极）或更大（同心电极）。的离子选择性微电极的前端将因此不仅在组织的其穿刺期间损伤组织，而且还扩大了ECS，有利于离子瞬变的低估。尽管有这些缺陷，响应于神经元活性细胞外离子瞬变不同实验室7,8，证明该方法的可靠性之间非常一致。 的离子选择性电极的性能和适用性取决于他们的灵敏度和选择性，这是由所用的传感器的鸡尾酒（&#39;液膜离子载体"）中所定义。传感器鸡尾酒含有一种特殊载体分子，例如缬氨霉素为K +选择性微电极显示高选择性钾27。尽管如此，交叉反应性的其它离子，可能会发生，并且必须进行测试。缬氨霉素表现出显著交叉反应性为铵，其具有解释结果时，需要考虑（例如11,12）。此外，由于离子载体的电压 - 响应遵循能斯脱行为（参见等式1），所述信噪比和检测阈值依赖于离子的浓度进行测量。因此，尽管小[K +] 0瞬变唤起对低基线K + 0，小[Na +]为大的电压变化Ø瞬变更难检测对喜GH基线[Na +]为O（ 参见图5和6）。 的离子选择性电极的性能也由时间分辨率，这在很大程度上是由它的电气时间常数支配确定。后者主要由传感器的轴向阻力来确定，并通过沿所述吸移管的长度的分布电容，其内部的解决方案与外部流体之间。在双管结构中，电阻高，由于回填离子传感器的长列。对于给定的绝缘电介质（在这种情况下，硼硅玻璃），该电容由电介质厚度决定。在双管的电极，在电介质宽度达移液管的玻璃墙。作为玻璃变薄接近尖端，电介质宽度下降，电容增大。这些因素结合起来，以产生具有响应时间电极，范围从几百毫秒到几秒，因为这些因素是多种多样的。 同心设计的一个主要优点是，无论是轴向电阻和电容，以浴都大大减少。同心吸管分流大部分的回填离子交换剂的阻力，而使在顶端之前的最后几微米只残余。此外，同心吸管内的填充液在物理上从浴隔开，由两个玻璃墙的厚度隔开，大大减少了电容。如前面所示10，降低的电阻和电容的共同影响是在两个数量级的时间分辨率的提高。在同心的 Ca 2+和pH微电极的情况下，90％的响应时间分别为低10-20毫秒10。同心设计的一个相关的优点是较低的噪声电平（参看图8）。从任何安眠药由于大大降低了电阻，电压瞬变T个噪声最小化。此外，从这些瞬态恢复是因为快速的时间常数迅速。这些文物是因此小而快，对生理记录（参见图8）不太破坏性影响。 也有同心技术的缺点。首先，它们的装配是较复杂的，并且费时。第二个缺点是需要放置一个单独的参考微电极以其尖端，将会导致使用的任一单独的显微或专用双操纵器。最后，双管的微电极可以扩展到一三管设计，允许检测两种不同的离子种的同时28，这是不可能的同心电极。 最常见的陷阱低效的硅烷化。 最重要的一步，并且在任何液体SENS制造主要障碍或基于离子选择性微电极是硅烷化过程。当电极不能响应的变化特定离子浓度，或与子能斯特响应（每10倍浓度差即井小于58毫伏）反应，硅烷化的疗效差是通常的原因。根据我们的经验，这可能，如果发生大气湿度过高或过低，一般的条件夏天，还是冬天的高度分别。如果可行施加过室内湿度一定的控制，这些问题可以克服。 电极电阻过高。 如果需要的话，在离子敏感圆筒的阻力可以通过斜削降低。为此，揭露其尖端的磨料悬浮在水中的几秒钟强大的喷射。这将导致其至上尖折断，降低到所期望的值的电阻。 盐桥。 盐的离子和参考桶之间的桥梁造成不良或无响应的电极，因此可以也大大变乱他们在校准性能。如上所述（见点1.6），这主要是在双管玻璃THETA选择的问题，而是一种罕见的发生使用此处描述的偏移，扭曲桶技术时。 轻松制作的初衷，往往可以有利地使用力士29的原双管设计。这种方法利用预填的离子和参考桶与盐溶液，一个快速曝光到由从前端吸气和驱逐硅烷溶液，通过离子交换剂掺入以下，也通过所述尖端（见30,31） 。这些电极可以制造在约10分钟，但它们的针尖大小一般为4微米或更多，他们更容易的实验过程中失败。与此相反，硅烷化的方法涉及暴露于硅烷蒸汽和热荷兰国际集团可以生产出更小的技巧，最后几天，有时几周电极。 综合起来，有几个协议和关于如何准备离子选择性微电极的方法。在这里，我们已经描述了两个主要程序的扭曲这在我们的实验室工作良好，可靠的双管以及同心微电极制造中，以接近100％的成功率。重要的是，这些技术将是转移到其他离子物种，包括pH或钙的测量，也将适用于其他制剂比脑，包括在一般流体填充腔或流体。最后，但并非最不重要，离子选择性微电极能够确定细胞内的离子浓度。因为它们的相对大的针尖大小（〜1微米）的，这将然而，很可能只在细胞与大细胞体，例如如在无脊椎动物制剂28,32找到。

An author's middle initial was omitted from the publication, <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/53058/double-barreled-concentric-microelectrodes-for-measurement">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue</a>. The author&#39;s name has been updated to:
Christine R. Rose
from:
Christine Rose

A correction was made to Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

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

Electrical signaling in the brain is based on the flux of ions across plasma membranes. Major ion movements into and from the extracellular space are not only mediated by passage through voltage-gated ion channels, but also by postsynaptic ionotropic receptors as well as ion transporters. Neuronal activity is thus accompanied by complex changes in the extracellular concentration of all major ions 1. For example, influx of sodium into neurons during excitatory activity has been shown to result in a decrease in the extracellular sodium concentration ([Na+]o) 2. The same holds true for the extracellular calcium concentration because calcium ions rapidly enter both pre- and postsynaptic structures 3. At the same time, potassium moves the opposite way and this mediates an increase in the extracellular potassium concentration ([K+]o) in the low mM range 4,5. Synaptic activity also causes changes in extracellular pH that are partly mitigated by concomitant glial membrane fluxes that change intraglial pH 6,7. These activity-related changes in extracellular ion concentrations have significant functional consequences. For example, even small increases in [K+]o depolarize neurons as well as glial cells thereby altering neuronal excitability, and several mechanisms exist to remove excess potassium 8. Failure of these may result in epileptiform activity of neurons or phenomena like spreading depression 1. 
Because of their critical importance, quantitative determination of extracellular ion concentrations is often necessary and required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. For decades, double-barreled ion-selective microelectrodes have proven to be excellent tools for such measurements in brain tissue 9. For many ions, highly specific sensors with low cross-reactivity for other ions are available. In addition to the classical double-barreled electrodes, so-called concentric electrodes were recently introduced. The latter provide a superior time resolution, but take a little more time and effort to construct 10. 
In the following, we will describe the preparation and calibration of these two types of ion-selective microelectrodes. We then show how these electrodes can be employed in brain slice preparations for measurement of changes in [K+]o or [Na+]o induced by excitatory activity following different stimulation paradigms including bath and pressure application of drugs.

<table><tbody><tr><td>Abrasive&amp;nbsp;&amp;nbsp;</td><td>MicroPolish&amp;nbsp;</td><td>Buehler GmbH</td><td>Dissolved in A.dest</td></tr><tr><td>Borosilicate-glass capillaries</td><td>1405059</td><td>Hilgenberg</td><td>Application pipette; 75 mm x 2 mm, wall thickness 0.3 mm</td></tr><tr><td>Borosilicate glass capillaries with filament</td><td>GC 150 F-15&amp;nbsp;</td><td>Clark Electromedical Instruments, Harvard Apparatus</td><td>For the sensor of double-barreled microelectrodes&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillaries with filament</td><td>GC100-F-15</td><td>Clark Electromedical Instruments, Harvard Apparatus</td><td>For the reference of double-barreled microelectrodes&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillaries with filament</td><td>GB-200TF-15&amp;nbsp;</td><td>Science Products</td><td>Concentric, outer channel. O.D. 2.0 mm&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillaries with filament</td><td>GB-120TF-10</td><td>Science Products</td><td>Concentric, inner channel. O.D. 1.2 mm</td></tr><tr><td>Digidata</td><td>1322A</td><td>Axon Instruments</td><td></td></tr><tr><td>Electrometer amplifier with headstage</td><td></td><td>Custom-made</td><td>Rin = 10 T&amp;Omega; and Ibias=50 fA-1pA (commercially available&amp;nbsp; alternatives: &lt;i&gt;e.g.&lt;/i&gt; Dagan IX2-700, with headstage (10 Gig feedback resistor) or EPMS-07, NPI, Tamm, Germany)</td></tr><tr><td>Experimental chamber</td><td></td><td>Custom-made</td><td>Commercially available from &lt;i&gt;e.g.&lt;/i&gt; &amp;nbsp; Warner Instruments,USA;&amp;nbsp; Scientifica, UK</td></tr><tr><td>Furnace</td><td></td><td>Heraeus</td><td>Must stay constant at 200 &amp;deg;C</td></tr><tr><td>Hard sticky wax / dental wax&amp;nbsp;</td><td>Deiberit 502&amp;nbsp;</td><td>Siladent Dr. Boehme &amp;amp; Schoeps GmbH</td><td></td></tr><tr><td>Hot plate</td><td></td><td>Custom-made</td><td>Must stay constant at 40 &amp;deg;C</td></tr><tr><td>Microelectrodes holder made of plexiglas</td><td></td><td>Custom-made</td><td>Double-barreled: O.D. &amp;nbsp;capillaries 1.5 mm, concentric: O.D.&amp;nbsp; capillaries 2 mm</td></tr><tr><td>Micromanipulator</td><td></td><td>Leitz</td><td></td></tr><tr><td>Micromanipulator</td><td>MD4R</td><td>Leica</td><td></td></tr><tr><td>Stereo microscope</td><td>M205C</td><td>Leica</td><td></td></tr><tr><td>Objective</td><td>Plan 0.8xLWD</td><td>Leica</td><td></td></tr><tr><td>Pipette puller</td><td>Model PP-830</td><td>Narishige</td><td>Concentric microelectrodes&amp;nbsp;</td></tr><tr><td>Pipette puller</td><td>Model P-97</td><td>Sutter Instruments&amp;nbsp;</td><td>Sensor of concentric microelectrodes&amp;nbsp;</td></tr><tr><td>Pneumatic drug ejection system</td><td>Picospritzter Type II</td><td>General Valve TM Corporation</td><td></td></tr><tr><td>Travel dovetail stage</td><td>DT 25/M</td><td>Thorlabs</td><td></td></tr><tr><td>Two-component glue</td><td>&amp;nbsp;Araldite</td><td>Huntsman advanced materials GmbH</td><td>One may also use a small stripe of aluminum foil to stick the capillaries together&amp;nbsp;</td></tr><tr><td>Silverwire</td><td>99.9%&amp;nbsp;</td><td>Wieland Edelmetalle</td><td></td></tr><tr><td>Slicer / Vibratome</td><td>Microm HM 650 V</td><td>Thermo Scientific</td><td></td></tr><tr><td>Software</td><td>AxoScope 8.1&amp;nbsp;</td><td>Axon Instruments</td><td></td></tr><tr><td>Vertical puller&amp;nbsp;</td><td>Type PE-2</td><td>Narishige Scientific Instruments</td><td>With a revolvable chuck for&amp;nbsp; double-barreled microelectrodes</td></tr><tr><td>x/y translational stage</td><td></td><td>Custom-made</td><td></td></tr><tr><td>1(S),9(R)-(&amp;minus;)-Bicuculline methiodide</td><td>Sigma aldrich</td><td>14343</td><td>Competitive antagonist of GABAA receptors (light-sensitive); CAUTION toxic</td></tr><tr><td>CNQX</td><td>Sigma aldrich</td><td>C-127</td><td>AMPA/kainate receptor antagonist; CAUTION toxic</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma aldrich</td><td>D5879</td><td></td></tr><tr><td>DL-AP5</td><td>Alfa Aesar</td><td>J64210</td><td>NMDA receptor antagonist; CAUTION toxic</td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Sigma aldrich</td><td>440191</td><td>CAUTION: Flammable, acute toxicity (oral, dermal, inhalation) and corrosive to metals and skin</td></tr><tr><td>L-Aspartic acid</td><td>Sigma aldrich</td><td>A9256</td><td>Activates NMDA and non-NMDA and EAATs</td></tr><tr><td>L-Glutamic acid monosodium salt hydrate</td><td>Sigma aldrich</td><td>G1626</td><td>Activates NMDA-R, AMPA-R, QA-R and KA-R),&amp;nbsp; mGluRs and EAATs</td></tr><tr><td>Potassium ionophore I - cocktail B</td><td>Fluka&amp;nbsp;</td><td>60403</td><td>Based on valinomycin; CAUTION toxic</td></tr><tr><td>Sodium ionophore II - cocktail A</td><td>Fluka&amp;nbsp;&amp;nbsp;</td><td>71178</td><td>Based on ETH 157</td></tr><tr><td>TTX</td><td>Ascent Scientific</td><td>Asc-055</td><td>Inhibitor of voltage-dependent Na+ channels; CAUTION toxic</td></tr><tr><td>Water, ultra pure</td><td>Sigma aldrich</td><td>W3500&amp;nbsp;</td><td></td></tr></tbody></table>

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.

<html>为了监测K + O和其中的变化响应兴奋的活动，双管钾选择性微电极插入到海马CA1区的辐射层 。穿刺几分钟后，电极的离子选择性桶达到稳定的基线（图7A）中，对应于一个K + 邻约2.8毫米，值接近所使用的ACSF的[K +]（ 2.5毫米）。为0.5mM谷氨酸浴应用10秒诱发[K +]的可逆增加ö由约4mM，接着下面的基线下冲达〜0.7毫米（ 图7A）。降低谷氨酸浓度为0.4％，0.3％和0.2毫引起相应减少两者的暂时增加，并在K + O（ 图7A）的下冲的幅度。 OAD / 53058 / 53058fig7.jpg"/&gt; 图响应于兴奋性活性7.胞外钾瞬变。（A）的[K +] 0瞬变，由增加接着下面基线下冲，诱发不同浓度谷氨酸盐下10秒的浴应用所指示的。上[K +] 0瞬变诱导0.5mM的谷氨酸浴应用10秒（B）一种灌注的影响与谷氨酸受体阻断剂（CNQX，100μM; APV，100μM）和河豚毒素（0.5μM的河豚毒素）。 （ 三）自发[K +] 0瞬变为0mg 2+ / BIC的存在。在交流中所示的实验用双管的电极进行。为了说明的目的，痕迹平滑用Sawitzky-格雷滤波器（宽度20）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">请CL</a>ICK此处查看该图的放大版本。 早期实验表明，这样的谷氨酸诱导的[K +] 0信号不显著应用河豚毒素的期间改变，并因此对电压门控钠通道和神经动作电位产生15,16的开口基本上独立。为了研究基础响应于谷氨酸观测[K +] 0变化的机制，我们采用谷氨酸受体阻断剂，即CNQX（100μM; AMPA受体的阻断剂）和APV（100μM; NMDA受体的阻断剂）在存在TTX（0.5μM）。洗澡后灌注与这些受体阻滞剂，谷氨酸诱导[K +] 0变化是几乎取消，确认其对离子型谷氨酸受体通道之前报道过的开幕依赖（ 如 17; 图7B）。 为了进一步证明牛逼对于外代他谷氨酸激励的相关性[K +]的信号，切片灌注名义上镁离子含有10μM荷包牡丹碘化物（0MG 2+ / BIC） -免费的盐水。这减轻NMDA受体的电压依赖性的Mg 2+ -嵌段和通过阻断GABA A受体，引起自发复发性癫痫样活动在网络中（例如，18,19）衰减抑制。正如预期的20，自发性，反复发作[K +] 0瞬变，金额约1.5毫米，为0mg 2+ / BIC盐水（ 图7C）进行检测。在这些反应之间，更小的[K +] 0瞬变约0.2的平均幅度毫米发生（ 图7C）。 在第二组实验中，我们使用了[Na +]为敏感的微电极来确定应用程序的诱发[钠+] 0的变化谷氨酸受体激动剂。在这里，我们也比较了采用两种不同的应用范式的双管和同心微电极的响应特性。 [Na +]为敏感的微电极定位在辐射层在约50微米的深度。经过一个稳定的基线达到，谷氨酸受体激动剂L-天门冬氨酸是每浴灌注（10毫米，120秒，浴灌注2.5毫升/分钟）应用。正如前面21观察，应用天冬氨酸了约15毫米，持续约5分钟，然后开始恢复到基线（ 图8A）造成一个缓慢的下降[钠+] 0。值得注意的是，在由两电极确定的[Na +]为O信号的峰值幅度和动力学是在这些条件下几乎相同，同心电极表现出更加稳定的基线和下的噪音水平（ 图8A）。 为了测试第r<html>下一个更快速的应用范例esponse的不同电极的特点，我们从离子选择性微电极的末端压力施加谷氨酸通过定位在辐射层细玻璃吸管在20-40微米的距离。如所预期的21，应用谷氨酸（10毫米）200毫秒引起的[Na +]为O（ 图8B）产生的压降。的[Na +]为Ø减少峰值幅度均在同一范围内对两种类型的电极（双管:4.5 - 13.5毫米，N = 14;同心:2.0 - 19.1毫米，N = 15）。然而，与此相反，以与慢浴灌注（见上文），不仅信噪比，而且通过这两种类型的电极检测出的[Na +]为O信号的时间过程中获得的结果显著不同。的平均时间峰值为3.5秒，双管，只有1.3同心微电极秒。 ve_content"&gt;因此，这些结果表明并确认的同心对双管的 Na +选择性微电极的更快的响应动力学，（参见图6C和图8B），由于还指出为钙离子和pH选择性同行10与此相反，前者的研究，其中短脉冲串突触刺激进行唤起快速突触诱导离子瞬变，只有一种倾向，但与我们的应用同心和双管电极之间的平均峰值幅度没有显著差异范例。这可能是由于这样的事实，该应用程序吸管从通过两个（20-40微米）的一个因素而变化的离子选择性微电极的前端，并因此的距离，即最大谷氨酸浓度在靶区域是不是在不同的实验一样，阻碍在峰值幅度的任何现有的差别。 （A）上部的曲线变化的沃尔特的双管电极（黑色虚线曲线）和一个同心电极（灰色曲线）响应于变化中的浴的 Na +浓度的参考桶（V REF）戈（[Na +]为二）所指示的。注意，这两种电极的电压响应几乎是一致的。底迹线:一个双管电极（黑色迹线），并且响应于变化的[Na +]为B A同心电极（灰迹线）的变化中的Na +的ζ-电势（V 的Na +）的电压。 （B）中的[Na +]为的半对数曲线B对两个电极的在V 的Na +。该数据的线性图揭示了大约48毫伏两个电极的斜率。 （C）响应双管（黑色线）的在V 的Na +和同心电极（灰迹），以快速变化中的[Na +]为b从 152毫米到70毫米。需要注意的是同心电极的响应时间是显著FAS之三。为了说明的目的，痕迹平滑用Sawitzky-格雷滤波器（宽度20）。 <img alt="图8" src="/files/ftp_upload/53058/53058fig8.jpg" /> 图8:由双管和同心电极所检测到细胞外的钠瞬变。 （A）在V REF瞬态变化和[Na +]为ö诱导浴灌注用10mM天冬氨酸120秒通过的条表示。上部迹线显示了带双管电极执行记录，下部迹线是用一同心电极记录。 （二）O引起的局部压力与应用10毫米谷氨酸0.2秒的箭头所指示的V REF和[Na +]为瞬时变化。上部迹线显示了带双管电极执行记录，下部迹线是用一同心电极记录。点缀红色线表示信号到它的最大的开始之间的时间段的线性拟合。注意，在此条件下的同心电极的响应时间是显著更快。为便于说明，痕迹平滑了Sawitzky -格雷过滤器（宽20）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

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

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 ...

double-barreled-concentric-microelectrodes-for-measurement

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

13.5K 次观看.  Heinrich Heine University Düsseldorf. 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. 

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

科研

神经科学

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

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 ...

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

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

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 ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

成人脑组织切片中钾离子选择性电极的制备、检测及应用

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

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 ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

细胞外离子流的使用离子选择性自引用的微电极技术测量

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

生物学

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 ...

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

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此视频中的章节

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

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

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

成人脑组织切片中钾离子选择性电极的制备、检测及应用

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

细胞外离子流的使用离子选择性自引用的微电极技术测量

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

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