Name: deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents
Uploaded: 2013-08-07T13:00:00
Description: Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

这项工作是由国家神经紊乱和中风院内研究计划（NS002986）。写的手稿和实施反褶积分析。 JSD反褶积分析，并开发了最初的版本上的文字进行了评论。

1。切片制备<ol><li>准备500毫升/存储解决方案（单位：mm）：119氯化钠，氯化钾2.5 1.3 0.5 氯化钙 ， 硫酸镁 ·7H 2 O，4 氯化镁 ，26.2的NaHCO 3，1的NaH 2 PO 4，和22葡萄糖，320切片毫渗量，pH值7.4 </li><li>使用250毫升的烧杯中，准备淹没室，为片，填充200毫升切片/存储解决方案，在水浴加热34°C和95％O2，5％CO2泡沫。 </li><li>保留剩下的切片/...

<ol>
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这里，我们描述的实验方法得到的星形胶质细胞的电生理记录，协议在星形胶质细胞谷氨酸转运电流隔离的分析和数学方法获得谷氨酸的空隙，星形细胞转运电流的时间过程。 分析的成功依赖于星形胶质细胞的能力，获得高品质的膜片钳记录，用来描述转运电流的拟合算法的准确性。反卷积分析依赖于以下两个假设，（1）的多个进程扭曲谷氨酸的间隙进入实验记录转?...

星形胶质细胞是大脑中的星形形态和细膜突起，扩展到整个神经纤维到达邻近的突触联系1,2最丰富的细胞类型之一。星形胶质细胞的细胞膜被密密麻麻的与谷氨酸转运分子3。谷氨酸转运体在生理条件下，迅速地结合在细胞外谷氨酸侧膜，并将其转移到细胞的细胞质中。通过这样做，转运保持较低的基底浓度的谷氨酸在细胞外空间4。精美的星形胶质细胞的过程相邻兴奋性突触的谷氨酸转运体在理想的位置，结合谷氨酸突触活动期间发布，因为它远离突触间隙扩散。通过这样做，运输也限制对围绝经期和额外的突触区域到相邻的突触的谷氨酸溢出，减少兴奋信号的空间传播5-7大脑S IN。 谷氨酸转运是一个生电的过程中化学计量的运动耦合到3 Na +和H +的电化学梯度沿它们的1 K + 8的逆运输。谷氨酸运输相关的（但不是化学计量）阴离子电导渗透到SCN - （硫氰酸盐）&gt; NO 3 - （硝酸）≈CLO 4 - （高氯酸盐）&gt; - &gt; - &gt;氯- &gt; F -不CH 3 SO 3 - （甲烷磺酸钠）和C 6 H 11 O 7 - （葡萄糖酸钙）9-11。这两个电流（化学计量的和非化学计量的），可记录，可通过获得全细胞膜片钳记录从星形胶质细胞，视觉识别根据DODT照明或者红外微分干涉对比（IR-DIC）ACUTê大脑切片12。当前与谷氨酸转运穿过膜的化学计量成分，可以分离通过使用CH 3 SO 3 - ，或C 6 H 11 O 7 -基于细胞内的解决方案，可诱发的星形胶质细胞的闪光uncaging谷氨酸对13,14，或从邻近的突触激活谷氨酸释放，无论是电12或有针对性的光遗传学控制。 的化学计量成分的转运电流的时间过程中被成形由谷氨酸浓度资料在星形胶质细胞的膜（ 即谷氨酸间隙）的生存期，谷氨酸转运体动力学的，被动的星形胶质细胞的膜性能，并在突触的刺激，由同步跨激活突触13谷氨 ​​酸的释放。在这里，我们描述的全部细节：（1）实验：约oach隔离电流全细胞膜片钳记录从使用急性小鼠海马脑片作为一个例子实验准备的星形胶质细胞谷氨酸转运体的化学成分;（2）分析法得出谷氨酸清除的时间当然从这些录音13， 14。这些方法可用于记录和分析在中枢神经系统的任​​何区域中星形胶质细胞谷氨酸转运电流。

<table border="1">
		<tbody>
		 <tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments </td>
		 </tr>
		 <tr>
			<td>CGP52432</td>
			<td>Tocris</td>
			<td>1246</td>
			<td></td>
		 </tr>
		 <tr>
			<td>(R,S)-CPP</td>
			<td>Tocris</td>
			<td>173</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DPCPX</td>
			<td>Tocris</td>
			<td>439</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LY341495 disodium salt</td>
			<td>Tocris</td>
			<td>4062</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MSOP</td>
			<td>Tocris</td>
			<td>803</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NBQX disodium salt</td>
			<td>Tocris</td>
			<td>1044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D,L-TBOA</td>
			<td>Tocis</td>
			<td>1223</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MNI-L-glutamate</td>
			<td>Tocris</td>
			<td>1490</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexa 594</td>
			<td>Life Technologies</td>
			<td>A10438</td>
			<td>Optional</td>
		 </tr>
		 <tr>
			<td>Matrix electrodes</td>
			<td>Frederick Haer Company</td>
			<td>MX21AES(JD3)</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG10165-4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual-stage glass micro-pipette puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Loctite 404 instant adhesive</td>
			<td>Ted Pella</td>
			<td>46551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xe lamp</td>
			<td>Rapp OptoElectronic</td>
			<td>FlashMic</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Igor Pro 6</td>
			<td>Wavemetrics</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents

发现大脑中的谷氨酸转运体密度最高的星形胶质细胞。谷氨酸转运体的移动耦合谷氨酸穿过细胞膜的与共同运输3的Na +和1 H +和1 K计数器运输+。运输过程中所产生的化学计量电流可以监视从星形胶质细胞与全细胞膜片钳记录。的时间过程中所记录的电流的时间过程中谷氨酸的浓度分布的星形胶质细胞暴露，谷氨酸转运体的动力学，和被动电张性能的星形胶质细胞的膜的形状。这里，我们描述的实验和分析的方法，可用于记录在星形胶质细胞谷氨酸转运体的电流和隔离的谷氨酸间隙从所有的其他因素而形成波形的星形胶质细胞的转运电流的时间过程。这里描述的方法可以被用来估计的生命周期F闪光灯开锁和突触释放的谷氨酸在任何地区的中枢神经系统在健康和疾病中的星形胶质细胞的膜。

这里描述的分析方法的成功严重依赖于获得高质量转运电流从任何地区的中枢神经系统的神经胶质细胞的电生理记录。星形胶质细胞在急性小鼠海马脑片，可以很容易地确定下DODT照明或IR-DIC，因为他们的小细胞体（O = 10微米）和髓核突出（ 图1）。其独特的星形的形态与落射荧光，共焦或双光子激光扫描显微镜，可以理解，当加入类似的Alexa 594荧光团（50μM）细胞内的溶液中，（ <stro...

Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

我们描述了一个分析估计寿命星形胶质膜谷氨酸在星形胶质细胞谷氨酸转运电流的电生理记录的方法。

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

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N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...