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/存储解决方案的玻璃瓶中，在4°C。 </li><li>使用氰基丙烯酸酯粘接剂vibratome的样本保持器上附加一个小的琼脂块（6％时，制备的人工脑脊液），并将其存储在4℃下</li><li>前30分钟开始切片，将切片/存储解决方案，在一个桶充满了冰，用95％O 2，5％CO 2的气泡的玻璃瓶中。 </li></ol><strong&gt;注：速度和精度是最重要的解剖步骤如下所述。 <ol start="6"><li>氟烷麻醉鼠标（P14-21，C57BL / 6）/异氟醚（异氟醚已上报增强星形胶质细胞谷氨酸摄取15），斩首，头浸在50毫升烧杯中含氧，冷切片/存储解决方案。 </li><li>用纸巾包裹，并储存于-20℃下在冰包进行大脑的解剖</li><li>使用手术刀作出头的背侧正中矢状切开皮肤，从正面尾端，暴露颅骨。 </li><li>将较低的剪切刀片，手术剪一双小枕骨孔和两个切口，向左侧和右手边（45°角）。 </li><li>沿正中矢状线切割颅骨，从尾至额叶结束。 </li><li>取出大脑，用锅铲蘸在含氧，冷的切片/存储的解决方案上。 </li><li>用手术刀，五年削减：（1）取出嗅球和额叶皮层（2）删除小脑;（3）删除（4）左右颞叶（5）分开的两个大脑半球与中矢状切。 </li><li>轻拍大脑的两个部分用纸巾，以消除任何多余的溶液。 </li><li>胶各脑切片的侧表面冷vibratome的基板的大脑背侧应朝向vibratome的叶片腹侧的大脑应该是在琼脂块的接触，相差的vibratome的叶片。 注意：取得最高品质的片，重要的是，这两个部位的脑牢固地粘在vibratome的基板。要做到这一点，使用氰基丙烯酸酯胶粘剂，是不是太液不干燥太快。 </li><li> vibratome的底板固定解剖室，设置片日ickness至250μm的宽度，并调整刀片运行。继续与切片。 注意：如果一切正确的方向，vibratome的应切割矢状切片从背侧向腹侧和从内侧向大脑的横向侧。 </li><li>一旦通过大脑皮层和海马的刀刃，用手术刀切的脑皮层/海马淹没室将每个切片在34°C。 </li><li>丢弃的第一对夫妇的切片。通常情况下，可以得到12片（厚度为250μm），从P14-21小鼠脑。 </li><li>片在34℃保持30分钟，并让它们冷却至室温30分钟，然后使用这些电录音。 </li></ol> 2。星形胶质细胞的识别和录音<ol><li>准备的内部溶液含有（以mM计）：120许3 SO 3，10 EGTA，20 HEPES，2 MgATP，0.2 NaGTP5 QX-314Br的，氯化钠，290毫渗量，pH值7.2。 </li><li>准备外的录音解决方案（单位：mm）：119氯化钠，2.5氯化钾， 氯化钙 2.5 1.3硫酸镁4·7H 2 O，1 氯化镁 ，26.2的NaHCO 3，1 NaHPO 4，22葡萄糖，300毫渗量，pH值7.4入95％O 2，5％CO 2，饱和。 </li><li>添加下列药物的细胞外记录溶液，来阻止激活GluA GluN，mGluRII，mGluRIII，γ-氨基丁酸，γ-氨基丁酸乙甲 ，和A1腺苷受体（以μM）：10 2,3 -二氧代-6 -硝基-1， 2,3,4 -四氢苯并[ƒ]喹喔啉-7 -磺酰胺钠盐（NBQX），10（RS）-3 - （2 - carboxypiperazin -4 -基） -丙基-1 -膦酸（CPP），（2S ）2 - 氨基-2 - [（1S，2S）-2-carboxycycloprop -1 - 基] -3 - （呫吨-9 - 基）丙酸二钠盐（LY341495），100（R，S）-α-甲基丝氨酸-O-磷酸（MSOP），100印防己毒素，5 3 - [[（3,4 - 二氯苯基）甲基]氨基]丙基]二乙氧基甲基）次膦酸（CGP52432），1个8 - 环戊基-1,3 - 二丙基黄嘌呤（DPCPX）。 </li><li>设置的录音室中的细胞外液的温度，典型的温度在34 - 36℃。 </li><li>准备膜片钳电极硼硅玻璃毛细管（R≈2.5MΩ）采用双级，玻璃微吸管车夫。 </li><li>径片之一，并将其​​放置在录音室。按住它采用了金属与铂丝和尼龙弦的竖琴。 </li><li>目视检查片下DODT照明或IR-DIC。星形胶质细胞可以通过细胞小体（直径= 10微米）和（ 图1）突出的髓核。 </li><li>突触的刺激，放置一个双极不锈钢焊条，星形胶质细胞，你打算修补远离〜100微米。 </li><li>修补星形胶质细胞，并打破整个单元配置，通过应用一个非常温和的吸力。 注：星形胶质细胞通常具有较低的输入线电阻Ë（〜10MΩ），极化静息膜电位（〜-90毫伏），以及没有烧成活动。星形胶质细胞保持在电压钳模式下（ 即保持电流应读为0 pA的）在整个实验中，其静息膜电位。每次刺激前，一个10毫秒的超极化电压阶跃（-3毫伏）是用来监视的星形胶质细胞的串联输入电阻。 </li><li>如果串联电阻的变化大于20％，或者如果星形胶质细胞的膜电位去极化，丢弃录音。星形胶质细胞的膜电位，可以直接测量，通过切换电压钳电流钳模式。或者，膜电位，而在电压钳模式下，可以监视由读数保持电位的值，结果在0 pA的保持电流。 </li><li>如果突触的刺激，候补单配对刺激（ 如相距100毫秒），每次10 - 20秒。 </li><li>对于UV光解实验的树人，连接uncaging的的氙灯落射荧光显微镜的端口，并添加笼化合物外液。转运电流〜100 pA的幅度时，可以得到闪存uncaging的100微米MNI-L-谷氨酸在整个视野（直径= 662.5微米时，使用40X的目标14）。可替代的的刺激光路打开和堵塞，每10 - 20秒。 </li><li>唤起谷氨酸的瞬态和记录转运电流在控制条件下，在一个子的饱和浓度的广谱谷氨酸转运拮抗剂D，L- 苏式 - β-Benzyloxyaspartic的酸（TBOA;10μM）的存在下，以减少转运电流的幅值到至少30％的控制值（见第4，5）的存在下，或在高浓度的TBOA（50 - 100μM）完全阻断转运电流和隔离持续的K +电流（见第3节） 。 </li></ol> 3。 PHARMAC生理隔离的K +电流的持续<ol><li>在控制条件下和在高饱和浓度的TBOA（50 - 100μM）的存在下，星形细胞电流记录。 </li><li>一般至少20次在TBOA（50 - 100微米）。 </li><li>飞度平均获得3.2的功能的痕迹： <img alt="公式1" fo:src="/files/ftp_upload/50708/50708eq1highres.jpg" src="/files/ftp_upload/50708/50708eq1.jpg" /> 注意：的不敏感TBOA的成分（即在TBOA（50 - 100μM））的存在下被记录的一方表示持续的K +电流。上述单指数函数近似它的时间过程。 </li><li>重复这个适合在不同的星形胶质细胞，并推导出平均值τ 上升 （ 即 K +电流的持续时间平均上升（见第5节））。 </li></ol> 4。我染料溶液的突触活性炭运输车电流的（fSTCs促进部分） <ol><li>平均至少20次扫描，获得与配对的刺激，在控制条件和TBOA的（10μM）（ 图2a左侧）。 </li><li>平均有相同数目的扫描得到的单一的刺激，在控制条件和TBOA的（10μM）（ 图2a中）。 </li><li>的平均电流的四个平均值的痕迹（单脉冲控制，控制成对的脉冲，单脉冲TBOA，TBOA配对脉冲）-3 mV的电压阶跃的响应的振幅进​​行比较。 </li><li>如果在所有四个痕迹的电流响应的振幅是相同的，则继续到步骤4.7。 </li><li>如果振幅的平均电流的响应是不同的，在四条迹线的任何检查，所有单独的轨迹适当地包括在平均。 </li><li>如果振幅的平均电流是不同的，在任何的四个痕迹，但都在灵敏度独立的痕迹是适当的要包含在平均中，验证在每个跟踪FSTC的大小呈线性变化关系的测试电压阶跃的电流响应的大小。如果是这样的话，相对于彼此缩放的所有痕迹，从而使测试电压阶跃的电流响应，都幅度相等。 注：在这里描述拍摄条件（即细胞内CH 3 SO 3 -或C 6 H 11 O 7 - ），生成星形胶质细胞的兴奋性神经轴突突触的刺激与复杂波形的电流由一个快速上升的瞬态向内化学计量电流和缓慢上升，持续内向K +电流反射K +沿周边轴突动作电位的传播后，在细胞外空间的重新平衡。这是至关重要删除此持续的K +电流，任何剩余电流将导致高估谷氨酸寿命。 </li><li>减去单刺激获得的平均轨迹与配对的刺激（ 图2a右）从得到的平均轨迹。这个步骤可让隔离的化学计量和持续的K +电流由第二刺激诱 ​​发。 </li><li>移位单刺激由双脉冲（ 即 100毫秒）（ 图2b），用于提供相匹配的脉冲间的时间间隔的时间间隔得到的平均轨迹。 </li><li>减去时移单刺激得到的平均响应从4.7（ 图2c）中得到的第二个刺激的平均响应。此步骤将隔离转运电流诱发的第二个刺激（FSTC）促进部分。 </li><li>在大多数情况下，在上一步完全消除持续的K +电流。如果是这样的情况下，隔离FSTC完成，可以PROCE反褶积分析，并得出星形胶质细胞谷氨酸清除的时间过程。然而，在某些情况下，一个小的持续的K +电流仍然存在（ 图2d），并需要进一步分析（见第5节）。 注意：请记住，在得到控制的条件和在TBOA（10微米）的平均痕迹分析在4.7-4.10必须执行。 </li></ol> 5。减法的的剩余zdustained K表+电流从fSTCs <ol><li>测量持续执行第4条中所描述的分析后剩余的K +电流的振幅。 </li><li>缩放单指数函数的振幅在3.3节中的剩余持续Ķ+电流的幅度在5.1（ 图2d左边和中间的）测量。要做到这一点，在方程3.3，设置项A等于幅度持续的K +电流5.1 asured的的术语Τ 上升等于3.4Τ 上升估计的平均值。 </li><li>中减去得到的单指数函数从+电流的的FSTC持续Ķ获得在4.10（ 图2d权利）。这一步完成FSTC隔离。 </li></ol> 6。隔离的闪光激活运输车电流（外贸公司） <ol><li>平均至少20次扫描得到的光通路开放，在控制条件和TBOA的（10μM）。 </li><li>平均有相同数目的扫描获得的光路关闭时，在控制条件和在TBOA（10μM）。 </li><li>的四个平均值的痕迹（单脉冲控制，控制成对的脉冲，单脉冲TBOA，TBOA配对脉冲）-3 mV的电压阶跃的电流响应的振幅进​​行比较。 </li><li>如果在所有四个痕迹的电流响应的振幅是相同的，则继续到步骤6.7。 </li><li&gt;如果振幅的平均电流的响应是不同的，在四条迹线的任何检查，所有单独的轨迹适当地包括在平均。 </li><li>如果在任何四个痕迹的平均电流的振幅是不同的，但是所有个体的痕迹都适当地包括在平均，验证，在光路中每个跟踪打开FTC的秤的大小的大小呈线性测试电压阶跃的电流响应。如果是这样的话，相对于彼此缩放的所有痕迹，从而使测试电压阶跃的电流响应，都幅度相等。 注：在这里描述拍摄条件（即细胞内CH 3 SO 3 -或C 6 H 11 O 7 - ），闪光刺激产生星形胶质细胞仅包括快速崛起的瞬时计量内向电流的电流。 </li><li>轻拍了拍获得减去平均轨迹ħ阻止从得到的平均轨迹与光通路开放。这一步可以去除刺激伪迹和隔离外贸公司。 注：在6.7中描述的分析，必须执行控制条件和在TBOA（10μM）中得到的平均的痕迹。 </li></ol> 7。解卷积分析<ol><li>适合转运电流（FSTC或FTC）记录，可在控制条件下（ 图3a左）和TBOA（10μM）（ 图3a右）和隔离的第4 - 6中所描述的，与多指数函数： <img alt="公式（2）" fo:src="/files/ftp_upload/50708/50708eq2highres.jpg" src="/files/ftp_upload/50708/50708eq2.jpg" /></li><li>创建一个瞬间上升的衰变函数，根据函数的单成倍： <img alt="等式（3）" fo:src="/files/ftp_upload/50708/50708eq3highres.jpg" src="/files/ftp_upload/50708/50708eq3.jpg" /> 和最能说明（10微米）（ 图3b）TBOA记录在转运电流衰减阶段。 注意：在7.2中描述的功能（即H（t））的代表从TBOA（10μM）的存在下的星形胶质细胞的谷氨酸清除的时间过程。 </li><li>反卷积单指数在7.2（ 图3B和3C中间）函数从转运电流记录在TBOA（10微米）7.1（ 图3a右，C左）获得的拟合。 注：反褶积中包含许多分析软件包，如（如MATLAB，蟒蛇）是一种数学运算。编程代码在IgorPro，我们通常使用的执行这种分析，卷积操作可以有效的计算方法如下所述，通过使用离散快速傅立叶变换。首先，使用了FFT运算，来计算二7.2中得到的单指数函数拟合获得的记录，可在TBOA 7.1转运电流screte快速傅立叶变换。接下来，使用的IFFT运算的逆离散快速傅立叶变换的两个FFT函数的比例来计算。由此产生的函数I（t）的可以被描述如下： <img alt="式（4）" fo:src="/files/ftp_upload/50708/50708eq4highres.jpg" src="/files/ftp_upload/50708/50708eq4.jpg" /> 第7.3节中描述的步骤允许导出电流TBOA（10μM）（ 图3c右）转运的过滤器。该过滤器代表的失真因素，转换谷氨酸的寿命星形细胞的膜转运电流隔离在第4 - 6（即FSTC或FTC）。 </li><li>反卷积滤波器（ 图3c吧， 图3d中间）在控制条件下适合转运电流（ 图3a </STRONG&gt;左， 图3d左）。 注：此步骤可以推导在控制条件下（ 图3d权）星形胶质细胞谷氨酸清除的时间过程。假设此解卷积步骤是在控制条件保持不变，并在TBOA（10μM），过滤器的时间的档案。 </li><li>要获得整体谷氨酸清除的时间过程的定量估计，在第7.4步得到的波形计算质心（&lt;T&gt;），。这是通过计算&lt;T&gt;为： <img alt="方程（5）" fo:src="/files/ftp_upload/50708/50708eq5highres.jpg" src="/files/ftp_upload/50708/50708eq5.jpg" /> 其中G（t）被在步骤7.1中得到的波形。在上述方程中，术语t对应的时间的积分计算。 注意：当成立之初的方法，&lt;T&gt;计算随着时间的推移被留下的窗户，不受约束的13。这种方法可以被使用，只要整合窗口被设置为宽于谷氨酸的间隙的间隙波形衰减的持续时间完全恢复到基线。然而，如果不是这种情况下，这估计&lt;T&gt;的方法遇到一定的局限性。例如，如果间隙波形不完全衰减回基线，然后按&lt;t&gt;集成窗口的宽度增加。以避免任何潜在的误差源为&lt;T&gt;估计，我们现在计算超过转运电流的峰值的10％相对应的一个时间窗口，其发病14之前和之后。后者的做法，提高细胞之间的一致性与&lt;T&gt;测量。这派上用场，特别是当药物治疗效果分析小间隙的时间过程。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+fluxes+associated+with+excitatory+amino+acid+transport.">Ion fluxes associated with excitatory amino acid transport.</a> Neuron. 15, 721-728 (1995).</li><li>Wadiche, J. I., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+and+microscopic+properties+of+a+cloned+glutamate+transporter/chloride+channel.">Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel.</a> J. Neurosci. 18, 7650-7661 (1998).</li><li>Bergles, D. E., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activation+of+glutamate+transporters+in+hippocampal+astrocytes.">Synaptic activation of glutamate transporters in hippocampal astrocytes.</a> Neuron. 19, 1297-1308 (1997).</li><li>Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+the+glutamate+clearance+time+course+from+transporter+currents+in+CA1+hippocampal+astrocytes:+transmitter+uptake+gets+faster+during+development.">Deriving the glutamate clearance time course from transporter currents in CA1 hippocampal astrocytes: transmitter uptake gets faster during development.</a> J. Neurosci. 25, 2906-2916 (2005).</li><li>Scimemi, A., Tian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+transporters+regulate+glutamate+clearance,+NMDA+receptor+activation,+and+synaptic+plasticity+in+the+hippocampus.">Neuronal transporters regulate glutamate clearance, NMDA receptor activation, and synaptic plasticity in the hippocampus.</a> J. Neurosci. 29, 14581-14595 (2009).</li><li>Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+enhances+glutamate+uptake+via+glutamate+transporters+in+rat+glial+cells.">Isoflurane enhances glutamate uptake via glutamate transporters in rat glial cells.</a> Neuroreport. 12, 1077-1080 (2001).</li><li>Barbour, B., Brew, H., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogenic+uptake+of+glutamate+and+aspartate+into+glial+cells+isolated+from+the+salamander+(Ambystoma)+retina.">Electrogenic uptake of glutamate and aspartate into glial cells isolated from the salamander (Ambystoma) retina.</a> J. Physiol. 436, 169-193 (1991).</li><li>Bergles, D. E., Tzingounis, A. V., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+coupled+and+uncoupled+currents+during+glutamate+uptake+by+GLT-1+transporters.">Comparison of coupled and uncoupled currents during glutamate uptake by GLT-1 transporters.</a> J. Neurosci. 22, 10153-10162 (2002).</li><li>Diamond, J. S., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+glutamate+does+not+overwhelm+transporters+on+hippocampal+astrocytes+during+high-frequency+stimulation.">Synaptically released glutamate does not overwhelm transporters on hippocampal astrocytes during high-frequency stimulation.</a> J. Neurophysiol. 83, 2835-2843 (2000).</li><li>Benediktsson, A. M., 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=Neuronal+activity+regulates+glutamate+transporter+dynamics+in+developing+astrocytes.">Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.</a> Glia. 60, 175-188 (2012).</li><li>Hires, S. A., Zhu, Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurement+of+synaptic+glutamate+spillover+and+reuptake+by+linker+optimized+glutamate-sensitive+fluorescent+reporters.">Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4411-4416 (2008).</li><li>Scimemi, A., Meabon, J., Woltjer, R. L., Sullivan, J. M., Diamond, J. S., Cook, D. 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作者宣称没有利益冲突。

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 ...

deriving-time-course-glutamate-clearance-with-deconvolution-analysis

Research

JoVE Journal

Neuroscience

10.2K 次观看.  National Institutes of Health. 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。

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

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

科研

神经科学

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

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

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

微胶质细胞抗体介导的陶氏清除研究

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...