韦恩州立大学国家酒精滥用和酒精中毒研究所（NIAAA; AA - 016967和AA016967 - 01S1;谭）提供的资金，启动资金，和韦恩州立大学研究资助计划。内容完全是作者的责任，并不代表NIAAA或国立卫生研究院的官方意见。

1。实验要领电极的制作<ul type="circle"><li>有无数的碳纤维微电极的制备方法，因为大多数是在内部。通常是什么决定了电极制造细节的电化学技术应用于电极（如安培与FSCV）。对于FSCV，微电极可以在内部使用以下三个步骤的过程。对于碳纤维电极的制作更完整的描述，可以看到最近的朱庇特的第1条。但是请注意，下面描述的电极是圆柱形的碳纤维微电极，它需要较少的步骤与上述协议安培碳纤维微电极制造。这种简化的协议不需要沸点在丙酮中的碳纤维，火抛光的玻璃毛细管，或用环氧密封玻璃纤维的交界处。 </li><li>使用真空吸aspir吃了碳纤维（直径7微米;古德费洛Oakdale，PA）成硼硅玻璃毛细管与微丝（长10厘米，直径1.2毫米，内径0.68毫米;上午系统，Carlsborg，西澳）。 </li><li>将螺纹毛细管毛细管拉到一半到电极拉马（Narishige，日本东京）。电极拉马的输出设置不同从实验室到实验室。作为参考，我们的车夫输出设置主磁铁90.7，23.2分磁铁，加热器53.4。输出设置应该是经验的提炼，生成约4.4毫米长，与周围的碳纤维密封，玻璃锥。 </li><li>在显微镜下（奥林巴斯，日本东京），内饰，碳纤维（使用手术刀刀片）从玻璃允许约50-200微米的碳纤维伸出密封界面提示延伸。 </li></ul>溶液的制备三种类型的人造蜡需要提前做好准备，在超纯水（18MΩ厘米）水，brospinal液（ACSF）解决方案。 <ul type="circle"><li>蔗糖，学联可以准备至少一天前的切片。蔗糖学联缓冲区由（毫米）：180蔗糖，30氯化钠，4.5氯化钾，1.0 MgCl 2的3碳酸氢钠，1.2的NaH 2 PO 4，和10个D -葡萄糖（pH值7.4），26，应使用95含氧％O 2 / 5％CO 2 15分钟2。如果提前准备的时间，该解决方案可冷藏保存于4 ° C为1周。 </li><li>学联为伏安录音解决方案应做好实验的一天。学联的解决方案，包括（毫米）：126氯化钠，2.5氯化钾，2.4，1.2氯化钙2 MgCl 2 的 25碳酸氢钠3，1.2的NaH 2 PO 4，11个D -葡萄糖，0.4抗坏血酸（pH值7.4）。在实验中，含氧95％O2 / 5％，在室温下的CO 2冒泡过程中。 </li><li>改性学联电极校准包含解决方案（毫米）：2.5氯化钾，126氯化钠，1.2的NaH 2 PO 4，2.4，1.2氯化钙氯化镁2，和25 3碳酸氢钠（pH值7.4），并且可以保持长达一个星期，没有制冷或充氧。 </li></ul>电极校准<ul type="circle"><li>前和校准后，分别是由电极的可行性和敏感性，使用甲流“T细胞”，其中包含一个密封的参比电极（Ag / AgCl电极）的3个端口。所制作的微电极是降低成“T细胞”的流动。注射泵进气口连接，允许在2毫升/分钟的流速不断流动的改性学联。的“T细胞”的第三个端口连接到3微米多巴胺在修改，学联，填补了注射器。 </li><li>预校准，允许修改学联溶液流量约3 - 7秒，然后手动注入1-2毫升的多巴胺3微米标准。对于每一个电极预校准，重新校准至少3次，平均每个获得的最大电流。 </li><li> （见第3节），切片后立即伏安实验的电极以同样的方式后校准如上所述。 </li><li>校正因子（后来在数据分析），是由多巴胺标准浓度除以平均多巴胺的氧化电流（NA）。例如，目前的反应是除以3，当使用3微米多巴胺标准。 </li></ul> 2。切片制备<ol><li>倒入小烧杯中，并在一个冰桶，10毫升蔗糖学联。此外，瞬间粘合剂（乐泰404）冰桶内达到。 </li><li>准备刀片和解剖所需的必要工具，如钳，铲，剪刀，擦拭干净用酒精垫。 </li><li>在一个小的气茶牺牲二氧化碳窒息鼠标mber，立即斩首使用锋利的剪刀。迅速取出整个大脑。约10分钟，放置在冰冷的蔗糖学联烧杯的大脑。 </li><li>在此期间，准备切片Vibratome。首先，将一些碎冰标本浴。标本室的位置，并拧紧到位坚决持有。添加更多的样品室周围的冰，以填补空白，确保没有冰室得到。广场清洁的刀片在刀片上Vibratome的持有人，并填写标本室，与冰冷的蔗糖学联。 </li><li>成立了一个工作准备的大脑表面，倒有些冷蔗糖学联已上翘的培养皿上放置了一块纸巾。使用镊子准备培养皿转移到大脑。对于冠状切片，切小脑内侧横向轴沿用刀片和丢弃。这将创建一个平底，可贴Vibratome阶段。 </li><li>地点（放置在冰桶在第1步）标本舞台上的乐泰胶粘剂下降。随即，贴上准备在舞台上脑的平头，保持尽可能直立。在标本室的阶段，并拧紧螺丝，确保大脑是完全沉浸在会议厅内的蔗糖学联。 </li><li> Vibratome前使用的控制，调整阶段，使刀片与大脑的顶部。 Vibratome的最佳参数，通过设置的频率和速度低。较低的速度是首选的挤压大脑，这是在更高的速度观察，以尽量减少刀片的力量。层厚设置为400微米。 </li><li>最初的几片不会包含纹状体。重复，直到获得片含纹状体切片。一旦纹状体区域是达到解剖标志肯定，使用油漆刷解除切片，并在烧杯含氧，室温学联。通常情况下，可以得到3至4片含有使尾壳和伏隔核包括纹状体复杂。 </li><li>允许切片至少1小时，以适应环境，在室温含氧学联在实验中使用。 </li></ol> 3。从片伏安录音虽然是孵化切片，切片录音室准备。 <ol><li>以油管连接淹没录音室（自定义科学，丹佛，CO）和充氧，房间温度学联地点。灌注泵（沃森马洛有限公司，法尔茅斯，英国）设置到1毫升/分钟的流量。温度控制器设定到32 ° C。学联填充定制的片持有人（修改丝网光盘阶段）后，准备切片删除任何气泡，用针注射器（BD脊髓针）。 </li><li>总理片按图纸上的真空浴流出管（玻璃液废液瓶）用注射器开始流动。放置一个kimwipe作为一个切片持有人控制的缓冲区溢出边缘的灯芯。 </li><li>培养1小时后，转让切片片在录音室的持有人，这是不断与95％的O 2 / 5％CO 2的室温学联灌注。 </li><li>淹没在片人的银/氯化银参比电极（录音片室盖）和连接电极使用弹簧夹头阶段。 <ul type="circle"><li>的Ag / AgCl参比电极可以在阳极氧化（+1 V）为5分钟，250微米的银线（AM系统，Carlsborg，WA），1 M盐酸沉积在表面的一层薄薄的AgCl的房子银线。 </li></ul></li><li>钨刺激​​电极（塑料，罗阿诺克，弗吉尼亚州）下纹状体脑片表面。刺激电极接触片，因为它在顶部在于的，但不应穿刺切片。在我们的实验装置是由一个Neurolog刺激，刺激。 </li><li>碳纤维工作电极回填用150毫米氯化钾使用BD脊髓针的解决方案。一个引线，然后插入（大地主电子，科尼利厄斯，或），这是连接到低噪声ChemClamp电位（矸团，明尼苏达州明尼阿波利斯）使用弹簧夹头阶段。工作电极置于约100 - 200微米远离双极刺激电极，成片的深约75微米。 </li><li>交付使用或者单（单相，350μA，60赫兹，4毫秒脉冲宽度）或多个（如5个脉冲，350μA，20赫兹，宽4毫秒）脉冲，电刺激，引起神经递质刺激电极第3版。 </li><li>要衡量使用FSCV电诱发的多巴胺，一个三角波形应用于电极。 DOP的典型参数胺检测：碳纤维微电极的潜力是举行-0.4 V与一个银/氯化银参比电极，然后带回扫描速度在400 -0.4 V至+1.2 V的正极限斜坡，V / S 。 </li><li>切片电刺激，每5分钟产生多巴胺流出伏安测量15秒。 </li><li>至少有三个稳定的电刺激多巴胺的释放录音（峰高之间的差异是&lt;10％）后，学联含有药理代理人的利益是灌注在流速为1 mL /分钟超过30分钟，以获得最大的效果切片。电刺激多巴胺录音的药理灌注期间每隔5分钟。 </li></ol> 4。数据分析由此产生的电流随时间片获得的痕迹，可以适合非线性回归了一套基于米氏方程，由怀特曼和他的同事编写的软件在LabVIEW（美国国家仪器公司，美国德克萨斯州奥斯汀 ） 4-6。在这个软件中，电流随时间的痕迹，可以安装不同的两个参数，V最大（nm / s时，相应递减阶段的跟踪表明，摄取的多巴胺转运率）和多巴胺浓度每脉冲（NM对应的最大峰高）。设置为160纳米的K m值，反映了多巴胺的多巴胺转运蛋白的亲和力，并没有改变。电极的校准后的实验后确定的因素是需要装修。 LabVIEW软件中包含的决定系数（R）参数来确定的拟合优度（R 2的值&gt; 0.8） 。 5。代表性的成果 FSCV用于检查单脉冲电刺激多巴胺的释放和摄取尾把阿门（CPU），伏隔核（NAC）的核心，NAC在小鼠壳。图1A所示的代表结果表明电流（或浓度）随时间变化的地块。红色箭头表示电刺激时，在相应的电流量在碳纤维微电极，在多巴胺浓度的变化应占上升切片。在电刺激的主要过程是多巴胺的释放，但整体观察峰的高度，以及其他如吸收和扩散的进程作出贡献。降的高峰阶段，主要是由于其转运的神经递质再摄取由因为神经刺激已经停止7。然而，峰值衰减不仅限于再摄取，扩散和新陈代谢，也有助于减少电流。它一直推测，由于电化学测量的时间尺度是几秒钟的事，FSCV太快措施contributio从代谢的NS 7 。在这些电流随时间的痕迹，Y轴转换为使用后的实验校正因子的浓度（微米）。图1A中的插图是各自的背景，减去循环伏安电流随时间的痕迹。测量电流（Y轴）绘制的电极（X轴）的应用潜力，多巴胺的化学发现与观察到+0.6 V的氧化峰，相应减少了多巴胺的邻醌的高峰期是观察 - 的Ag / AgCl参比电极为0.2 V与。第三代表性的数据，使用一个三维伪彩色图（图1B）相结合的电流随时间的痕迹和循环伏安的信息，形成一个单一的情节。代表的伪彩色图，绘制沿x轴秒的时间，碳纤维工作电极施加电压是沿Y轴图表，和目前的FAL代表沿Z轴本身的颜色。由于碳纤维微电极（直径〜7微米）体积小，电刺激多巴胺的动态可检测纹状体（CPU和NAC的核心与NAC壳图2）分立的解剖区域。 使用纹状体冠状切片的一个好处是，它消除了多巴胺的细胞体的贡献，并允许调查突触前多巴胺动态。突触前多巴胺的释放和吸收控制没有严格限制多巴胺autoreceptor或转运功能，正如其他人16，17所示。 Heteroreceptors其他神经递质系统也调节多巴胺动态18，19。代表电流随时间的变化如图3a所示的痕迹表明，当片1微米quinpirole（D2/D3受体激动剂）30分钟，电诱发多巴胺的释放减少治疗观察。另一方面，当基板多巴胺转运蛋白，如甲基安非他明，是通过30分钟的片灌注，多巴胺的释放无差异（图3B）。峰值衰减转移到右侧，通常是与多巴胺转运动力学的改变（的K m）3 。最后，图3C是一个代表电流随时间的跟踪跟踪，一旦切片已在沐浴着一个100毫微克/毫升的脑源性神经营养因子（BDNF）的解决方案，其中已假设影响多巴胺的释放，21动态20。从这个代表性的跟踪，可以看出，BDNF的能力，增强电诱发多巴胺的释放。两者合计，这些药物治疗强调FSCV公用事业探测纹状体内多巴胺的动态。 使用大脑切片调查FSCV突触前多巴胺动态的主要限制因素是，从一个完整的大脑neurocircuitry丢失。随着片FSCV是不可能的研究来自其他脑区神经递质的影响，很难理解这些系统的贡献，对被调查地区的功能（如纹状体）或评估无刺激多巴胺的水平。然而，在FSCV最近的技术进步使多巴胺的瞬态测量（与无电刺激），在自由活动大鼠药理操纵，自我管理，或新奇 22 - 24。总体而言，片FSCV突触前多巴胺动态提供有价值的信息，并提供互补的神经化学物质的技术，如微透析，电，和/或自由活动FSCV耦合片FSCV结果在大脑中的神经递质的运作有更全面的观点。 <img alt="图1" src="/files/ftp_upload/3464/3464fig1.jpg" /> 电诱发多巴胺图1。Ë发布使用FSCV以下单脉冲刺激C57BL/6J小鼠背CPU片。 （一）浓度随时间变化的轨迹，在单脉冲（红色箭头）诱发多巴胺的释放。一个星号代表的因素，有助于浓度的上升，这主要是多巴胺的释放，但也有利于吸收和扩散。两个星号代表的峰值信号返回基线，主要是由于摄取，也有助于扩散。插图显示了相应的循环伏安。 （二）从背CPU的显示时间（X轴）的代表色图，碳纤维微电极的Ag / AgCl参比电极（Y轴）与应用潜力，并在当前伪彩色。 <img alt="图2" src="/files/ftp_upload/3464/3464fig2.jpg" /> 图2：由单一的电刺激脉冲（红色箭头表示）在NAC核心诱发多巴胺的释放。C57BL/6J小鼠和外壳。 （A和C）浓度随时间变化的轨迹和其相应的循环伏安曲线（插图）从NAC的核心和壳。 （B和D）如前所述，从NAC的核心和壳的代表色图。 <img alt="图3" src="/files/ftp_upload/3464/3464fig3.jpg" /> 图3切片后的代表跟踪治疗了30分钟的药理剂;在所有情况下，单脉冲被用来唤起CPU中的多巴胺的释放。 （一）多巴胺D2受体激动剂的应用，quinpirole（红色曲线）相比，治疗前（​​黑色曲线）。 （二）甲基安非他明灌注（紫色曲线）相比，治疗前（​​黑色曲线）。 （三）脑源性神经营养因子的能力，影响多巴胺的动态（蓝线）相比，治疗前（​​黑色曲线）。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+release+evoked+by+short+electrical+stimulations+in+striatal+brain+slices+is+primarily+activity+dependent.">Adenosine release evoked by short electrical stimulations in striatal brain slices is primarily activity dependent.</a> A.C.S. Chem. Neurosci. 1, 775-787 (2010).</li><li>Sanford, A. L., Morton, S. W., Whitehouse, K. L., Oara, H. M., Lugo-Morales, L. Z., Roberts, J. G., Sombers, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltammetric+detection+of+hydrogen+peroxide+at+carbon+fiber+microelectrodes.">Voltammetric detection of hydrogen peroxide at carbon fiber microelectrodes.</a> Anal. Chem. 82, 5205-5210 (2010).</li><li>Park, J., Kile, B. M., Wightman, R. M. <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+voltammetric+monitoring+of+norepinephrine+release+in+the+rat+ventral+bed+nucleus+of+the+stria+terminalis+and+anteroventral+thalamic+nucleus.">In vivo voltammetric monitoring of norepinephrine release in the rat ventral bed nucleus of the stria terminalis and anteroventral thalamic nucleus.</a> Eur. J. Neurosci. 30, 2121-2133 (2009).</li><li>Hashemi, P., Dankoski, E. C., Petrovic, J., Keithley, R. B., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltammetric+detection+of+5-hydroxytryptamine+release+in+the+rat+brain.">Voltammetric detection of 5-hydroxytryptamine release in the rat brain.</a> Anal. Chem. 81, 9462-9471 (2009).</li><li>Borue, X., Cooper, S., Hirsh, J., Condron, B., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+serotonin+release+and+clearnece+in+drosophila.">Quantitative evaluation of serotonin release and clearnece in drosophila.</a> J. Neuroscience. Methods. 179, 300-308 (2009).</li><li>Vickrey, T. L., Condron, B., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+endogenous+dopamine+changes+in+drosophila+melanogaster+using+fast-scan+cyclic+voltammetry.">Detection of endogenous dopamine changes in drosophila melanogaster using fast-scan cyclic voltammetry.</a> Anal. Chem. 81, 9306-9313 (2009).</li><li>Makos, M. A., Han, K. A., Heien, M. L., Ewing, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+in+vivo+electrochemistry+to+study+the+physiological+effects+of+cocaine+and+other+stimulants+on+the+drosophila+melanogaster+dopamine+transporter.">Using in vivo electrochemistry to study the physiological effects of cocaine and other stimulants on the drosophila melanogaster dopamine transporter.</a> A.C.S. Chem Neurosci. 1, 74-83 (2009).</li><li>Budygin, E. A., John, C. E., Mateo, Y., Daunais, J. B., Friedman, D. P., Grant, K. A., Jones, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+ethanol+exposure+alters+presynaptic+dopamine+function+in+striatum+of+monkeys:+a+preliminary+study.">Chronic ethanol exposure alters presynaptic dopamine function in striatum of monkeys: a preliminary study.</a> Synapse. 50, 266-268 (2003).</li><li>Jones, S. R., Gainetdinov, R. R., Jaber, M., Giros, B., Wightman, R. M., Caron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+neuronal+plasticity+in+response+to+inactivation+of+the+dopamine+transporter.">Profound neuronal plasticity in response to inactivation of the dopamine transporter.</a> PNAS. 95, 4029-4034 (1998).</li><li>Kennedy, R. T., Jones, S. R., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+observation+of+dopamine+autoreceptor+effects+in+rat+striatal+slices.">Dynamic observation of dopamine autoreceptor effects in rat striatal slices.</a> J. Neurochem. 59, 449-445 (1992).</li><li>Rice, M. E., Cragg, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+amplifies+reward-related+dopamine+signals+in+striatum.">Nicotine amplifies reward-related dopamine signals in striatum.</a> Nat. Neurosci. 7, 583-584 (2004).</li><li>Zhang, L., Doyon, W. M., Clark, J. J., Phillips, P. E., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+tonic+and+phasic+dopamine+transmission+in+the+dorsal+and+ventral.">Controls of tonic and phasic dopamine transmission in the dorsal and ventral.</a> 76, 396-404 (2009).</li><li>Paredes, D., Grnaholm, A. C., Bickford, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+NGF+and+BDNF+on+baseline+glutamate+and+dopamine+release+in+the+hippocampal+formation+of+the+adult+rat.">Effects of NGF and BDNF on baseline glutamate and dopamine release in the hippocampal formation of the adult rat.</a> Brain. Res. 11141, 56-64 (2007).</li><li>Goggi, J., Puller, I. A., Carney, S. L., Bradford, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+involved+in+the+short-term+potentiation+of+dopamine+release+by+BDNF.">Signalling pathways involved in the short-term potentiation of dopamine release by BDNF.</a> Brain. Res. 968, 156-161 (2003).</li><li>Cheer, J. F., Wassum, K. M., Heien, M. L., Phillips, P. E., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+enhance+subsecond+doapmine+relese+in+the+nucleus+accumbens+of+awake+rats.">Cannabinoids enhance subsecond doapmine relese in the nucleus accumbens of awake rats.</a> J. Neurosci. 24, 4393-4400 (2004).</li><li>Phillips, P. E., Stuber, G. D., Heien, M. L., Wightman, R. M., Carelli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsecond+dopamine+release+promotes+cocaine+seeking.">Subsecond dopamine release promotes cocaine seeking.</a> Nature. 422, 614-618 (2003).</li><li>Robinson, D. L., Heien, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+dopamine+concentration+transients+increases+in+dorsal+and+ventral+striatum+of+male+rats+duing+introduction+of+conspecifics.">Frequency of dopamine concentration transients increases in dorsal and ventral striatum of male rats duing introduction of conspecifics.</a> J. Neurosci. 22, 10477-10486 (2002).</li></ol>

没有利益冲突的声明。

这里介绍的协议，演示了如何准备和使用FSCV实验小鼠冠状脑切片。虽然这种方法是具体 ​​的获取和测量多巴胺的动态，其他神经递质如腺苷，过氧化氢 ​​，去甲肾上腺素和血清素已在体内或体外监测与FSCV 3，8 - 11 。 FSCV可以用来监视其他神经化学物质的一些简单的修改适用于工作电极3，11的波形。由于这些神经化学物种中，有很多类似的氧化电位，产生的循环伏安每...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> CAS号码 </td></tr><tr><td width="274"> 试剂 </td><td></td><td></td></tr><tr><td width="274">氯化钾</td><td>费舍尔</td><td width="142" valign="top"> 7447407 </td></tr><tr><td width="274">氯化钠</td><td> EMD化学品</td><td width="142" valign="top"> 764714​​5 </td></tr><tr><td width="274">氯化镁</td><td width="194" valign="top">费舍尔</td><td width="142" valign="top"> 7791186 </td></tr><tr><td width="274">氯化钙</td><td width="194" valign="top">费舍尔</td><td width="142" valign="top"> 10035048 </td></tr><tr><td width="274">碳酸氢钠</td><td width="194" valign="top"> EMD化学品</td><td width="142" valign="top"> 144558 </td></tr><tr><td width="274">磷酸钠，二元</td><td width="194" valign="top">电解二氧化锰化学小号</td><td width="142" valign="top"> 7558794 </td></tr><tr><td width="274"> D -葡萄糖</td><td width="194" valign="top">费舍尔</td><td width="142" valign="top"> 50997 </td></tr><tr><td width="274">抗坏血酸</td><td width="194" valign="top">费舍尔</td><td width="142" valign="top"> 50817 </td></tr><tr><td width="274">蔗糖</td><td width="194" valign="top">费舍尔</td><td> 57501 </td></tr><tr><td width="274">&nbsp;</td><td width="194" valign="top">&nbsp;</td><td>&nbsp;</td></tr><tr><td width="274"> 材料 </td><td width="194" valign="top"> 供应商 </td><td> 目录编号 </td></tr><tr><td width="274">碳纤维</td><td>古德费洛Oakdale，PA </td><td></td></tr><tr><td width="274">玻璃毛细管</td><td>上午系统Carlsborg WA。 </td><td> 602000 </td></tr><tr><td width="274">银线</td><td> A -M系统Carlsborg西澳。 </td><td> 787000 </td></tr><tr><td width="274">钨刺激​​电极</td><td>塑料之一，弗吉尼亚州罗阿诺克</td><td></td></tr><tr><td width="274">铂丝</td><td></td><td></td></tr><tr><td width="274">导线</td><td>斯夸尔斯电子，科尼利厄斯，或</td><td></td></tr><tr><td width="274">乐泰404瞬间粘合剂</td><td> Hankal公司洛基山 CT检查。 </td><td></td></tr><tr><td width="274">刀片</td><td>世界精密仪器公司的佛罗里达州。 </td><td></td></tr><tr><td width="274"> BD脊髓针</td><td> BD医疗系统， 位于美国新泽西州Franlin湖， </td><td>楼盘405234 </td></tr><tr><td width="274">手术刀片</td><td>羽毛安全剃刀有限公司。日本</td><td></td></tr><tr><td width="274"></td><td></td><td></td></tr><tr><td width="274"> 软件 </td><td width="194" valiGN =“顶”&gt; 供应商 </td><td> 目录编号 </td></tr><tr><td width="274"> TH软件</td><td>欧空局的公司，切姆斯福德，MA </td><td></td></tr><tr><td width="274"> 仪器 </td><td width="194" valign="top"> 供应商 </td><td> 目录编号 </td></tr><tr><td width="274">浸没录音室</td><td>自定义科学，丹佛，CO </td><td></td></tr><tr><td width="274"> Neorolog刺激隔离器</td><td>英国赫特福德郡，Digitmeter， </td><td></td></tr><tr><td width="274">自动温度控制器</td><td>华纳仪器公司</td><td></td></tr><tr><td width="274">显微镜（SZX7） </td><td>奥林巴斯</td><td></td></tr><tr><td width="274">显微镜</td><td>费舍尔</td><td></td></tr><tr><td width="274"> 3000 Vibratome切片系统</td><td>圣楼是，莫。 </td><td></td></tr><tr><td width="274">灌注泵</td><td>沃森马洛有限公司，康沃尔郡法尔茅斯 - 英格兰</td><td> H110708 </td></tr><tr><td width="274">微管拉拔</td><td> Narishige，东京，日本</td><td></td></tr><tr><td width="274"> ChemClamp电位</td><td>大矸法团， 明尼苏达州明尼阿波利斯。 </td><td></td></tr></tbody></table>

presynaptic dopamine dynamics in striatal brain slices with fast-scan cyclic voltammetry

广泛的研究主要集中于神经递质多巴胺，因为其在滥用药物（如可卡因和安非他明），发挥的作用，在精神病（如精神分裂症，多动症），和其在退行性参与的行动机制的重要性障碍像帕金森氏症和亨廷顿氏病。在正常生理条件下，多巴胺是众所周知的规范自发活动，认知，学习，情感的影响，和神经内分泌激素的分泌。多巴胺神经元密度最大的一个是纹状体内，可分为两个不同的神经解剖区域，被称为伏隔核和尾壳。目的是要说明的小鼠纹状体内一般为片快速扫描循环伏安（FSCV）协议。 FSCV是一个定义良好的电化学技术在D实时提供机会来衡量多巴胺的释放和吸收iscrete大脑区域。在FSCV碳纤维微电极（直径〜7微米）用于检测多巴胺的氧化。使用FSCV检测多巴胺的分析优势，是100毫秒和空间分辨率小于10微米在体内微透析提供的补充信息，增强的时间分辨率。

Watch this Scientific Journal Video about Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry at JoVE.com

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

使用快速扫描循环伏安法测量纹状体脑片电刺激突触前多巴胺动态。

突触前多巴胺纹状体脑片的快速扫描循环伏安动态

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

presynaptic-dopamine-dynamics-striatal-brain-slices-with-fast-scan

Research

JoVE Journal

Neuroscience

21.7K 次观看.  Wayne State University. 使用快速扫描循环伏安法测量纹状体脑片电刺激突触前多巴胺动态。

视频: Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

科研

神经科学

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

偏置电压，循环伏安，神经接口电阻抗光谱

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

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

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

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

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

使用QNsim1.0从电刺激多巴胺神经传递数据建模快速扫描循环伏安数据

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...