Name: examination of rapid dopamine dynamics with fast scan cyclic voltammetry during intra-oral tastant administration in awake rats
Uploaded: 2015-08-12T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats at JoVE.com

Research reported in this publication was supported by an NSF Graduate Research Fellowship (RJW) and by the National Institute on Drug Abuse of the National Institutes of Health and FDA Center for Tobacco Products (CTP) under Award Number P50DA036151(EJN and NAA). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

所有的实验都是根据健康（NIH）指南实验动物的护理和使用全国学院进行，并批准了耶鲁大学机构动物护理和使用委员会（IACUC）。 1）预手术的准备<ol><li>口服溶液的制备<ol><li>创建10％蔗糖和0.005％L-薄荷醇在去离子水（pH 7.4）中的溶液。在密闭容器中储存这些溶液，在室温下，并重新制作每2周，以防止降解。 </li></ol></li><li>电极校准解决方案<ol><li>使Tris缓冲液（pH7.4）中，在校准之前的任何时间。该解决方案由12.0毫摩尔Tris盐酸，140 mM氯化钠，3.25毫米氯化钾，1.20毫氯化钙，1.25毫磷酸二氢钠，1.20毫的MgCl 2，和2.00毫摩尔的Na 2 SO 4。 </li><li>创建10mM的多巴胺（DA;盐酸多巴胺）的储备溶液在0.1M高氯酸。高氯酸有助于防止多巴胺的氧化即存储此原液在-20℃，并使用时间长达6个月。使DA原液存储使用每个等分试样只有一次的几个等分试样。 </li><li>从10毫米DA股票，稀释于Tris缓冲液为1微米，500纳米，250纳米，125纳米，62.5纳米，纳米31.25浓度。 </li><li>对于pH校准，准备的Tris解决方案7.2，7.3，7.5，和7.6的pH缓冲剂。这将提供了模仿体内实验过程中可能发生的pH值的变化的解决方案。 </li></ol></li><li>电极制造<ol><li>通过真空抽吸，吸一件T-650碳纤维（7微米直径）为硼硅玻璃毛细管（长10毫米，外径0.6mm时，内直径为0.4nm毫米）。 </li><li>将玻璃毛细管填充碳纤维成垂直的电极拉马。为初始参数，设置加热器以55.0和关闭磁铁。然而，进一步的优化可能是必要的，因为这两个加热器和磁设置的不同FROM实验室到实验室。 注意:电极应该是长（包括锥度），以便它可以容易地装配到显微和插入至少6.9毫米下面硬脑膜大鼠脑内至少5.5厘米。如果电极太短，录音从伏隔核的腹侧部将无法实现。另一方面，总电极长度不应超过6.5厘米否则会太长装配到显微操纵。 </li><li>使用光学显微镜，切开露出的碳纤维，使得它延伸75-100微米超出玻璃的末端。验证电极也非常紧张，几乎看不出来，玻璃电极和碳纤维，这将随后的录制过程中产生的噪音极小之间的密封。丢弃的电极，如果有任何明显的裂纹，在玻璃，或者如果密封件松动，其表示一个密封不良。有关更详细的说明，请参见[12]和[13]。 </li></ol></li><li>组装电极放入麦克风romanipulator <ol><li>获取沿导线长度的一半绝缘的3，30G导线和用小刷子施加一薄层的银直接绘制到电线。立即施加银涂料后，将导线插入在电极的顶部的孔，以提供相对于碳纤维的物理和电连接。在显微镜下，保证了银线，使与碳纤维接触。 </li><li>固定银线和电极用热缩微操作。 </li><li>将制备的碳纤维电极插入纯化异丙醇（IPA）为至少10分钟，以清洗电极表面并提高后续敏感性多巴胺。浸泡在IPA后，冲洗碳纤维用自来水除去IPA。 </li></ol></li><li>参比电极的制造<ol><li>以含有约6mm银/银氯目（7毫米独自银线，6个筛孔，0.4毫米直径）13毫米的银线，切银部向下到大约原来的一半长度和焊接导线的银部分的金销。 </li><li>环氧树脂应用在引脚上的焊料。 </li><li>浸银/银氯啮合端入5％聚四氟乙烯和的共聚物的全氟-3,6-二氧杂-4-甲基7octene磺酸5次，每次浸渍之间30分钟空气干燥周期。 </li><li>让涂银/银氯目空气干燥O / N。 </li><li>固化在烘箱中在120ºC1小时。 </li></ol></li></ol> 2）联合口内手术和颅内手术插管（约75分钟） <ol><li>口腔导管制造<ol><li>使口服导管，和通过环氧乙烷灭菌消毒，至少10天手术前。 </li><li>切PE管材100为6厘米长的段。 </li><li>使用烙铁，熔化的聚乙烯管的一端，并立即压在固体表面，以冷却的PE管上。结果应该创建一个小的，扁平的圆盘在PE管的一端，而不应是法兰。使用的针（18 G）或推销来创建在该盘的中心的孔。 </li><li>关于在PE管的另一端，紧贴地插入一个18G的针头插入管的钝端。 </li><li>使用标准纸打孔器，冲头几个环形件一个聚四氟乙烯薄片（3.18毫米厚，6毫米直径）来创建聚四氟乙烯垫圈。 </li><li>创建在垫圈的中心孔。取附着在PE管针和它完全通过垫圈推。一旦针是通过，滑动垫圈到PE管的底部，以便它是对垫圈平齐。 </li></ol></li><li>口腔外科<ol><li>消毒的外科手术工具，套管（修剪至2.5mm灭菌前基座下方），和参考电极后，麻醉大鼠腹膜内注射氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）的。 <ol><li>自动对焦第5分钟，施加一个有害刺激试验（英尺或尾巴捏）来评估麻醉的水平。如果受试者显示任何物理反应的有害刺激试验（退缩反应，增加呼吸或心脏速率），辖10％氯胺酮升压到原始剂量的15％，并重复上述有害刺激协议。 </li></ol></li><li>处理后大鼠完全麻醉，并显示没有反应的有害刺激测试中，使用动物毛发推剪，以从眼睛剃鼠的皮毛到耳朵后面约2毫米。然后放置在薄的循环水加热垫的大鼠在手术过程中保持体温。敷眼润滑剂。施用非甾体抗炎（NSAID）的药物，卡洛芬（5毫克/千克），通过腹膜内注射之前，执行任何切口并插入口内的插管之前。使用无菌技术，在任何时候都。 </li><li>应用优碘，接着用70％乙醇清洁皮肤。重复3次。 </li><li>解放军CE在它的后面的大鼠，并使用无菌棉签张口。找到第一个上臼齿。 </li><li>针插入脸颊和第一上臼齿之间的组织，直到针退出后面和内侧的耳朵。 </li><li>皮尔斯通过皮肤，直到在PE管针离开皮肤和可访问。 </li><li>上的套管（由套管本身，而不是针把持）拉起并固定聚四氟乙烯垫圈插入臼齿使得导管保持在适当位置。 注:切割垫片成梯形形状，确保其针对与平面侧对着面颊臼齿，使这一步容易。 </li><li>再举一个聚四氟乙烯垫片和滑动在暴露的针，顺着塑料管，并紧靠切口。 </li><li>以固定的聚四氟乙烯垫圈，使用灭菌胶带来创建一个三角形的"标志"，使垫圈不能向上滑动。固定洗尔对大鼠的皮肤和灭菌胶带。 </li><li>切开露出导管以允许2-4厘米管要高于鼠的头部露出。 </li><li>为口的另一侧重复步骤2.2.1至2.2.10。 </li></ol></li><li>颅内手术伏安<ol><li>放置在立体定位框架的大鼠和使用两阶段擦洗清洁动物的头皮（一个优碘擦洗，接着用70％的乙醇擦洗，用3周期重复执行）。 </li><li>使用消毒尖嘴镊子和手术剪剪去头皮组织的15×15mm的部分。 </li><li>轻轻擦拭消毒用棉尖涂药头骨的表面。通过将2〜3滴的3％的过氧化氢进一步清洁头骨。 </li><li>用立体演练，使3小（直径1毫米）的孔中头骨的螺丝后续插入，1孔为引导套管，1孔为刺激电极，和1孔为基准选骑着。 </li><li>对于记录在NAC核心，定位引导套管为1.2mm前，1.4毫米外侧前囟门。降低插管设备，直到塑料底座是与颅骨平。 </li><li>将灭菌的参考电极在对侧半球的导向导管。降低参考硬脑膜下面约2毫米。 </li><li>定位刺激电极在5.2毫米后至1.0mm外侧囟和降低8.0毫米下面硬脑膜靶向腹侧被盖区（VTA）。 </li><li>用灭菌小的手术螺​​丝刀拧紧螺丝的头骨。然后，将参比电极，刺激电极，引导插管。最后，使用cranioplastic水泥固定所有组件。 </li><li>对于最初的48小时的术后护理，通过皮下注射（5毫克/公斤）施用非甾体抗炎药（NSAID）卡洛芬。留出至少1周手术完全恢复执行voltam前metry记录。 <ol><li>在手术后的护理，口腔冲洗导管日常用水。提供食品中水饱和的2天，以帮助动物咀嚼和进食。提供日常监测疼痛的应力（由于轻度感染或脱水）潜在标志以及提供所需的适当的干预（包括施用流体，防腐剂的局部给药，和或NSAID卡洛芬施用5毫克/千克）。 </li></ol></li></ol></li></ol> 3）伏安录音清醒，自由移动鼠<ol><li>降低了电极和收购DA训练集。 <ol><li>在实验的当天，通过注入200微升水，并观察口颌面响应检查口腔插管的通畅（以验证大鼠品尝水）。 </li><li>通过将刺激电极（在探头）插入双极刺激电极cannu附加FSCV探头与大鼠拉（在大鼠）。因此，探头（小型化的电流 - 电压转换器）直接连接到所述双极刺激电极。 </li><li>从插管装置除去虚设插管并应用两滴50％肝素溶液进入插管。然后轻轻插入虚设套管（稍长的虚设套管先前移除）以清除任何血块或神经胶质疤痕可能已发生，这将打破随后将在实验过程中被插入的碳纤维的电极。延伸用于清除血块3-4毫米颅骨下方（至少2毫米以上的记录位点）的套管。 </li><li>将显微，与电极，到引导套管，并将欧米茄环在"锁定"的位置。 </li><li>从探头到相应的引脚连接参比电极丝。然后工作电极导线从显微连接到所述探头的相应导线。 </li><li>降低ELEC处接上下面的头骨大约4-4.5毫米。 </li><li>使用恒电位施加三角波形（400 V /秒，-0.4到1.3 V）。适用的波形到电极60赫兹，至少10分钟，然后在波形施加频率改变为10赫兹用于随后的实验记录。 注:60 Hz的波形应用程序步骤产生一个稳定的碳纤​​维表面，将有助于防止电气漂移</li><li>应用电刺激VTA中使用不同频率（10至60Hz）的脉冲（10至30个脉冲）和电流（50〜150微安）所有以2毫秒/相的脉冲宽度，以唤起不同浓度DA释放和pH值转变。获得至少5种不同浓度的DA释放的和5种不同的pH值的变化。等待（2 - 3分钟最小）之间的刺激，以便水泡重装[14]。 注意:得到能产生跨越，将在随后被收集的整个范围的DA浓度的刺激是重要实验中，因为它们将被用来作为主成分分析（参见5.1节）的训练组。 </li><li>降低电极进入伏隔核（6.3毫米腹侧硬膜）。接着降低电极的伏隔核内100μm递增直到瞬态DA释放事件被以至少1每分钟的频率观察到。 </li><li>管（内径1/32"，外径3/32"）连接到20ml的注射器，连接于注射器泵。在管的另一端，用一个斜面23政针的管路连接到老鼠的插管。确保油管与所需促味剂完全填充，并验证在管路中不存在气泡。 注意:通常，施用少量促味剂的到大鼠的嘴的记录，以确保在第一次输注用于记录提供的全部输液量而不是任何残留的水或空气中的管道之前。此外，这允许动物有一些以前的经验机智^ h小说以来的促味呈味物质，甚至是那些食欲，可以开始厌恶由于其新颖性。 </li><li>数据收集，注入200μl的促味剂的（6.5秒，使用一个泵和先前描述的管件）。注入的呈味物质每隔1-3分钟。注入总共每促味剂的25倍。每次输注提供200微升溶液。 </li><li>如果多个促味剂被交付在一个实验中，冲洗口腔导管，水收集数据对于一个呈味物质后，等待至少20分钟，注入新的呈味物质之前。 </li><li>在DA记录会议结束后，通过创建一个小的病灶在录制现场准备组织核查。为了保持对后续校准的碳纤维电极，先拉回电极，并删除微操作。然后用一个新的显微操作，用玻璃微电极和一个钨丝（延伸为100μm以外会使玻璃前端部）至（下面硬脑膜）相同的深度与原始碳˚Fiber微电极。 </li><li>对于组织学检验，通过施加3伏的钨丝和增加的电压在1伏的增量创建一个小的病灶，每10秒，直到10 V设置为止。 </li><li>如果一个标准浓度曲线已经使用多个碳纤维的电极产生，通过应用直接的潜在的碳纤维电极进行病变步骤上述（3.1.14）。 注意:此病变方法将损坏的碳纤维和防止后续校准与该特定电极。 </li><li>安乐死使用致命注射戊巴比妥（150毫克/千克，IP）的动物。 </li><li>打跑使用钳子通过在headcap直接拉动向上插管的headcap。不要扭曲headcap，因为这会导致大脑造成不必要的损害，并使其具有挑战性的组织学期间确定电极的位置。 <ol><li>取出大脑并放入4％的福尔马林进行3天，然后30％苏crose 1周。创建使用低温恒温器30微米切片，安装在盖玻片和检查切片，用光学显微镜，以确定碳纤维或钨病变的位置。 注:灌注是可选的3.1.17。 </li></ol></li></ol></li></ol> 4）电极校准<ol><li>用流动池来创建一个电流 - 浓度换算因子。 <ol><li>为了确定电极的灵敏度，校准用流动的"T细胞"装置每次实验后电极。降低电极进入"叔细胞"，而Tris缓冲液，pH值溶液，或DA溶液洗涤在电极表面（以2ml /分钟的速率）。使用六位置空气螺线管致动器，以缓冲和pH或DA溶液之间来回切换。 </li><li>对于每个校准，在此期间的Tris缓冲液（基线），接着pH值或DA溶液5秒应用5秒应用收集伏安数据，接着20秒的pplication的Tris缓冲液（30秒总文件）。每次重复经营的3次，每次校准标准浓度，不同浓度的各标准之间的制衡。 </li><li>对于每一个样品收集，测量峰DA或pH值诱发的电流。平均峰值电流在每个试验，以获得相对浓度峰值电流的关系。有关更详细的说明，请参见[12]和[13]。 </li></ol></li></ol> 5）数据分析<ol><li>使用训练在主成分分析集（PCA） 注意:在一个伏安实验中，输出被产生作为电流，这是多巴胺氧化和还原，pH值的变化，和电化学噪声的结果。 PCA允许这些因素分离，使得可以分离所需的组分（DA和pH下），并删除可能扭曲所需信号附加组分（为一个更详细的说明，请参阅第15，16）。 <ol><li>分配校正​​系数为thë输出电流（大约5-10 nA的/μM）的PCA之后，以允许要确定的分析物的浓度值。 </li><li>使用一个实验期间获得的训练集来"训练"的PCA模型中分离出的DA，pH和其他因素。 PCA取训练集循环伏安（收集在节3.1.8），并确定其伏安的本质特征可以被用来确定在体内伏安录音收集每种分析物。 </li><li>为了确定主成分保持的数目，使用一个斯基F-检验。通常情况下，只保留2或3通常在数据解释约95-99％的变异的主要组成部分。有关实现的马林诺夫斯基F检验的进一步讨论，请参见第17。 注意:一旦元件的数量被确定，在PCA分析有效将投射的测量伏安从DA和pH值训练集到该已保留的主要构成。其结果是，已被过滤，以便只有（DA或pH）的数据保持，并且不象DA或pH值的残留数据被除去（见预期输出代表结果）的数据集。有关使用PCA进行伏安分析进一步的详细说明，并分步说明见材料表，16，17。 </li></ol></li></ol>

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F., 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=Real-time+chemical+responses+in+the+nucleus+accumbens+differentiate+rewarding+and+aversive+stimuli).">Real-time chemical responses in the nucleus accumbens differentiate rewarding and aversive stimuli).</a> Nature Neuroscience. 11 (12), 1376-1377 (2008).</li><li>Cheer, J. F., 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=Phasic+dopamine+release+evoked+by+abused+substances+requires+cannabinoid+receptor+activation.">Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation.</a> J Neurosci. 27 (4), 791-795 (2007).</li><li>Owesson-White, C. A., 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=Neural+encoding+of+cocaine-seeking+behavior+is+coincident+with+phasic+dopamine+release+in+the+accumbens+core+and+shell.">Neural encoding of cocaine-seeking behavior is coincident with phasic dopamine release in the accumbens core and shell.</a> Eur J Neurosci. 30 (6), 1117-1127 (2009).</li><li>Phillips, P. E., 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=Subsecond+dopamine+release+promotes+cocaine+seeking.">Subsecond dopamine release promotes cocaine seeking.</a> Nature. 422 (6932), 614-618 (2003).</li><li>Chen, C., 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=Levels+of+mint+and+wintergreen+flavorants:+smokeless+tobacco+products+vs.+confectionery+products.">Levels of mint and wintergreen flavorants: smokeless tobacco products vs. confectionery products.</a> Food Chem Toxicol. 48 (2), 755-763 (2010).</li><li>Manning, K. C., Kelly, K. J., Comello, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavoured+cigarettes,+sensation+seeking+and+adolescents'+perceptions+of+cigarette+brands.">Flavoured cigarettes, sensation seeking and adolescents' perceptions of cigarette brands.</a> Tob Control. 18 (6), 459-465 (2009).</li><li>Rainey, C. L., Conder, P. A., Goodpaster, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characterization+of+dissolvable+tobacco+products+promoted+to+reduce+harm.">Chemical characterization of dissolvable tobacco products promoted to reduce harm.</a> J Agric Food Chem. 59 (6), 2745-2751 (2011).</li><li>Phillips, P. E., 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=Real-time+measurements+of+phasic+changes+in+extracellular+dopamine+concentration+in+freely+moving+rats+by+fast-scan+cyclic+voltammetry.">Real-time measurements of phasic changes in extracellular dopamine concentration in freely moving rats by fast-scan cyclic voltammetry.</a> Methods Mol Med. 79, 443-464 (2003).</li><li>Robinson, D. 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=.">.</a> Rapid Dopamine Release in Freely Moving Rats. Electrochemical Methods for Neuroscience. , (2007).</li><li>Montague, P. 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P., 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=Healthy+Young+Adults:+description+and+use+of+an+innovative+health+insurance+program.">Healthy Young Adults: description and use of an innovative health insurance program.</a> J Adolesc Health. 41 (4), 350-356 (2007).</li><li>Day, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+learning+mediates+dynamic+shifts+in+dopamine+signaling+in+the+nucleus+accumbens.">Associative learning mediates dynamic shifts in dopamine signaling in the nucleus accumbens.</a> Nature Neuroscience. 10 (8), 1020-1028 (2007).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catecholamines+in+the+bed+nucleus+of+the+stria+terminalis+reciprocally+respond+to+reward+and+aversion.">Catecholamines in the bed nucleus of the stria terminalis reciprocally respond to reward and aversion.</a> Biol Psychiatry. 71 (4), 327-334 (2012).</li><li>Wheeler, R. A., 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=The+neuroscience+of+pleasure.+Focus+on+'Ventral+pallidum+firing+codes+hedonic+reward:+when+a+bad+taste+turns+good.">The neuroscience of pleasure. Focus on 'Ventral pallidum firing codes hedonic reward: when a bad taste turns good.</a> J Neurophysiol. 96 (5), 2175-2176 (2006).</li><li>Bunin, M. A., 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=Release+and+uptake+rates+of+5-hydroxytryptamine+in+the+dorsal+raphe+and+substantia+nigra+reticulata+of+the+rat+brain.">Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of the rat brain.</a> J Neurochem. 70 (3), 1077-1087 (1998).</li><li>Zimmerman, J. 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=Simultaneous+electrochemical+measurements+of+oxygen+and+dopamine+in+vivo.">Simultaneous electrochemical measurements of oxygen and dopamine in vivo.</a> Anal Chem. 63 (1), 24-28 (1991).</li><li>Levy, A., 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=A+novel+procedure+for+evaluating+the+reinforcing+properties+of+tastants+in+laboratory+rats:+operant+intraoral+self-administration.">A novel procedure for evaluating the reinforcing properties of tastants in laboratory rats: operant intraoral self-administration.</a> J Vis Exp. (84), e50956 (2014).</li></ol>

The authors have nothing to disclose.

口腔内促味剂递送结合FSCV允许"实时"DA响应口服食用香料分析。但是也有一些需要成功的DA测量在协议三个关键步骤。首先，在口服导管的适当的注入是用于递送香味剂是至关重要的。确保导管插入第一磨牙后面和楔入就位防止导管从失去通畅，并防止意外删除由动物。在恢复后最初几天冲洗导管也很重要，因为在第一2或3天给予动物软食手术后会阻塞导管。第二，电极的质量提高信噪比以可自由?...

Erratum: Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Phasic DA release plays an important role in mediating reward-directed behavior [1-3]. However, isolating and studying how a primary reward alters phasic DA release is often complicated by co-occurring behavioral or cognitive processes that are also capable of altering phasic DA release - such as decision making processes or reward-directed motor behavior to acquire the reward. In the current work, we isolate phasic DA responses to tastants through the use of in vivo fast-scan cyclic voltammetry (FSCV) combined with tastant delivery through intraoral cannulae. This technique bypasses choice and action and allows us to examine extracellular DA release during direct infusion of a tastant to the palate of a rat.
FSCV is an electrochemical technique with high temporal and spatial resolution, permitting measurements of DA release in a discrete local area (approximately 100 &#181;m) on a sub-second scale (10 Hz resolution). The combination of intra-oral delivery and FSCV provides the advantage of observing rapid, 'real-time' DA responses to a tastant, which cannot be examined using conventional microdialysis methods. Furthermore, phasic DA release in response to either rewards or reward-associated cues occurs at concentrations between 20-100 nM, which is above the 10-20 nM detection threshold for FSCV [4]. The high spatial resolution of FSCV also permits recording from sub-regions of small brain areas, such as the nucleus accumbens core (NAc). Thus, FSCV combined with intraoral infusions of tastants is an ideal model for studying how tastants or other stimuli alter phasic DA release in an awake, behaving animals. Indeed, these techniques have permitted experiments investigating how rewarding and aversive tastants alter extracellular DA release [5]. 
Over the last decade, FSCV analyses have been successfully combined with intravenous drug delivery [6] and rat self-administration paradigms [7, 8] to identify the role of phasic DA mechanisms in drug addiction models. In addition, combined intraoral delivery with FSCV has been used to examine how tastant cues paired with cocaine availability modulate phasic DA release and behavioral responses that reflect emotional affect [3]. This combined intraoral and FSCV methodology can also be powerfully utilized to examine phasic DA responses to flavorants, such as menthol and oral sweeteners, that are added to cigarettes and dissolvable tobacco products [9-11]. Although many of the flavorants added to tobacco products are appetitive [9-11], it is unknown if these flavorants increase phasic DA release in a manner consistent with a rewarding hedonic valence. Indeed, flavorants added to cigarettes and to dissolvable tobacco products may have direct effects on the DA reward system and may act through dopaminergic mechanisms to influence the rewarding valence of cigarettes and other tobacco products. Thus, intraoral delivery combined with FSCV can provide new understanding on how flavorants modulate rapid DA release. The use of combined intra-oral and in vivo FSCV methodology, and the data obtained from such studies, can also facilitate future studies to determine how flavorants and nicotine interact to alter DA signaling and to potentially modulate nicotine reinforcement. Further, the data gained from such studies can be used to inform regulatory decisions about tobacco product flavorants.

<table><tbody><tr><td>Stimulating electrode</td><td>PlasticsOne</td><td>MS303/2-A/SPC</td><td>when ordering, request a 22 mm cut below pedestal</td></tr><tr><td>Cannula for electrode</td><td>BioAnalytical Systems</td><td>MD-2251</td><td></td></tr><tr><td>Glass capillary</td><td>A-M systems</td><td>624503</td><td></td></tr><tr><td>Carbon fiber</td><td>Thornel</td><td>T650</td><td></td></tr><tr><td>Electrode puller</td><td>Narishige International</td><td>PE-22</td><td></td></tr><tr><td>&lt;a href=&quot;http://digitimer.com/products/neurolog-system/&quot; target=&quot;_blank&quot;&gt;Neurolog stimulus isolator&lt;/a&gt;</td><td><a href="http://www.digitimer.com" target="_blank"><img src="/img/new_window.png" /> &lt;a href=&quot;http://digitimer.com&quot; target=&quot;_blank&quot;&gt;Digitimer Ltd.&lt;/a&gt;</a></td><td>&lt;a href=&quot;http://digitimer.com/products/research/stimulators/ds4-bi-phasic-stimulus-isolator/&quot; target=&quot;_blank&quot;&gt;DS4&lt;/a&gt;</td><td></td></tr><tr><td>Heat shrink</td><td>3M</td><td>FP-301</td><td></td></tr><tr><td>Insulated wires for electrodes</td><td>Squires electronics</td><td>Custom</td><td>L 3.000 x 1.000s x 1.000s UL1423 30/1 BLU</td></tr><tr><td>Micromanipulator</td><td>Univ. of Illinois at Chicago, Engineering Machine Shop</td><td></td><td></td></tr><tr><td>Epoxy</td><td>ITW Devcon</td><td>14250</td><td>&quot;5 minute epoxy&quot;</td></tr><tr><td>Copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid and tetrafluoroethylene</td><td>Ion Power</td><td>LQ-1105</td><td>&lt;em&gt;i.e.,&lt;/em&gt; Nafion</td></tr><tr><td>Silver paint</td><td>GC Electronics</td><td>22-023</td><td></td></tr><tr><td>Power supply</td><td>BK Precision</td><td>9110</td><td></td></tr><tr><td>Tubing for intra-oral catheters</td><td>Intramedic</td><td>427426</td><td>PE 100, I.D. = 0.86 mm; O.D. = 1.52 mm</td></tr><tr><td>Tubing for intra-oral infusion</td><td>Fischer Scientific</td><td>02-587-1A</td><td>I.D. 1/32&quot;; O.D. 3/32&quot;</td></tr><tr><td>Syringe pump for flow cell</td><td>Pump Systems Inc.</td><td>NE 1000</td><td></td></tr><tr><td>Surgical cement</td><td>Dentsply Caulk</td><td>675571 and 675572</td><td></td></tr><tr><td>Air actuator</td><td>VICI</td><td>A60</td><td>6 position digital valve interface</td></tr><tr><td>Digital valve interface</td><td>VICI</td><td>DVI</td><td>230 VAC</td></tr><tr><td>Quad headstage</td><td>Univ. of N. Carolina, Electronics Facility</td><td></td><td></td></tr><tr><td>UEI power supply</td><td>Univ. of N. Carolina, Electronics Facility</td><td></td><td></td></tr><tr><td>UEI breakout box</td><td>Univ. of N. Carolina, Electronics Facility</td><td></td><td></td></tr><tr><td>Power supply for tastant syringe pump</td><td>Med Associates</td><td>SG-504</td><td></td></tr><tr><td>Tastant syringe pump</td><td>Med Associates</td><td>PHM-107</td><td></td></tr><tr><td>Tungsten microelectrode</td><td>MicroProbes</td><td>WE30030.5A3</td><td></td></tr><tr><td>Silver wire reference with AgCl</td><td>InVivo Metric</td><td>E255A</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>80497</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Sodium chlroide</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Perchloric acid</td><td>Sigma</td><td>244252</td><td></td></tr><tr><td>Hydrochloric acid (4 M)</td><td>Sigma</td><td>54435</td><td></td></tr><tr><td>Soidum hydroxide</td><td>Sigma</td><td>306576</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma</td><td>H1009</td><td></td></tr><tr><td>Dopamine hydrochloride</td><td>Sigma</td><td>H8502</td><td></td></tr><tr><td>TarHeel HDCV Software</td><td>University of North Carolina-Chapel Hill</td><td></td><td>&lt;a href=&quot;http://www.chem.unc.edu/facilities/index.html?display=electronics&amp;amp;content=software&amp;amp;software_id=2&quot; target=&quot;_blank&quot;&gt;Must request software: click here for link to software request page&lt;/a&gt;</td></tr></tbody></table>

examination of rapid dopamine dynamics with fast scan cyclic voltammetry during intra-oral tastant administration in awake rats

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat &#8211; bypassing reward-directed behavior and voluntary drinking behavior &#8211; allowing for direct examination of DA responses to tastant stimuli.

FSCV结合口内导管植入用于研究如何蔗糖，食欲一个呈味物质，调节阶段性多巴胺释放在伏隔核。前促味剂输注，电刺激（150微安，60赫兹，24个脉冲;由红色条表示）VTA中的产生健壮增加相位DA释放在伏隔核（ 图1）图1示出一个彩色图上的潜力。 y轴，时间上的x轴，和电流（表示为伪色）上的z轴。下面的颜色情节DA浓度随时间变化的痕迹。浓度随时间的变化，使用的电流随时间跟踪在?...

Watch this Scientific Journal Video about Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats at JoVE.com

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

考试多巴胺快速与动态快速扫描循环伏安法在口内呈味物质管理清醒大鼠

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. ...

examination-rapid-dopamine-dynamics-with-fast-scan-cyclic-voltammetry

Universidad San Sebastian

Research

JoVE Journal

Behavior

12.6K 次观看.  Yale University. Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

视频: Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET:  Dopamine Movies of Cigarette Smoking

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8 that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7 to a conventional model 9. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

创建动态图像的短命多巴胺波动与LP-ntPET：多巴胺电影吸烟

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute ...

科研

行为学

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

一种新的方法为评估味质的实验室大鼠的补强性能：操作性口腔自我管理

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

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

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 amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

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 ...

神经科学

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

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 ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

雌性叙利亚仓鼠自然奖赏行为中细胞外神经递质的测量体内变化

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

联合输注和刺激与快速扫描循环伏安法（CIS-FSCV）评估腹侧被盖区域受体对相位多巴胺的调节

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

Source: Wickham, R. J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60886">Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine.</a> J. Vis. Exp. (2020).
This video demonstrates the implantation of electrodes and cannulas in an anesthetized rat to measure dopamine release in the nucleus accumbens using a potentiostat and the infusion of drugs to modulate dopaminergic activity in the ventral tegmental area.

使用 VTA 多巴胺能神经元受体作监测大鼠模型中的多巴胺释放

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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