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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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...

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.

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</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

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

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

清醒的行为小鼠慢性神经录音的微型硬盘阵列建设

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

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 ...

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

巴恩斯迷宫测试策略与小型和大型啮齿类动物模型

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

平地板的空气解禁平台：一种新方法与显微镜或电对清醒结合行为自由移动鼠害

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

一个过程来观察大鼠巴甫洛夫空调酒精寻求行为的背景下引发的重建

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, an understanding of how perceptions of social exclusion develop over the course of a social interaction is important for advancing treatments aimed at lessening the harmful costs of being excluded. To date, most scientific examinations of social exclusion have looked at exclusion after a social interaction has been completed. While this has been very helpful in developing an understanding of what happens to a person following exclusion, it has not helped to clarify the moment-to-moment dynamics of the process of social exclusion. Accordingly, the current protocol was developed to obtain an improved understanding of social exclusion by examining the patterns of event-related brain activation that are present during social interactions. This protocol allows greater precision and sensitivity in detailing the social processes that lead people to feel as though they have been excluded from a social interaction. Importantly, the current protocol can be adapted to include research projects that vary the nature of exclusionary social interactions by altering how frequently participants are included, how long the periods of exclusion will last in each interaction, and when exclusion will take place during the social interactions. Further, the current protocol can be used to examine variables and constructs beyond those related to social exclusion. This capability to address a variety of applications across psychology by obtaining both neural and behavioral data during ongoing social interactions suggests the present protocol could be at the core of a developing area of scientific inquiry related to social interactions.

Research on social exclusion has grown tremendously in recent years. As the field expands, it is imperative to develop sophisticated methodologies allowing for the simultaneous measurement of neural and behavioral outcomes during social exclusion. This protocol utilizes event-related brain potentials to record ongoing neural activity during computerized social interactions.

测量神经和行为活动期间持续计算机化的社会互动：对事件相关电位的考察

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

修改后的孔板 - 测量行为，认知和社会互动小鼠和大鼠

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.