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
Summary
该协议的目标是直接操纵腹侧被盖区域受体，以研究它们对亚秒多巴胺释放的贡献。
Abstract
相位多巴胺（DA）从腹侧被盖区（VTA）释放到伏隔核在奖励处理和强化学习中起着关键作用。了解VTA控制相位DA释放中的各种神经元输入如何能够更好地反映控制奖励处理和强化学习的电路。在这里，我们描述了一种将药物激动剂和拮抗剂的VTA插管内输注与刺激诱发的相位DA释放（联合输注和刺激，或CIS）相结合的方法，该方法通过体内快速扫描循环伏安法（FSCV）测量。在麻醉大鼠中使用CIS-FSCV，可以通过用装有套管的双极电极电刺激VTA来引起相位DA反应，同时记录在伏隔核核心中。药理激动剂或拮抗剂可以直接输注在刺激部位，以研究特异性VTA受体在驱动相位DA释放中的作用。CIS-FSCV的一个主要好处是VTA受体功能可以在体外研究的基础上在体内进行研究。
Introduction
相位多巴胺（DA）从腹侧被盖区（VTA）释放到伏隔核（NAc）在奖励相关行为中起着至关重要的作用。VTA DA神经元从强直性放电（3-8 Hz）切换到突发式放电（>14Hz）1，从而在NAc中产生相位DA释放。VTA表达多种躯体外胚层受体，这些受体处于有利地位，可以控制从强直到爆发发射的转变2，3，4，5。确定这些受体中的哪些以及它们各自的输入控制相位DA释放将加深我们对奖励相关电路如何组织的理解。这里描述的方法，结合输注和刺激与快速扫描循环伏安法（CIS-FSCV）的目的是快速而稳健地评估VTA受体在驱动相位DA释放方面的功能。
术语联合输注和刺激（CIS）是指在药理学上操纵一组神经元（此处为VTA）上的受体并刺激这些神经元以研究受体的功能。在麻醉的大鼠中，我们电刺激VTA以在NAc核心中唤起大相DA信号（1-2μM），如快速扫描循环伏安法（FSCV）测量的那样。在刺激部位输注药物（即受体激动剂/拮抗剂）可用于通过观察诱发的相位DA释放的后续变化来测量VTA受体的功能。FSCV是一种电化学方法，具有高空间（50-100μm）和时间（10 Hz）分辨率，非常适合测量与奖励相关的相位DA事件6，7。该分辨率比其他体内神经化学测量（如微透析）更精细。因此，CIS-FSCV一起非常适合评估VTA受体对相位多巴胺释放的调节。
研究VTA受体功能的一种常见方法是使用电生理学方法的组合来解决这些受体如何改变神经元的放电速率1，8。这些研究对于了解哪些受体参与驱动DA激活时放电非常有价值。然而，这些研究只能表明在轴突末端下游可能发生的情况（即神经递质的释放）。CIS-FSCV建立在这些电生理学研究的基础上，通过回答VTA爆裂放电，相位DA释放的输出如何受到位于VTA树突和细胞体上的受体的调节。因此，CIS-FSCV非常适合在这些电生理学研究的基础上进行。例如，烟碱受体激活可以在VTA9中诱导爆裂，并且使用麻醉大鼠中的CIS-FSCV来证明烟碱乙酰胆碱受体（nAChR）在VTA中的激活也控制NAc10，11中的相位DA释放。
相位DA调节的机制检查也通常使用切片制剂以及药物的浴液应用进行研究。这些研究通常集中在从多巴胺末端释放的相位DA的突触前调节上，因为细胞体通常从切片12中移除。这些制剂对于研究突触前受体对多巴胺末端的影响很有价值，而CIS-FSCV更适合研究躯体突触受体对多巴胺神经元的影响，以及VTA的突触前输入。这种区别很重要，因为VTA中的体外胚层受体激活可能与NAc突触前受体激活具有不同的效果。事实上，阻断NAc中的多巴胺能突触前nAChRs可以提高爆发13期间的相位多巴胺释放，而VTA somatodendritc nAChRs10，11则相反。
CIS-FSCV是研究VTA受体调节相位DA释放能力的理想方法。重要的是，这种方法可以在完整的大鼠中进行，无论是麻醉还是自由移动。这种方法适用于急性研究，研究基线状态10，14下的受体功能以及可以评估药物暴露或行为操作后受体功能变化的长期研究11，15。
Protocol
所有实验均根据美国国立卫生研究院（NIH）实验动物护理和使用指南进行，并得到伊丽莎白镇学院和耶鲁大学机构动物护理和使用委员会（IACUC）的批准。该协议特定于利用CIS-FSCV的麻醉大鼠制剂。 1. 术前准备 电极溶液制备 为了使电极回填溶液，制备4M醋酸钾与140mM氯化钾16的溶液。 电极制备 使用真空抽吸，将T-650碳纤维…
Representative Results
CIS-FSCV用于研究VTA N-甲基-D-天冬氨酸受体（NMDAR），烟碱乙酰胆碱受体（nAChRs）和毒蕈碱乙酰胆碱受体（mAChRs）在驱动NAc核心中相位DA释放中的功能。 图2 显示了阴性对照的代表性数据，输注0.9%盐水，在输注前（基线）和输注后9分钟（生理盐水）。 图2 显示了一个颜色图，y轴上有电位，x轴上有时间，z轴上有电流（表示为假色?…
Discussion
CIS-FSCV为研究相位DA释放的潜在VTA受体机制提供了独特的机会。为了确保正确录制，有两个关键步骤。首先，必须实现稳定的基线记录，并且诱发的DA信号漂移很小。增加建立稳定记录的可能性的一个重要方法是确保电极有足够的时间在60 Hz和10 Hz下循环（通常在60 Hz下为15分钟，在10 Hz下为10分钟）。当碳纤维被循环时，碳纤维本身氧化并变得蚀刻，减少表面积，但产生用于多巴胺吸附?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
工作得到了伊丽莎白镇学院（R.J.W，M.L.和L.M），NSF研究生奖学金（R.J.W.）和耶鲁大学医学院（N.A.）的支持。
Materials
| Electrode Filling Solution/Supplies | |||
| Micropipette | World Precision Instruments | MF286-5 (28 gauge) | |
| Potassium Acetate | Sigma | 236497-100G | |
| Potassium Chloride | Sigma | P3911-25G | |
| Electrode Supplies | |||
| Carbon fiber | Thornel | T650 | |
| Electrode puller | Narishige International | PE-22 | Note: horizontal pullers can be used as well |
| Glass capillary | A-M systems | 626000 | |
| Insulated wires for electrodes | Weico Wire and Cable Incorporated | UL 1423 | Length; 10 cm; diameter,0.4mm; must get custom made; insulated material should cover 5 cm of the wire |
| Light Microscope (for viewing and cutting electrode) | Fischer Scientific | M3700 | |
| Pin | Phoenix Enterprises | HWS1646 | To be soldered onto the insuled electrode wire and reference electrode; connects to headstage |
| Putty | Alcolin | 23922-1003 | Used to place electrode on while cutting the carbon fiber |
| Scalpal Blade | World Precision Instruments | 500239 | For cutting carbon fiber to the apprpriate length |
| Silver Wire | Sigma | 327026-4G | |
| FSCV Hardware/Software | |||
| Faraday Cage | U-Line | H-3618 (36" x 24" x 42") | |
| Potentiostat | Univ. of N. Carolina, Electronics Facility | ||
| Stimulating electrode | PlasticsOne | MS303/2-A/SPC | when ordering, request a 22 mm cut below pedestal |
| TarHeel HDCV Software | University of North Carolina-Chapel Hill | – | https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information |
| UEI breakout box | Univ. of N. Carolina, Electronics Facility | https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information | |
| UEI power supply | Univ. of N. Carolina, Electronics Facility | https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information | |
| Stimulator Hardware | |||
| Neurolog stimulus isolator | Digitimer Ltd. | DS4 | Neurolog 800A |
| Infusion/Stimulation Supplies | |||
| Infusion Pump | New Era Syringe Pump | NE-300 | |
| Internal Cannula | PlasticsOne | C315I/SPC INTERNAL 33GA | |
| Microliter Syringe | Hamilton | 80308 | |
| Tubing | PlasticsOne | C313CT/ PKG TUBING 023 X 050 PE50 | |
| Surgical Supplies | |||
| Cannula Holder | Kopf Instruments | 1776 P-1 | |
| Cotton Tip Applicators | Vitality Medical | 806 | |
| Electrode Holder | Kopf Instruments | 1770 | |
| Heating Pad | Kent Scientific | RT-0501 | |
| Povidone Iodine | Vitality Medical | 29906-004 | |
| Screws | Stoelting | Bone Anchor Screws/Pkg.of 100 | 1.59 mm O.D., 3.2 mm long |
| Silver wire reference with AgCl | InVivo Metric | E255A | |
| Square Gauze | Vitality Medical | 441408 | |
| Stereotax | Kopf Instruments | Model 902 (Dual Arm Bar) | |
| Histological Supplies | |||
| Formulin | Sigma | 1004960700 | |
| Power supply | BK Precision | 9110 | |
| Sucrose | Sigma | 80497 | |
| Tungsten microelectrode | MicroProbes | WE30030.5A3 | |
| Drugs for infusions | |||
| ((2R)-amino-5-phosphonovaleric acid | Sigma Aldrich | A5282 | |
| N-methyl-D-aspartate | Sigma Aldrich | M3262 | |
| Mecamylamine hydrochloride (M9020-5mg) | Sigma Aldrich | M9020 | |
| Scopolamine hydrobromide (S0929-1g) | Sigma Aldrich | S0929 |
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Cite This Article
Wickham, R. J., Lehr, M., Mitchell, L., Addy, N. A. Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine. J. Vis. Exp. (158), e60886, doi:10.3791/60886 (2020).