用解剖学特异性单细胞基因表达方法研究成瘾行为中抗奖励的驱动因素
Summary
激光捕获显微切割和微流体RT-qPCR的组合为测量单个神经元和神经胶质细胞中的转录组提供了解剖学和生物技术特异性。将创造性方法与系统生物学方法应用于精神疾病可能会导致理解和治疗的突破，例如成瘾中的神经炎症抗奖励假说。
Abstract
成瘾行为率的增加促使心理健康研究人员和临床医生都了解反奖励和康复。这种从奖励和开始的转变需要新的视角，范式和假设，以及用于调查成瘾的方法的扩展。在这里，我们提供了一个示例：一种系统生物学方法来研究抗奖励，该方法结合了激光捕获显微切割（LCM）和高通量微流体逆转录定量聚合酶链反应（RT-qPCR）。测量了基因表达网络动力学，并确定了酒精和阿片类药物戒断中神经内脏失调的关键驱动因素，即神经炎症。这种技术组合以单细胞分辨率提供解剖学和表型特异性，具有高通量灵敏度和特异性基因表达测量，产生假设生成数据集和机制可能性，为新的见解和治疗创造机会。
Introduction
成瘾在发达国家仍然是一个日益严峻的挑战1，2。尽管科学和临床取得了重大进展，但成瘾率继续增加，而既定治疗方法的疗效最多保持稳定3，4，5。然而，生物技术和科学方法的进步导致了进一步研究物质依赖病理生理学的新方法和假设6，7，8。事实上，最近的发展表明，新的概念和治疗范式可能会导致具有社会，经济和政治后果的突破9，10，11，12。
我们调查了酒精戒断和阿片类药物依赖中的抗奖励13，14，15，16。方法是这种范式的核心17，18。激光捕获显微切割（LCM）可以选择具有高解剖特异性的单个细胞。此功能是神经炎症抗奖励假说不可或缺的一部分，因为神经胶质细胞和神经元都可以从同一动物的同一神经元亚核中收集和分析13，14，15，16，19。然后可以使用高通量微流体逆转录定量聚合酶链反应（RT-qPCR）测量所选细胞转录组的相关部分，为计算分析提供高维数据集，从而深入了解功能网络20，21。
测量特定脑核中神经元和神经胶质细胞中的转录组子集会生成一个数据集，该数据集在样本数量和测量的基因方面都是稳健的，并且具有敏感性和特异性。这些工具是系统治疗精神疾病的神经科学方法的最佳选择，因为神经胶质细胞，主要是星形胶质细胞和小胶质细胞，在过去十年中已在神经和精神疾病中发挥核心作用22，23。我们的方法可以测量神经胶质细胞和神经元在参与局部旁分泌信号传导的众多受体和配体上的表达反应。实际上，可以使用各种定量方法（例如模糊逻辑24）从这些数据集中推断信号。此外，识别神经元或神经胶质细胞中的细胞亚表型及其功能可以深入了解特定细胞核中的脑细胞如何在单细胞水平上组织、响应和失调。该功能系统的动力学也可以通过时间序列实验16进行建模。最后，动物模型可以在解剖学或药理学上受到干扰，为该系统的方法提供机械条件。
代表性实验：
下面，我们提供了这些方法的应用示例。本研究调查了大鼠神经元和小胶质细胞基因在孤立核（NTS）中对酒精依赖和随后的戒断的反应16。大鼠队列包括1）对照，2）乙醇依赖性（EtOH），3）8小时戒断（Wd），4）32小时Wd和5）176小时Wd（图1A）。快速斩首后，将脑干与前脑分离并冷冻切片，并对酪氨酸羟化酶阳性（TH +）神经元和小胶质细胞切片进行染色（图1B）。LCM用于收集TH+和TH-神经元以及小胶质细胞。所有细胞均来自NTS，并作为10个细胞池的样本进行分析。在测量65个基因的RT-qPCR平台上运行四个96 x 96微流体RT-qPCR动态阵列（图1B-C）。使用-ΔΔCt方法对数据进行归一化并使用R进行分析，并使用分子标记验证单细胞选择（图1D-E）。通过单批次和跨批次分析的技术重复进一步验证了技术验证（图2和图3）。TH+和TH-神经元组织成不同的亚表型，具有相似的炎症基因簇，但γ-氨基丁酸（GABA）受体（R）簇不同（图4和图5）。炎症基因簇表达升高的亚表型在32小时Wd时过度代表，而GABA受体（GABAR）表达在长期酒精戒断（176小时Wd）中保持低。这项工作有助于酒精和阿片类药物依赖的反奖励假说，该假说推测戒断时来自内脏的拦截反馈有助于内脏 – 情绪神经元核（即NTS和杏仁核）的失调，导致更严重的自主神经和情绪后遗症，从而导致物质依赖（图6）。
Protocol
Representative Results
Discussion
酒精使用障碍仍然是一种具有挑战性的疾病。我们的团队通过从系统神经科学的角度研究反奖励过程来研究这种疾病。我们测量了酒精戒断时间序列16中单个NTS神经元和小胶质细胞的基因表达变化。选择NTS是因为其在酒精戒断综合征中发生的自主神经失调中的突出作用。我们将LCM与单细胞微流体RT-qPCR相结合，允许以低成本测量稳定数量的样品和基因，具有解剖学和分子敏感性和?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这里介绍的工作由NIH HLB U01 HL133360授予JS和RV，NIDA R21 DA036372授予JS和EVB，T32 AA-007463授予Jan Hoek以支持SJO’S，以及国家酒精中毒和酒精滥用研究所：R01 AA018873资助。
Materials
| 20X DNA Binding Dye | Fluidigm | 100-7609 | NA |
| 2x GE Assay Loading Reagent | Fluidigm | 85000802-R | NA |
| 96.96 Dynamic Array IFC for Gene Expression (referred to as qPCR chip in text) | Fluidigm | BMK-M-96.96 | NA |
| Anti-Cd11β Antibody | Genway Biotech | CCEC48 | Microglia Stain |
| Anti-NeuN Antibody, clone A60 | EMD Millipore | MAB377 | Neuronal Stain |
| Anti-tyrosine hydroxylase antibody | abcam | ab112 | Stain for TH+ neurons |
| ArcturusXT Laser Capture Microdissection System | Arcturus | NA | NA |
| Biomark HD | Fluidigm | NA | RT-qPCR platform |
| Bovine Serum Antigen | Sigma-Aldrich | B4287 | |
| CapSure Macro LCM Caps | ThermoFisher Scientific | LCM0211 | NA |
| CellDirect One-Step qRT-PCR Kit | ThermoFisher Scientific | 11753500 | Lysis buffer solution components |
| CellsDirect Resuspension & Lysis Buffer Kit | ThermoFisher Scientific | 11739010 | Invitrogen |
| DAPI | ThermoFisher Scientific | 62248 | Nucleus Stain |
| DNA Suspension Buffer | TEKnova | T0221 | |
| Donkey anti-Rabbit IgG (H+L) ReadyProbe Secondary Antibody, Donkey anti-Rabbit IgG (H+L) ReadyProbe Secondary Antibody, Alexa Fluor 488 | ThermoFisher Scientific | R37118 | Seconadry Antibody |
| Exonuclease I | New Englnad BioLabs, Inc. | M0293S | NA |
| ExtracSure Sample Extraction Device | ThermoFisher Scientific | LCM0208 | NA |
| FisherbrandTM Superfrost Plus Microscope Slides | ThermoFisher Scientific | 22-037-246 | Plain glass slides |
| GeneAmp Thin-Walled Reaction Tube | ThermoFisher Scientific | N8010611 | |
| Goat anti-Mouse IgG (H+L), Superclona Recombinant Secondary Antibody, Alexa Fluor 555 | ThermoFisher Scientific | A28180 | Seconadry Antibody |
| IFC Controller | Fluidigm | NA | NA |
| RNaseOut | ThermoFisher Scientific | 10777019 | |
| SsoFast EvaGreen Supermix with Low Rox | Bio-Rad | PN 172-5211 | NA |
| SuperScript VILO cDNA Synthesis Kit | ThermoFisher Scientific | 11754250 | Contains VILO and SuperScript |
| T4 Gene 32 Protein | New Englnad BioLabs, Inc. | M0300S | NA |
| TaqMan PreAmp Master Mix | ThermoFisher Scientific | 4391128 | NA |
| TE Buffer | TEKnova | T0225 | NA |
| TempPlate Semi-Skirted 96-Well PCR Plate, 0.2 mL | USA Scientific | 1402-9700 | NA |
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