光谱共焦成像的荧光标记的尼古丁受体敲在慢性尼古丁政府小鼠
Summary
我们已经开发出一种量化烟碱型乙酰胆碱受体亚地区的中枢神经系统神经元的具体亚型内的变化，更好地了解使用爆震的方法和光谱用荧光蛋白标记的受体相结合的办法，包括对尼古丁成瘾的机制的新技术共聚焦成像。
Abstract
配体门控离子通道，在中枢神经系统（CNS）有牵连的有严重的医疗和社会后果的许多条件。例如，通过吸烟尼古丁成瘾的首要原因是全球过早死亡（世界卫生组织）和可能造成的离子通道分布在大脑中的变更1。慢性尼古丁暴露于啮齿动物和人类的烟碱乙酰胆碱受体（nAChRs）的脑组织1-3人数增加的结果。同样，在谷氨酸GluN1或GluA1渠道，改变已牵连到其它能使人成瘾的药物，如可卡因，安非他明和鸦片4-6引发过敏。
因此，映射和量化特定的离子通道的分布和表达模式的能力是极为重要的理解成瘾的机制。研究大脑的特定区域的EF个别药物fects先进的技术，如放射性配体的问世。然而，低放射性配体结合的空间分辨率，防止量化在特定的神经元亚型配体门控离子通道的能力。
基因编码的荧光记者，如绿色荧光蛋白（GFP）和它的许多颜色的变种，7生物学领域的一个革命性的基因标记内源性蛋白，可以可视化的蛋白质在体内 7-10荧光记者。用探针荧光标记蛋白质的优势之一是消除抗体的使用，有目标蛋白的非特异性和无障碍的问题。我们已经用这种战略荧光标签nAChRs，这使福斯特共振能量转移（FRET）在转染细胞培养11受体装配研究。最近，我们已经使用了KNOCK-工程师与黄色荧光蛋白标签胆碱受体α4亚基（α4YFP）的，能够精确量化的亚微米决议通过光谱共焦显微镜12中枢神经系统的神经元的受体体外小鼠的方法。针对性的荧光敲在突变中的内源性的轨迹和其本地子的控制之下，成立生产受体的表达和调控的正常水平相比，在野生型小鼠无标记的受体时。这个连锁反应中的方法可以扩展到其他离子通道的荧光标记，并提供了一个可视化和量化受体在中枢神经系统的功能强大的方法。
在本文中，我们描述了一种方法来量化在特定的中枢神经系统神经元暴露后慢性尼古丁胆碱受体表达的变化。我们的方法包括微型渗透泵植入术，心内灌注固定，成像和荧光标记的烟碱REC分析eptor亚基从α4YFP敲入小鼠（图1）。我们已经优化了固定技术，从固定脑tissue.We的，以尽量减少自体荧光，在详细描述我们的成像方法，利用光谱共聚焦显微镜结合线性光谱分离算法减去autofluoresent为了准确地获取α4YFP荧光信号的信号。最后，我们表明慢性尼古丁诱导的海马内侧perforant的路径α4YFP受体上调的结果。
Protocol
1。泵植入术泵植入前，填写，并准备在Alzet微型渗透泵（Alzet，型号2002年，库珀蒂诺，美国），小心不要引入气泡。这种微型渗透泵的模型提供了0.5微升/小时的速度在14天的解决方案。保证无菌的条件。权衡空和填充泵。在实验（移植后10天）结束，剩余的液体在泵可去除注射器和针头，称重，计算容积泵。 泵与控制解决方案包含生理盐水（0.9％W / V，Teknova，S5819，霍利斯特，美国…
Discussion
<p class="jove_content">在敲在小鼠模型，以确定一个特定的离子通道的数量和本地化的荧光受体的使用提供了许多优势。相反，如肌动蛋白，它是无处不在所有细胞中表达的蛋白质，离子通道是目前人数要少得多，并作出准确的分析，通过免疫组化技术挑战传统的神经元亚型之间的表达变化。在α4YFP基因产物表达野生型水平相同的启动子，增强和贩卖机制的控制下，<sup> 12</sup>。荧光受体也显示了相同的WT离子通…
Disclosures
The authors have nothing to disclose.
Acknowledgements
安东尼仁达支持由维多利亚大学研究生奖学金奖。这项研究是由自然科学和工程研究理事会，加拿大发现格兰特，NARSAD青年研究者奖（RN）的维多利亚基金会 – Myre和王佩瑜SIM基金，创新补助金，加拿大不列颠哥伦比亚省知识发展基金，基金会的支持一个自然的科学和工程研究理事会，加拿大研究工具和仪表格兰特。我们感谢优秀的鼠标畜牧业吉利安麦基，克里斯蒂娜巴恩斯，阿里尔·沙利文，珍妮弗·麦克唐纳和丹尼尔·莫尔加多。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| mini-osmotic pumps | Alzet | model 2002 | |
| saline | Teknova | S5819 | |
| (-)-nicotine hydrogen tartrate salt | Sigma | N5260 | |
| eye drops | Novartis | Tear-Gel | |
| Vetbond glue | 3M | 1469SB | |
| heparin sodium salt | Sigma | H4784 | |
| 10x PBS | Invitrogen | 70011 | |
| ketamine | Wyeth Animal Health | 0856-4403-01 | |
| medatomidine hydrochloride | Pfizer | 1950673 | |
| 23G butterfly needle | Becton Dickinson | 367253 | |
| paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| plastic embedding mold | VWR | 18986-1 | |
| O.C.T. Mounting Compound | Tissue-Tek | 4583 | |
| Mowiol 4-88 | EMD-Calbiochem | 475904 | pH 8.5 |
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Tags
Spectral Confocal ImagingFluorescently Tagged Nicotinic ReceptorsKnock-in MiceChronic Nicotine AdministrationLigand-gated Ion ChannelsCentral Nervous System (CNS)Addiction To NicotineIon Channel DistributionChronic Nicotine ExposureNicotinic Acetylcholine Receptors (nAChRs)Glutamatergic GluN1GluA1 ChannelsSensitization To Addictive DrugsMap And Quantify DistributionExpression PatternsSpecific Ion ChannelsMechanisms Of AddictionBrain Region-specific EffectsRadioactive LigandsLow Spatial ResolutionLigand-gated Ion Channels In NeuronsGenetically Encoded Fluorescent Reporters
Cite This Article
Renda, A., Nashmi, R. Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration. J. Vis. Exp. (60), e3516, doi:10.3791/3516 (2012).