在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应
Summary
犁鼻器（VNO）检测种内的化学信号，传达社会和生殖信息。我们已经进行钙离子成像实验，利用转基因小鼠犁鼻组织中表达的G – CaMP2。这种方法可以让我们来分析复杂的犁鼻器神经元的反应模式，以大量的信息素的刺激。
Abstract
犁鼻器（VNO）检测，化学感应信号在同一物种的1,2个人的社会，性和生殖状态信息。这些种内的信号，信息素，以及来自一些食肉动物3的信号，激活高水平的特异性和灵敏度 4犁鼻感觉神经元（VSNS） 。 G蛋白偶联受体V1R，V2R和FPR 5-14，至少有三个不同的家庭表示在犁鼻神经元的调解检测的化学感应线索。为了了解信息素的信息是如何编码的VNO，它是分析个人VSNS响应各种刺激，并确定这些反应的特异性受体介导的关键。
VNO neuroepithelia括在一对犁骨头。 VNO半盲管状结构有一个开口端（犁鼻管）连接到鼻腔。 VSNS延长树突的犁鼻器信息素线索接触表达的受体在树突状旋钮管腔部分。 VSNS的细胞体的形式与V1R和V2R伪分层表示层分别6-8在根尖和基底层。几项技术已被用于监察VSNS 4,12,15-19感官刺激的反应。急性切片准备在这些技术中，有几个优点。首先，3,17分离VSNS相比，切片的筹备工作保持在其原生形态和细胞树突的神经元保持相对完好。二，VSNS胞体很容易在冠状片的VNO访问，允许电生理研究和成像实验相比，整个上皮细胞和整个安装准备工作 12,20 。第三，这种方法可以结合分子克隆技术，让受体识别。
感官刺激会引起强烈的Ca 2 + VSNS涌入，表明受体激活 4,21 。因此，我们开发的转基因小鼠的嗅感觉神经元，其中包括VSNS 15,22表达的G – CaMP2。探针的灵敏度和遗传性，极大地促进钙离子成像实验。这种方法消除了染料4,21以前的研究中使用的加载过程。我们还采用了配体传递系统，可应用各种刺激的VNO片。这两种技术的结合使我们能够监测多个神经元，同时响应大量的刺激。最后，我们已经建立了一个半自动化分析的管道，以协助图像处理。
Protocol
1。溶液的配制根据下表，准备R1的10X，10X的R2和10X R3的解决方案。 R1 化学品兆瓦（克/摩尔） 毫米（1X） 10倍的股票（G / L） 氯化钠 58.44 125 73.05 氯化钾 74.55 2.5 1.86 MgCl 2的 1中号股票 1 10毫升氯化钙2 …
Discussion
大多数的犁鼻器受体（VRS）保持作为，因为他们发现由杜拉克和Axel 5的孤儿受体。这些化学感应受体和他们在调解动物行为中的作用的信息素配体不能很好地理解。到现在为止，只有一对配体/受体，ESP1肽及其受体，Vmn2r116（V2Rp5），已确定并传达特定的社会信息，19,23。另一个受体，V1rb2，已被证明回应2 -庚酮，这大概是从雌性小鼠24尿液丰富。然而，此配体所传达的具体?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢安德烈莫兰连同实验动物服务设施（LASF）的成员，他们对畜牧业和技术服务的优秀支持在斯托瓦斯研究所。这项工作是支持，从斯托瓦斯研究所和国立卫生研究院（NIDCD 008003）资金，哭了。内容完全是作者的责任，并不一定代表美国国立耳聋与其他交流障碍研究所或国立卫生研究院的官方意见。美国正在申请专利的斯托瓦斯研究所，哭和LM的tetO – G CaMP2小鼠。
References
- Birch, M. C. . Pheromones. , (1974).
- Wyatt, T. D. Pheromones and animal behaviour : communication by smell and taste. , (2003).
- Papes, F., Logan, D. W., Stowers, L. The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. Cell. 141, 692-703 (2010).
- Leinders-Zufall, T. Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature. 405, 792-796 (2000).
- Dulac, C., Axel, R. A novel family of genes encoding putative pheromone receptors in mammals. Cell. 83, 195-206 (1995).
- Herrada, G., Dulac, C. A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution. Cell. 90, 763-773 (1997).
- Matsunami, H., Buck, L. B. A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell. 90, 775-784 (1997).
- Ryba, N. J., Tirindelli, R. A new multigene family of putative pheromone receptors. Neuron. 19, 371-379 (1997).
- Pantages, E., Dulac, C. A novel family of candidate pheromone receptors in mammals. Neuron. 28, 835-845 (2000).
- Zhang, X., Rodriguez, I., Mombaerts, P., Firestein, S. Odorant and vomeronasal receptor genes in two mouse genome assemblies. Genomics. 83, 802-811 (2004).
- Liberles, S. D. Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ. Proc. Natl. Acad. Sci. U.S.A. 106, 9842-9847 (2009).
- Riviere, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors. Nature. 459, 574-577 (2009).
- Yang, H., Shi, P., Zhang, Y. P., Zhang, J. Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats. Genomics. 86, 306-315 (2005).
- Rodriguez, I., Punta, D. e. l., Rothman, K., Ishii, A., T, ., Mombaerts, P. Multiple new and isolated families within the mouse superfamily of V1r vomeronasal receptors. Nat. Neurosci. 5, 134-140 (2002).
- He, J., Ma, L., Kim, S., Nakai, J., Yu, C. R. Encoding gender and individual information in the mouse vomeronasal organ. Science. 320, 535-538 (2008).
- Holy, T. E., Dulac, C., Meister, M. Responses of vomeronasal neurons to natural stimuli. Science. 289, 1569-1572 (2000).
- Chamero, P. Identification of protein pheromones that promote aggressive behaviour. Nature. 450, 899-902 (2007).
- Leinders-Zufall, T., Ishii, T., Mombaerts, P., Zufall, F., Boehm, T. Structural requirements for the activation of vomeronasal sensory neurons by MHC peptides. Nat. Neurosci. 12, 1551-1558 (2009).
- Kimoto, H., Haga, S., Sato, K., Touhara, K. Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons. Nature. 437, 898-901 (2005).
- Holekamp, T. F., Turaga, D., Holy, T. E. Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy. Neuron. 57, 661-672 (2008).
- Leinders-Zufall, T. MHC class I peptides as chemosensory signals in the vomeronasal organ. Science. 306, 1033-1037 (2004).
- He, J. Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ. J. Neurosci. 30, 7473-7483 (2010).
- Haga, S. The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor. Nature. 466, 118-122 (2010).
- Boschat, C. Pheromone detection mediated by a V1r vomeronasal receptor. Nat. Neurosci. 5, 1261-1262 (2002).
- Hendel, T. Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in. 28, 7399-7411 (2008).
- Pologruto, T. A., Yasuda, R., Svoboda, K. Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J. Neurosci. 24, 9572-9579 (2004).
- Jayaraman, V., Laurent, G. Evaluating a genetically encoded optical sensor of neural activity using electrophysiology in intact adult fruit flies. Front Neural Circuits. 1 (3), (2007).
- Tian, L. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods. 6, 875-881 (2009).
Tags
ImagingNeuronal ResponsesSlice PreparationsVomeronasal OrganGenetically Encoded Calcium SensorChemosensory SignalsSocialSexualReproductive StatusSpeciesPheromonesPredatorsVomeronasal Sensory NeuronsSpecificitySensitivityG-protein Coupled ReceptorsV1RV2RFPRDetection Of Chemosensory CuesPheromone Information EncodingResponse ProfilesStimuliSpecific ReceptorsNeuroepithelia Of VNOVomer BonesSemi-blind Tubular StructureVomeronasal DuctNasal CavityDendritesLumen Part Of VNOPheromone Cues Contact With ReceptorsCell Bodies Of VSNs Form Pseudo-stratified Layers
Cite This Article
Ma, L., Haga-Yamanaka, S., Yu, Q. E., Qiu, Q., Kim, S., Yu, C. R. Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor. J. Vis. Exp. (58), e3404, doi:10.3791/3404 (2011).