鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录：神经元表达鉴定的气味受体功能分析
Summary
Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.
Abstract
当与特定的配体刺激的判断嗅感觉神经元的生理反应（OSN）关键是理解嗅觉驱动行为和其调制的基础。这些编码特性在很大程度上依赖于臭味分子和嗅觉受体（OR）之间的初始相互作用表达在嗅觉神经元。的表达，或者关键的身份，特异性和配体谱。找到或配体的可能性表示在内的上皮随机选择一个OSN很低。为了应对这一挑战，这个协议使用基因表达定义的OR的启动子控制下的荧光蛋白标记的小鼠。嗅觉神经元位于在紧张的和有组织的上皮衬里鼻腔，与邻近细胞影响他们的成熟和功能。这里，我们描述的方法来分离完整嗅上皮，并通过膜片钳记录记录嗅觉神经元e的特性xpressing定义的气味受体。该协议允许一个同时保持相邻组织的影响表征OSN膜特性。膜片钳的结果分析产生的配体/或相互作用，转导途径和药理学，嗅觉神经元“编码性质和它们在膜级调制的精确定量。
Introduction
嗅觉感觉神经元（OSN）代表嗅觉感知的第一步骤。位于嗅觉上皮衬里啮齿动物的鼻腔，他们变换气味物质的化学信息转换成通过他们的轴突传送到大脑的动作电位。为了更好地理解嗅觉编码机制，有必要表征嗅觉神经元的转导和膜特性。直到最近，大多数用于表征哺乳动物的嗅觉神经元的性能的技术进行了分离上1-4嗅觉神经元。解离过程中使用的各种机械和化学（ 即酶）进程从环境释放的嗅觉神经元。这些过程可引起细胞的记录数量较少。这种低数量可以甚至在GFP标记的细胞的情况下更加重要。离解也除去嗅觉神经元和嗅觉上皮的可以提高surviv其它细胞之间的局部细胞 – 细胞相互作用人与嗅觉神经元“性质的调制。为了绕过分离程序，一个完好的制剂被开发5。
每个OSN表达一种嗅觉受体（OR）从一个大的多基因家族6中选择。有〜表示在小鼠的主要嗅觉上皮1000 OR值。由于在野生型动物的大量或，机会来记录嗅觉神经元表达相同或非常低。为了克服这些限制，基因靶向的小鼠可在其中表达所识别或标记有荧光蛋白7-9所有嗅觉神经元。这些标记的嗅觉神经元被用来做功能分析分离的筹备与7,10,11前面提到的缺点。因此，一个完整的上皮细胞的准备从5标记基因小鼠规避这些问题。它允许嗅觉神经元表达精确定义的OR 值的环境中尽可能接近在vi的活动的监控沃越好。此外，嗅觉神经元的膜片钳记录也允许膜性能，转导途径药理学，配体/或相互作用的精确分析。所有这些话题几乎不能使用外记录进行分析。我们使用这种技术来监控嗅觉神经元表达气味受体SR1和MOR23 12,13的响应。该技术的可行性，通过MOR23其他群体表达嗅觉神经元14以及上表达神经元15,16等手术室证实。限定嗅觉神经元的人口的监视可以导致它们的性质在许多不同的情况下，例如显影14的分析，老化17，臭气物质诱发塑性18，和变化中的气味受体的序列中的气味编码15的作用。这个协议从而提供了一个有力的工具在膜级监测定义嗅觉神经元的功能特性。
Protocol
该协议遵循UNIVERSITE勃艮第的动物护理准则和批准了UNIVERSITE勃艮第伦理委员会。 1.动物使用可在杰克逊实验室基因工程OR-IRES-tauGFP小鼠。这些小鼠以分析轴突靶向嗅觉系统19的和发展开发彼得Mombaerts博士的实验室。例如，MOR23-IRES-tauGFP行，股票号码006643，耐官方应变名B6; 129P2- Olfr16 tm2Mom / MomJ;同样，SR1-IRES-tauGFP行，股票编号6717负有正式名称B6; 129P2-…
Representative Results
本协议的结果取决于解剖的质量。此清扫步骤必须短（少于10至15分钟）和精确的（ 即，避免了上皮的损害）。 图1说明了一个理想的准备看起来像在不同的放大级别。在明亮的下一个领域低倍率不同的细胞类型（如嗅觉神经元的旋钮，支持细胞）是有区别的（ 图1A）。在最高放大级别，通常为80X 160X，在明亮的领域，嗅觉神经元的树突状旋钮应该是从支持细胞（ <s…
Discussion
这个协议的正确监测健康嗅觉神经元的性质的能力在很大程度上依赖于制剂的质量。因此，清扫步骤是关键的。首先关键是要注重质量（pH值，渗透压摩尔浓度），氧和温度（冰冷的，但不冻结）夹层介质。第二，与解剖工具上皮的操作必须尽可能限制，以避免损坏。最后，这是至关重要的，以获得以访问最大OSN人口尽可能制剂尽可能平坦。
解剖质量是根本，获得一个健康的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil Régional de Bourgogne (FABER and PARI grants), by Université de Bourgogne (BQR program).
Materials
| heavy equipment | |||
| vibration table with Faraday cage | TMC | 63-500 SERIES | required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment |
| optics | |||
| microscope | Olympus | BX51WI | upright microcope equipped with epifluorescence; fixed or moving stage depending on the user's preference |
| objectives | Olympus | LUMPLFL40XW | at least 2 objectives required: a 4x or 10x for coarse approach to the cell; and a 40x immersion long distance example Olympus LUMPLFL40XW/IR/0,8/WD:3.3 MM |
| magnifier | Olympus | U-TVCAC | ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode |
| camera | Olympus | DP72 | a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary |
| filters | Olympus/Chroma | depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m | |
| recording electrodes /system | |||
| amplifier | HEKA | EPC10 USB | monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer |
| software | HEKA | Patchmaster | controls the amplifier during the experiment |
| micromanipulator | Sutter | MP225 | precision micromanipulator, allows precise movements down to 1/1Oth of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift |
| electrode puller | sutter | P97 | with a FT345-B wide trough filament; to prepare recording pipets of about 2µm diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution |
| glass | sutter | BF120-69-10 | in our recording conditions, this glass is ideal for recording pipets |
| recording chamber | warner instruments | RC-26G | a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used. |
| stimulation | |||
| glass | WPI | TW100F-4 | attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes |
| multibarrel puller | MDI | PMP-107-Z | by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels |
| precision pressure injector | Toohey Company | P/N T25-1-900 Single Channel | this precision pressure injector controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V TTLs |
| micromanipulator | Narishige | YOU-1 | a coarse manipulator is enough to bring the puffing electrode close to the recording site |
| tubings | N/A | tygon tubing to bring the pressure from the puffer to the puffing pipette | |
| solutions/perfusion/chemicals | |||
| vacuum pump | gardner denver | 300 series | a vibrating membrane pump is more quiet and efficient than other types of pumps |
| perfusion system | N/A | N/A | gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber |
| nystatin | Sigma-Aldrich | N3503 | mandatory to perpare internal solution for perforated patch clamp |
| DIMETHYL SULFOXIDE | Sigma-Aldrich | D5879 | used to disolve nystatin for internal solution for perforated patch |
| Sodium chloride | Sigma-Aldrich | S9625 | extracellular solution |
| Potassium chloride | Sigma-Aldrich | P4504 | intracellular/extracellular solution |
| Calcium chloride di hydrate | Sigma-Aldrich | C7902 | extracellular solution |
| Sodium phosphate monobasic monohydrate (NaH2PO4) | Sigma-Aldrich | S9638 | extracellular solution |
| Magnesium sulfate heptahydrate (MgSO4 7H2O) | Sigma-Aldrich | 63140 | extracellular solution |
| Glucose | Sigma-Aldrich | G8270 | extracellular solution |
| Sodium bicarbonate | Sigma-Aldrich | S6297 | extracellular solution |
| EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) | Sigma-Aldrich | E3889 | internal solution |
| Potassium hydroxyde | Sigma-Aldrich | P1767 | internal solution |
| MethylSulfoxide | Sigma-Aldrich | 47,135-6 | intracellular solution |
| Hepes-Na | Sigma-Aldrich | H7006 | intracellular solution |
| Sucrose | Sigma-Aldrich | S0389 | intracellular solution |
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Cite This Article
Jarriault, D., Grosmaitre, X. Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor. J. Vis. Exp. (101), e52652, doi:10.3791/52652 (2015).