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 R&#233;gional de Bourgogne (FABER and PARI grants), by Universit&#233; de Bourgogne (BQR program).

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<ol><li>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- Olfr124 tm1Mom / MomJ。 </li><li>关于动物的年龄：用于协议的一个更好的结果，使用2和4周龄之间的动物。在这个年龄组中，清扫容易（较软的骨头，更坚定的嗅上皮）和树突状旋钮都是双向gger比较年长的动物。 </li></ol> 2.制备电极和解决方案<ol><li>对于刺激移液器：购买prepulled刺激移液器。否则，手动准备他们。 <ol><li>使用火焰，弯曲61毫米玻璃吸管从尖端约1厘米。插入这六个移液器加直7 日在1.5厘米热缩管通过小孔加强。 </li><li>热缩管，以维护连接在一起的桶。附加一个额外的热缩管到的桶的另一端。用多管拉出器拉出此刺激吸管。 </li><li>添加一些白色液体胶水周围的小孔，以加强弯曲的提示。让干燥O / N。 </li></ol></li><li>制备1或2升的正常林格氏胞外溶液（以mM计：氯化钠124，氯化钾3，用MgSO 4 1.3， 氯化钙 2， 碳酸氢钠 26，将NaH 2 PO 4 1.25，葡萄糖15; pH值7.6和305毫渗透摩尔浓度）。保持在4℃直至使用。 </li><li>制备细胞内储备溶液（以mM计）：氯化钾70，KOH 53，甲磺酸30，EGTA 5，HEPES 10，蔗糖70; pH为7.2（KOH）和310毫渗透摩尔浓度。保持在4℃直至使用。好几个星期。 </li><li>准备用制霉菌素实验前细胞内的录音解决方案即兴（在最后一分钟）。 <ol><li>称取3毫克制霉菌素，加入50微升的DMSO;涡旋20秒然后超声处理2-3分钟，直到完全稀释。 </li><li>加入20微升的DMSO-制霉菌素溶液在5毫升细胞内贮备液。涡旋20秒，然后超声处理3分钟。保持此解决方案，在4°C和光线直射保护，制霉菌素是光敏感。此溶液可用于几个小时。更换每一天。 </li><li>一旦嗅觉上皮的准备是在显微镜下，需要一定的该溶液以1毫升注射器用火焰细长黄色尖端以填充电极;从光线直射保护。一旦在室温，制霉菌素液是不是稳定的;更换溶液在1ml注射器每小时或保持在冰上。 </li></ol></li><li>拉记录电极与一个车夫来获得长长的脖子和小头（〜2微米）以15-20MΩ与内部制霉菌素液阻力。 </li><li>制备在0.5M的在DMSO下通风柜加臭剂溶液;等分和保持在-20℃直到使用。稀臭气物质在林格氏溶液中，直至所希望的浓度。填写刺激吸管。 </li></ol> 3.准备嗅觉上皮注意：或-IRES-tauGFP小鼠表达的tauGFP的或启动子的控制之下。在这些小鼠中，表达感兴趣的或所有神经元都GFP标记的。该协议适用于表示在所有区域手术室。然而，解剖和录音是更容易表达在背区手术室。 <ol><li>通过注射氯胺酮和赛拉嗪（150毫克/公斤和10毫克/公斤生化需氧量的混合物麻醉动物Ÿ重，分别）。断头可以用锋利的剪刀对年轻小鼠或用适当的维护啮齿动物为铡老年小鼠进行。 <ol><li>采用环形解剖剪刀作一纵切口内侧通过背部皮肤。拉它拆开去除皮肤。使用剪刀，剪断下颚处下颚关节。由平行于牙齿冠状切口取出上前牙。 </li><li>使后面的眼睛头部的冠状切，并保持头部的仅前部。浸在了管制范围内的解剖冰冷的林格氏液。 </li></ol></li><li>范围下剖析：在腹侧，使沿上颚/牙齿的纵向切割。切背侧骨头纵向，鼻腔的dorso-侧面以下。除去大部分骨骼和味觉;在RT传送它相连的隔膜和上皮中含氧林格。 </li><li>对于最终的解剖：在开始录制前右会议上，剥离上皮从底层隔钳。分离钳和具有两个4-5毫米剪刀切口（使用microvannas剪刀）在隔膜，其中粘合力更强的前部的上皮。 <ol><li>小心地切割出来沿其背部连接到隔上皮去除犁鼻器。转移上皮至记录室朝上粘液层;保持平与竖琴室。 </li></ol></li><li>一个堂堂正正的显微镜配备荧光光学系统和感光摄像头安装在室内。可视化通过40X水浸物镜（数值孔径0.8）和一个额外的2X或4X放大率由放大透镜取得的制备在计算机屏幕上以高倍率。用含氧林格在室温（1-2毫升/分钟）连续灌注。 </li></ol> 4.记录会话<ol><li>搜索荧光细胞：激发准备在480纳米EGFP的，发射的光在530-550纳米范围内;针对一个树突状旋钮，这是在荧光下亮场可靠可见。 </li><li>填写与制霉菌素液内电极;轻轻拍打在电极去除气泡。 </li><li>在插入电极支架电极，适用于移​​液器正压;电阻应为15〜20兆欧。 </li><li>把电极接近细胞;一旦电阻达到〜40MΩ，释放正压和应用轻微负压达到千兆欧密封。 </li><li>一旦密封达到，设置膜电位约为-75毫伏; </li><li>一旦进入细胞被打开，继续刺激方案，药物治疗。为了测量响应单一的加臭剂的刺激，记录200-500毫秒自发性活动，刺激为500毫秒，并测量细胞的长达10秒13的响应。对于药物治疗，灌注药物制剂在所需的浓度20。 </li></ol> 5.数据分析<ol><li>在50％的最大幅值，上升时间（必要的时间，以达到90％的最大振幅的，在毫秒），总电流（在PAS的曲线下面积），时间：作为接着分析电流通过加臭剂刺激引起（发作和响应在最大振幅的50％的偏移量，在毫秒之间的时间）。 <ol><li>使用峰值传导电流（最大振幅达到）在不同浓度，绘制并使用希尔方程拟合剂量响应曲线为：I = I最大/（1+（K 1/2 / C）n），其中I表示峰值电流，IMAX在饱和浓度的最大响应，K 1/2的浓度在该半最大反应的达到，C加臭剂的浓度和n希尔系数。 </li></ol></li><li>在电流钳的最大去极化分析膜电位的录音中，托塔升势引起（在mVsec曲线下面积）;记录自发扣球活动在30秒到1分钟的录音;通过尖峰通过注入电流（5-10 PA）的记录引起兴奋。 </li></ol>

<ol>
	<li>Lowe, G., Gold, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+amplification+by+calcium-dependent+chloride+channels+in+olfactory+receptor+cells.">Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells.</a> Nature. 366 (6452), 283-286 (1993).</li><li>Ponissery Saidu, S., Dibattista, M., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-induced+responses+recorded+from+olfactory+receptor+neurons+using+the+suction+pipette+technique.">Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique.</a> J Vis Exp. (62), e3862 (2012).</li><li>Moss, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+biochemical+responses+of+mouse+vomeronasal+receptor+cells+to+urine-derived+compounds:+possible+mechanism+of+action.">Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action.</a> Chem Senses. 23 (4), 483-489 (1998).</li><li>Kaur, A., Dey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+calcium+imaging+of+dissociated+vomeronasal+neurons.">Live cell calcium imaging of dissociated vomeronasal neurons.</a> Methods Mol Biol. 1068, 189-200 (2013).</li><li>Ma, M., Chen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation.">Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation.</a> J Neurosci Methods. 92 (1-2), 31-40 (1999).</li><li>Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li><li>Bozza, T., Feinstein, P., Zheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+expression+defines+functional+units+in+the+mouse+olfactory+system.">Odorant receptor expression defines functional units in the mouse olfactory system.</a> J Neurosci. 22 (8), 3033-3043 (2002).</li><li>Bozza, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+class+I+and+class+II+odorant+receptors+to+glomerular+domains+by+two+distinct+types+of+olfactory+sensory+neurons+in+the+mouse.">Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse.</a> Neuron. 61 (2), 220-233 (2009).</li><li>Vassalli, A., Rothman, A., Feinstein, P., Zapotocky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minigenes+impart+odorant+receptor-specific+axon+guidance+in+the+olfactory+bulb.">Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb.</a> Neuron. 35 (4), 681-696 (2002).</li><li>Feinstein, P., Bozza, T., Rodriguez, I., Vassalli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+of+mouse+olfactory+sensory+neurons+by+odorant+receptors+and+the+beta2+adrenergic+receptor.">Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor.</a> Cell. 117 (6), 833-846 (2004).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat Rev Neurosci. 5 (4), 263-278 (2004).</li><li>Grosmaitre, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SR1,+a+mouse+odorant+receptor+with+an+unusually+broad+response+profile.">SR1, a mouse odorant receptor with an unusually broad response profile.</a> J Neurosci. 29 (46), 14545-14552 (2009).</li><li>Grosmaitre, X., Vassalli, A., Mombaerts, P., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+responses+of+olfactory+sensory+neurons+expressing+the+odorant+receptor+MOR23:+a+patch+clamp+analysis+in+gene-targeted+mice.">Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice.</a> Proc Natl Acad Sci U S A. 103 (6), 1970-1975 (2006).</li><li>Lam, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+responsiveness+of+embryonic+mouse+olfactory+sensory+neurons+expressing+the+odorant+receptors+S1+or+MOR23.">Odorant responsiveness of embryonic mouse olfactory sensory neurons expressing the odorant receptors S1 or MOR23.</a> Eur J Neurosci. 38 (2), 2210-2217 (2013).</li><li>Zhang, J., Huang, G., Dewan, A., Feinstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+stimulus+specificity+and+glomerular+position+in+the+mouse+olfactory+system.">Uncoupling stimulus specificity and glomerular position in the mouse olfactory system.</a> Mol Cell Neurosci. 51 (3-4), 79-88 (2012).</li><li>Zhang, J., Pacifico, R., Cawley, D., Feinstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+detection+of+amines+by+a+trace+amine-associated+receptor.">Ultrasensitive detection of amines by a trace amine-associated receptor.</a> J Neurosci. 33 (7), 3228-3239 (2013).</li><li>Lee, A. C., Tian, H., Grosmaitre, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice.">Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.</a> Chem Senses. 34 (8), 695-703 (2009).</li><li>Cadiou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+odorant+exposure+induces+peripheral+olfactory+plasticity+at+the+cellular+level.">Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.</a> J Neurosci. 34 (14), 4857-4870 (2014).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+wiring+in+the+mouse+olfactory+system.">Axonal wiring in the mouse olfactory system.</a> Annu Rev Cell Dev Biol. 22, 713-737 (2006).</li><li>Grosmaitre, X., Santarelli, L. C., Tan, J., Luo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+functions+of+mammalian+olfactory+sensory+neurons+as+odor+detectors+and+mechanical+sensors.">Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors.</a> Nat Neurosci. 10 (3), 348-354 (2007).</li><li>Duchamp-Viret, P., Chaput, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+response+properties+of+rat+olfactory+receptor+neurons.">Odor response properties of rat olfactory receptor neurons.</a> Science. 284 (5423), 2171-2174 (1999).</li><li>Heydel, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-binding+proteins+and+xenobiotic+metabolizing+enzymes:+implications+in+olfactory+perireceptor+events.">Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events.</a> Anat Rec (Hoboken). 296 (9), 1333-1345 (2013).</li><li>Pelosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perireceptor+events+in+olfaction).">Perireceptor events in olfaction).</a> J Neurobiol. 30 (1), 3-19 (1996).</li><li>Spehr, M., Wetzel, C. H., Hatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-phosphoinositides+modulate+cyclic+nucleotide+signaling+in+olfactory+receptor+neurons.">3-phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons.</a> Neuron. 33, 731-739 (2002).</li><li>Chen, S., Lane, A. P., Bock, R., Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+adenylyl+cyclase+inhibits+olfactory+generator+currents+induced+by+'IP(3)-odors.">Blocking adenylyl cyclase inhibits olfactory generator currents induced by 'IP(3)-odors.</a> J Neurophysiol. 84 (1), 575-580 (2000).</li><li>Savigner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+spontaneous+and+odorant-evoked+activity+of+rat+olfactory+sensory+neurons+by+two+anorectic+peptides,+insulin+and+leptin.">Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin.</a> J Neurophysiol. 101 (6), 2898-2906 (2009).</li><li>Ukhanov, K., Brunert, D., Corey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide+3-kinase-dependent+antagonism+in+mammalian+olfactory+receptor+neurons.">Phosphoinositide 3-kinase-dependent antagonism in mammalian olfactory receptor neurons.</a> J Neurosci. 31 (1), 273-280 (2011).</li></ol>

The authors declare that they have no competing financial interest.

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...

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。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">heavy equipment</td>
		</tr>
		<tr>
			<td>vibration table with Faraday cage</td>
			<td>TMC</td>
			<td>63-500 SERIES</td>
			<td>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</td>
		</tr>
		<tr>
			<td colspan="4">optics</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#39;s preference</td>
		</tr>
		<tr>
			<td>objectives</td>
			<td>Olympus</td>
			<td>LUMPLFL40XW</td>
			<td>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</td>
		</tr>
		<tr>
			<td>magnifier</td>
			<td>Olympus</td>
			<td>U-TVCAC</td>
			<td>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</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td>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</td>
		</tr>
		<tr>
			<td>filters</td>
			<td>Olympus/Chroma</td>
			<td></td>
			<td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">recording electrodes /system</td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>HEKA</td>
			<td>EPC10 USB</td>
			<td>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</td>
		</tr>
		<tr>
			<td>software</td>
			<td>HEKA</td>
			<td>Patchmaster</td>
			<td>controls the amplifier during the experiment</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter</td>
			<td>MP225</td>
			<td>precision micromanipulator, allows precise movements down to 1/1Oth of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td>
		</tr>
		<tr>
			<td>electrode puller</td>
			<td>sutter</td>
			<td>P97</td>
			<td>with a FT345-B wide trough filament;&#160; to prepare recording pipets of about 2&#181;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>sutter</td>
			<td>BF120-69-10</td>
			<td>in our recording conditions, this glass is ideal for recording pipets</td>
		</tr>
		<tr>
			<td>recording chamber</td>
			<td>warner instruments</td>
			<td>RC-26G</td>
			<td>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.</td>
		</tr>
		<tr>
			<td colspan="4">stimulation</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td>
		</tr>
		<tr>
			<td>multibarrel puller</td>
			<td>MDI</td>
			<td>PMP-107-Z</td>
			<td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td>
		</tr>
		<tr>
			<td>precision pressure injector&#160;</td>
			<td>Toohey Company</td>
			<td>P/N T25-1-900 Single Channel&#160;&#160;&#160;</td>
			<td>this precision pressure injector&#160; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&#160; TTLs</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>YOU-1</td>
			<td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td>
		</tr>
		<tr>
			<td>tubings</td>
			<td>N/A</td>
			<td></td>
			<td>tygon tubing to bring the pressure from the puffer to the puffing pipette</td>
		</tr>
		<tr>
			<td colspan="4">solutions/perfusion/chemicals</td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>gardner denver</td>
			<td>300 series</td>
			<td>a vibrating membrane pump is more quiet and efficient than other types of pumps</td>
		</tr>
		<tr>
			<td>perfusion system</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber</td>
		</tr>
		<tr>
			<td>nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td>mandatory to perpare internal solution for perforated patch clamp</td>
		</tr>
		<tr>
			<td>DIMETHYL SULFOXIDE</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>used to disolve nystatin for internal solution for perforated patch&#160;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td>intracellular/extracellular solution</td>
		</tr>
		<tr>
			<td>Calcium chloride di hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4 7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>Potassium hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>MethylSulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>47,135-6</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Hepes-Na</td>
			<td>Sigma-Aldrich</td>
			<td>H7006</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>intracellular solution</td>
		</tr>
	</tbody>
</table>

perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor

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。

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...

Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

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.

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

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

perforated-patch-clamp-recording-mouse-olfactory-sensory-neurons

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Neuroscience

26.6K 次观看.  Université de Bourgogne. 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.

视频: Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor