这项工作是支持的研究补助金（NIH / NIDCD 009269，012831和ARRA行政补充剂NIH补助）维宏林。我们要特别感谢UMBC蒂姆·福特先生在他的偷拍和处理的技术援助。我们还要感谢达芙妮布隆伯格博士UMBC的的CHERE小资女士和尼古拉斯·麦科勒姆先生从奥林巴斯美国公司，他们的设备援助偷拍。

1。鼠标鼻子准备在本研究中，我们使用成人的C57BL / 6背景的小鼠。所有动物护理和程序批准的动物护理和使用委员会（IACUC）美国马里兰大学巴尔的摩县。 1.1获取鼻子从paraformaldahyde固定小鼠<img src="/files/ftp_upload/50538/50538fig1.jpg" alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50538/50538fig1highres.jpg"/> 图1。鼠标头骨骨骼的头骨背视图。B：除去下颌骨的头骨与腹面观。从40日龄小鼠头骨。个别骨头的颜色更好的可视化。 <a href="https://www.jove.com/files/ftp_upload/50538/50538fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <ol><li>灌注transcardially解决个别的小鼠日É林， 等。协议，（2008）16。简单地说，小鼠深度麻醉，三溴乙醇（圣阿韦坦250微克/克体重），心脏灌注，用0.1M磷酸缓冲液（PB，30〜50毫升）中，然后由磷酸盐缓冲固定液中含有3％多聚甲醛，19 mM的L-赖氨酸单盐酸盐，0.23％的钠米，高碘酸（约35-50毫升）。也可以按照动物灌注35的朱庇特的文章中的步骤。 </li><li>使用一把剪刀切断的下颌骨（或下颚）和去除皮肤的头。 </li><li>从身体的其余部分分开，整个头部。 </li><li>取出的口感。此外，清洗，除去残留的结缔组织和肌肉的头骨取得试样，在图1A和1B所示的表面上。 </li><li>在解剖显微镜下，取出脑和嗅球颅骨覆盖。剪掉多余的组织和骨骼。没有特扩展的组织制剂，其中大脑和鼻子保持连接，只有头骨都被删除。对于免疫组化实验中，将组织后固定1.5小时，然后转移到0.1M磷酸盐缓冲盐水（PBS）与25％蔗糖过夜。鼻组织应保持加湿整个剥离，通过浸渍在缓冲​​的蔗糖溶液数次。 </li></ol> 1.2获取鼻子从新鲜安乐死小鼠<ol><li>个别小鼠转移到一个干净的笼中，并暴露在CO 2气体，随后用颈脱位5分钟后，最后的呼吸。为了减少血液中的鼻子组织，用一把剪刀开胸切心脏，使血液漏。 </li><li>重复步骤1.1.2至1.1.5，除了25％的蔗糖后的固定和冷冻保护。应保持的检体加湿保持与台氏盐含有（以mM计）：140钠氯化钾，氯，5 1的MgCl 2，1的 CaCl 2，10的丙酮酸钠，10个D-葡萄糖，10 N-2-羟乙基哌嗪-N&#39;-2-乙磺酸缓冲液（HEPES，在pH 7.4）调整。或者，折片的Kimwipes，浸泡，用台氏溶液，和这里下方的检体解剖时，偶尔浸入试样的台氏溶液或一些解决办法拖放到组织上，以保持它加湿以维持细胞和组织的可行性。 </li></ol> 2。门齿，前犁骨和上颌骨骨去除<ol><li>开始腹观点。找到犁骨和牙齿咬骨钳或镊子打破最腹侧部分骨，沿其长度打破犁骨。 </li><li>使用锯齿钳轻轻行走碎骨离犁鼻器（VNO），取下破裂的犁骨段。请注意，这些老鼠不到一个月以前，或者如果只有兴趣在教育部，两个STEps的，可以跳过。 </li><li>牢牢握住头钳。使用咬骨钳打破两个门牙的前部的两个门牙和犁骨之间的接合区域的前上颌骨。 </li><li>额的右侧上颌骨腹侧部上面的区域只是前颧弓颧板的背侧的水平。 </li><li>在鼻子翻转背的观点。用咬骨钳打破背颧板的右侧上颌骨前壁。整个右上颌前颧板要宽松。用细镊子轻轻任何相关颌骨鼻组织分开，然后轻轻抬起了骨头碎片。 </li><li>重复步骤2.4和2.5，，松开左门牙和左上颌前牙区，，鼻骨后已被删除，并删除它们。 </li></ol> 3。鼻骨和背颧板拆除<ol><li>使用锯齿钳或咬骨钳重新将剩余的额骨鼻甲骨只是尾椎。取出这块骨头，后尾部分的鼻骨可以使用镊子夹住。 </li><li>使用咬骨钳打破颧弓的前部，这是连接到颧板。 </li><li>颧板的背端外侧缘和细镊子轻轻翻转骨，并删除它。如果骨头不松，用咬骨钳轻轻松开去除。 </li><li>用细镊子或刀片松动之间的左，右鼻甲骨内侧缝合。 </li><li>使用锯齿镊子夹住尾端右鼻骨。轻轻地移动，从一侧到另一侧的骨到相关组织分开。移动骨一侧到另一侧移动钳沿尾第三鼻骨它是有用的。由于鼻骨分离，慢慢抬起骨，从尾端。 </li><li>而T他鼻骨轻微抬起，横向倾斜骨的骨内衬薄呼吸道上皮组织揭示了横向露头。用细镊子轻轻松开这个组织从鼻腔骨。继续解除鼻骨。当骨完全分离皮下组织，用剪刀切断鼻骨喙端。 </li><li>左鼻骨去除重复步骤3.1，3.5和3.6。 </li><li>鼻子的左侧，重复步骤3.2和3.3。 </li></ol> 4。颧骨外侧板拆除<ol><li>删除颧板一侧的时间。无论是颧板可以拆下第一。 </li><li>从腹侧的观点，打破休息，直到达到颧弓颧弓上颌骨逊色。 </li><li>从背部的角度看，轻轻地抓住颧弓和正向和侧向抬起。如果颧板仍连接到任何组织，采取罚款钳轻轻地切断的组织和骨之间的连接。 </li><li>鼻颧板的另一侧重复上述步骤。 </li></ol> 5。轨道骨去除<ol><li>从腹侧的观点，打破巴伦丁的磨牙骨之间的鼻子。 </li><li>使用咬骨钳的每一侧的鼻子打破3臼齿和上颌骨。 </li><li>打破和消除任何剩余的腹厚的骨片和后每一侧鼻孔的鼻甲。 </li></ol> 6。筛骨去除<ol><li>中断任何部分筛骨突出尾鳍鼻甲。这是必要的，以避免损失的鼻甲组织，除去薄件时，筛骨覆盖鼻甲。 </li><li>对于鼻子的右侧，将筛骨前缘细镊子轻轻取出。如果骨的部分仍然存在，重复这个过程，直到所有薄骨覆盖鼻甲已被删除。鼻甲骨不需要除去大部分的制剂。在老年小鼠，筛板变得脆弱。如果需要鼻腔组织cryosectioning是，用细镊子取出小块板，以减少潜在的损害，引起骨。 </li><li>重复步骤6.2），鼻子的左侧。 </li><li>切片之前删除任何剩余的碎骨。注：在动物超过一岁，隔骨posterodorsal地区是有点厚和硬。人们可以用细镊子取出这部分。镊子的尖端插入到骨背侧部的隔垫骨和衬里上皮组织分离的两侧。使用一对镊子，抢骨头和保持标本。使用另一双钳打破上层部分的中隔软骨下骨部分，轻轻取出。 </li></ol><ol><li>设置抽真空泵。 </li><li>将鼻子嵌入模具。淹没鼻子媒体华侨城。 </li><li>使用真空以除去空气气泡内鼻组织。这个过程最多需要5分钟。 </li><li>除去气泡后，将组织在所需的方向。 </li><li>冻结在模具使用干冰华侨城和组织。然后包埋组织立即cryosectioned，或储存于-80℃以备将来使用。 </li></ol>

<ol><li>Naclerio, R. M., Pinto, J., Assanasen, P., Baroody, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Observations+on+the+ability+of+the+nose+to+warm+and+humidify+inspired+air%5BTitle%5D)+AND+%22Rhinology%22%5BJournal%5D)">Observations on the ability of the nose to warm and humidify inspired air.</a> Rhinology. 45, 102-111 (2007).</li>
<li>Bjermer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nose+as+an+air+conditioner+for+the+lower+airways%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">The nose as an air conditioner for the lower airways.</a> Allergy. 54, Suppl 57. 26-30 (1999).</li>
<li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+the+olfactory+system+makes+sense+of+scents%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">How the olfactory system makes sense of scents.</a> Nature. 413, 211-218 (2001).</li>
<li>Bryant, B., Silver, W. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBryant%2C+B+%22Chemisthesis%3A+The+common+chemical+sense%22">Chemisthesis: The common chemical sense</a>. , 2nd, Wiley-Liss. (2000).</li>
<li>Gross, E. A., Swenberg, J. A., Fields, S., Popp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+morphometry+of+the+nasal+cavity+in+rats+and+mice%5BTitle%5D)+AND+%22J.+Anat%22%5BJournal%5D)">Comparative morphometry of the nasal cavity in rats and mice.</a> J. Anat. 135, 83-88 (1982).</li>
<li>Halpern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+organization+and+function+of+the+vomeronasal+system%5BTitle%5D)+AND+%22Annu.+Rev.+Neurosci%22%5BJournal%5D)">The organization and function of the vomeronasal system.</a> Annu. Rev. Neurosci. 10, 325-362 (1987).</li>
<li>Rodolfo-Masera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Su+l%27esquoestizenza+di+un+particulare+organo+olfacttivo+nel+setto+nasale+della+cavia+e+di+altri+roditori%5BTitle%5D)+AND+%22Arch.+Ital.+Anat.+Embryol%22%5BJournal%5D)">Su l'esquoestizenza di un particulare organo olfacttivo nel setto nasale della cavia e di altri roditori.</a> Arch. Ital. Anat. Embryol. 48, 157-212 (1943).</li>
<li>Levai, O., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Projection+pattern+of+nerve+fibers+from+the+septal+organ%3A+DiI-tracing+studies+with+transgenic+OMP+mice%5BTitle%5D)+AND+%22Histochemistry+and+Cell+biology%22%5BJournal%5D)">Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice.</a> Histochemistry and Cell biology. 120, 483-492 (2003).</li>
<li>Storan, M. J., Key, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Septal+organ+of+Gruneberg+is+part+of+the+olfactory+system%5BTitle%5D)+AND+%22J.+Comp.+Neurol%22%5BJournal%5D)">Septal organ of Gruneberg is part of the olfactory system.</a> J. Comp. Neurol. 494, 834-844 (2006).</li>
<li>Restrepo, D., Arellano, J., Oliva, A. M., Schaefer, M. L., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+views+on+the+distinct+but+related+roles+of+the+main+and+accessory+olfactory+systems+in+responsiveness+to+chemosensory+signals+in+mice%5BTitle%5D)+AND+%22Horm.+Behav%22%5BJournal%5D)">Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice.</a> Horm. Behav. 46, 247-256 (2004).</li>
<li>Breer, H., Fleischer, J., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+sense+of+smell%3A+multiple+olfactory+subsystems%5BTitle%5D)+AND+%22Cell+Mol.+Life+Sci%22%5BJournal%5D)">The sense of smell: multiple olfactory subsystems.</a> Cell Mol. Life Sci. 63, 1465-1475 (2006).</li>
<li>Munger, S. D., Leinders-Zufall, T., Zufall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subsystem+organization+of+the+mammalian+sense+of+smell%5BTitle%5D)+AND+%22Annu.+Rev.+Physiol%22%5BJournal%5D)">Subsystem organization of the mammalian sense of smell.</a> Annu. Rev. Physiol. 71, 115-140 (2009).</li>
<li>Finger, T. E., St Jeor, V. L., Kinnamon, J. C., Silver, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+of+substance+P-+and+CGRP-immunoreactive+nerve+fibers+in+the+nasal+epithelium+of+rodents%5BTitle%5D)+AND+%22J.+Comp.+Neurol%22%5BJournal%5D)">Ultrastructure of substance P- and CGRP-immunoreactive nerve fibers in the nasal epithelium of rodents.</a> J. Comp. Neurol. 294, 293-305 (1990).</li>
<li>Papka, R. E., Matulionis, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+substance-P-immunoreactive+nerves+with+the+murine+olfactory+mucosa%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Association of substance-P-immunoreactive nerves with the murine olfactory mucosa.</a> Cell Tissue Res. 230, 517-525 (1983).</li>
<li>Baraniuk, J. N., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nasonasal+reflexes%2C+the+nasal+cycle%2C+and+sneeze%5BTitle%5D)+AND+%22Curr.+Allergy+Asthma+Rep%22%5BJournal%5D)">Nasonasal reflexes, the nasal cycle, and sneeze.</a> Curr. Allergy Asthma Rep. 7, 105-111 (2007).</li>
<li>Lin, W., Ogura, T., Margolskee, R. F., Finger, T. E., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPM5-expressing+solitary+chemosensory+cells+respond+to+odorous+irritants%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">TRPM5-expressing solitary chemosensory cells respond to odorous irritants.</a> J. Neurophysiol. 99, 1451-1460 (2008).</li>
<li>Ogura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholinergic+microvillous+cells+in+the+mouse+main+olfactory+epithelium+and+effect+of+acetylcholine+on+olfactory+sensory+neurons+and+supporting+cells%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells.</a> J. Neurophysiol. 106, 1274-1287 (2011).</li>
<li>Finger, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solitary+chemoreceptor+cells+in+the+nasal+cavity+serve+as+sentinels+of+respiration%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8981-8986 (2003).</li>
<li>Gulbransen, B. D., Clapp, T. R., Finger, T. E., Kinnamon, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nasal+solitary+chemoreceptor+cell+responses+to+bitter+and+trigeminal+stimulants+in+vitro%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">Nasal solitary chemoreceptor cell responses to bitter and trigeminal stimulants in vitro.</a> J. Neurophysiol. 99, 2929-2937 (2008).</li>
<li>Zhao, K., Dalton, P., Yang, G. C., Scherer, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Numerical+modeling+of+turbulent+and+laminar+airflow+and+odorant+transport+during+sniffing+in+the+human+and+rat+nose%5BTitle%5D)+AND+%22Chemical+Senses%22%5BJournal%5D)">Numerical modeling of turbulent and laminar airflow and odorant transport during sniffing in the human and rat nose.</a> Chemical Senses. 31, 107-118 (2006).</li>
<li>Ressler, K. J., Sullivan, S. L., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+zonal+organization+of+odorant+receptor+gene+expression+in+the+olfactory+epithelium%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A zonal organization of odorant receptor gene expression in the olfactory epithelium.</a> Cell. 73, 597-609 (1993).</li>
<li>Vassar, R., Ngai, J., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+segregation+of+odorant+receptor+expression+in+the+mammalian+olfactory+epithelium%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium.</a> Cell. 74, 309-318 (1993).</li>
<li>Fulle, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+receptor+guanylyl+cyclase+expressed+specifically+in+olfactory+sensory+neurons%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 3571-3575 (1995).</li>
<li>Juilfs, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+subset+of+olfactory+neurons+that+selectively+express+cGMP-stimulated+phosphodiesterase+%28PDE2%29+and+guanylyl+cyclase-D+define+a+unique+olfactory+signal+transduction+pathway%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, 3388-3395 (1997).</li>
<li>Lin, W., Arellano, J., Slotnick, B., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Odors+detected+by+mice+deficient+in+cyclic+nucleotide-gated+channel+subunit+A2+stimulate+the+main+olfactory+system%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+The+Official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Odors detected by mice deficient in cyclic nucleotide-gated channel subunit A2 stimulate the main olfactory system.</a> The Journal of Neuroscience: The Official journal of the Society for Neuroscience. 24, 3703-3710 (2004).</li>
<li>Ishii, T., Omura, M., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocols+for+two-+and+three-color+fluorescent+RNA+in+situ+hybridization+of+the+main+and+accessory+olfactory+epithelia+in+mouse%5BTitle%5D)+AND+%22J.+Neurocyt%22%5BJournal%5D)">Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse.</a> J. Neurocyt. 33, 657-669 (2004).</li>
<li>Lee, A. C., Tian, H., Grosmaitre, X., Ma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice%5BTitle%5D)+AND+%22Chemical+Senses%22%5BJournal%5D)">Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.</a> Chemical Senses. 34, 695-703 (2009).</li>
<li>Packard, A., Schnittke, N., Romano, R. A., Sinha, S., Schwob, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DeltaNp63+regulates+stem+cell+dynamics+in+the+mammalian+olfactory+epithelium%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 31, 8748-8759 (2011).</li>
<li>Matthews, J. B., Mason, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+decalcifying+agents+on+immunoreactivity+of+formalin-fixed%2C+paraffin-embedded+tissue%5BTitle%5D)+AND+%22Histochem+J%22%5BJournal%5D)">Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue.</a> Histochem J. 16, 771-787 (1984).</li>
<li>Athanasou, N. A., Quinn, J., Heryet, A., Woods, C. G., McGee, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+decalcification+agents+on+immunoreactivity+of+cellular+antigens%5BTitle%5D)+AND+%22J.+Clin.+Pathol%22%5BJournal%5D)">Effect of decalcification agents on immunoreactivity of cellular antigens.</a> J. Clin. Pathol. 40, 874-878 (1987).</li>
<li>Hegg, C. C., Irwin, M., Lucero, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+store-mediated+signaling+in+sustentacular+cells+of+the+mouse+olfactory+epithelium%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium.</a> Glia. 57, 634-644 (2009).</li>
<li>Spehr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essential+role+of+the+main+olfactory+system+in+social+recognition+of+major+histocompatibility+complex+peptide+ligands%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Essential role of the main olfactory system in social recognition of major histocompatibility complex peptide ligands.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 26, 1961-1970 (2006).</li>
<li>Ma, M., Chen, W. R., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation.</a> J. Neurosci. Methods. 92, 31-40 (1999).</li>
<li>Cygnar, K. D., Stephan, A. B., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analyzing+responses+of+mouse+olfactory+sensory+neurons+using+the+air-phase+electroolfactogram+recording%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording.</a> J. Vis. Exp. (37), e1850(2010).</li>
<li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+animal+perfusion+fixation+for+rodents%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Whole animal perfusion fixation for rodents.</a> J. Vis. Exp. (65), e3564(2012).</li>
<li>Lin, W., Margolskee, R., Donnert, G., Hell, S. W., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Olfactory+neurons+expressing+transient+receptor+potential+channel+M5+%28TRPM5%29+are+involved+in+sensing+semiochemicals%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 2471-2476 (2007).</li>
<li>Lin, W., Ezekwe, E. A., Zhao, Z., Liman, E. R., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPM5-expressing+microvillous+cells+in+the+main+olfactory+epithelium%5BTitle%5D)+AND+%22BMC+Neurosci%22%5BJournal%5D)">TRPM5-expressing microvillous cells in the main olfactory epithelium.</a> BMC Neurosci. 9, 114(2008).</li>
<li>Ogura, T., Krosnowski, K., Zhang, L., Bekkerman, M., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemoreception+regulates+chemical+access+to+mouse+vomeronasal+organ%3A+role+of+solitary+chemosensory+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Chemoreception regulates chemical access to mouse vomeronasal organ: role of solitary chemosensory cells.</a> PLoS One. 5, e11924(2010).</li>
<li>Sathyanesan, A., Feijoo, A. A., Mehta, S. T., Nimarko, A. F., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+profile+of+G-protein+%CE%B2%CE%B3+subunit+gene+transcripts+in+the+mouse+olfactory+sensory+epithelia%5BTitle%5D)+AND+%22Frontiers+in+Cellular+Neuroscience%22%5BJournal%5D)">Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia.</a> Frontiers in Cellular Neuroscience. 7, 84(2013).</li>
</ol>

作者宣称，他们有没有竞争经济利益。

在这里，我们展示了一个隔离完好鼠标鼻子的嗅觉和呼吸道组织的顺序取出骨头，同时保留周围组织的一步一步的过程。我们发现，小心骨去除的全部，甚至可以保留最微妙的组织。我们也分享洞察可能修改这项技术，我们在其中分离大脑和鼻子组织共同维护神经连接。这种新方法提供了一个隔离装置，用于在一个单一的免疫组化，RNA 原位杂交，以便进一步处理和生理实验标本的整个嗅觉...

哺乳动物鼻腔包含各种类型的组织和器官，服务于不同的功能。鼻腔，使上呼吸道的入口部分，它允许空气流入和流出肺部旅行。吸入的空气通过鼻腔的温度和湿度进行调节1，以及作为清洁或过滤除去刺激性和毒性的物质和感染性微生物的2。这两种治疗方法进行鼻上皮及上皮下的组织，包括腺体和血管，是至关重要的保护下呼吸道和肺部。除了它的作用在呼吸和上皮防御，鼻组织也包含外周感觉的嗅觉和三叉神经系统，使空气中的化学物质的检测范围广泛的设备。根据该系统被激活，在鼻子的化学品的感官检测既可以引起感的气味，刺激或疼痛3,4。 的外周的嗅觉系统是复杂的，由几个解剖分离鼻腔内的嗅觉器官。其中，主要的嗅觉上皮细胞（MOE）是最大的，它构成了约45-52％的鼻上皮细胞在啮齿类动物中5和位于后方区域。在腹侧区域中，有一对管状结构被称为犁鼻器6，坐在沿鼻中隔的每一侧。两个额外的嗅感觉神经元的小分组，称为室间隔器官马塞拉7,8和在Gruneberg神经节9，位于沿腹隔垫和背进入区域的鼻腔。这些外周器官中含有神经上皮细胞形态特色鲜明，细胞标记物的表达，和生理功能。他们共同检测成千上万的气味分子与精致的灵敏度10-12。 除了嗅觉器官，鼻腔内还设有其他感官系统。它是已知的，三 ​​叉神经肽能神经纤维出现在鼻腔上皮细胞，特别是呼吸道上皮13,14。这些纤维检测刺激性和有毒化学品，并负责启动保护性反射，如咳嗽和打喷嚏4,15。有刺激性的异味和苦味化合物也可以被检测到最近发现的孤化学感受细胞（SCCS），其中有许多是由三叉神经纤维16-19支配人口。这些的SCC位于鼻腔及犁鼻进入管道的入口区域中的高密度，暗示它们可能也有助于保护功能16-18。因此，鼻上皮细胞可显着不同的功能，形态，取决于它们的细胞的组合物解剖位置。 即使在一个单一的和专门的上皮细胞，存在地区差异。教育部就是这样一个例子。各种鼻甲教育部线，这是很复杂的卷曲结构。因为他们，不同地区的教育部体验不同的空气流速，因此，不同的扩散，空气中的气味分子的清除率20。此外，它被称为表达一种特定气味受体的嗅感觉神经元（OSNS）位于四规避教育部21,22区。这个位置如何差异会影响OSN气味的反应主要是不知道的。此外，一些的OSN种群具有区域偏好。鸟苷酸环化酶-D（GC-D）表达的OSN有带状分布，有利于地区的ectoturbinates 23,24的小路，小路。最近，我们发现了一个典型的嗅觉神经元亚群表达瞬时受体电位通道M5（TRPM5），优先地位于在横向和腹侧区域25。这些结果表明，本部不统一。然而，如何使这些区域的差异影响嗅编码的不理解。这部分是保存完好的解剖组织，用目前的方法获得完整的鼻上皮的难度，因为彻底的生理教育部的调查和鼻子已经有限。 鼻腔上皮细胞主要是四周前，包括鼻，上颌骨，腭骨，颧骨，筛骨颅骨。在成年老鼠和其它啮齿类动物模型，这些骨头是硬而难以去除，不破坏密切相关的鼻部组织，特别是细腻鼻甲。通常，使用的化学物质为基础的脱钙软化骨骼以允许cryosectioning鼻腔组织形态，免疫组织化学， 原位杂交研究，然而，依赖丁动物的年龄，脱钙过程可以持续7天过夜24,26-28。这种治疗方法也是有限的，因为它需要组织固定液保存。此外，化学脱钙可恶劣影响的一些敏感的抗体免疫标记29,30。对于生理研究，活组织是必需的，因此，这些实验往往是孤立的OSN或MOE切片得到的新生儿的头骨薄而软17,31,32进行。生理研究也可以利用整装准备分裂头25,33,34，但通常只有内侧表面的鼻子是方便，限制了对其他方面的生理记录。 在这里，我们描述了一个有效的，手动的的去骨方法来准备完整的鼻腔组织保存了原有的解剖组织形态。我们依次取出骨头的前在解剖显微镜下，几乎完全完整的鼻上皮暴露同时保持薄鼻甲骨骼完好除非老鼠很旧，cryosectioning的头骨是必要的。我们还可以延长保存鼻组织嗅球之间的连接，以及大脑的其余部分，从而促进外周和中枢的电路的同时检查方法。我们的方法可以被用于制备多聚甲醛固定，以及新鲜的，活的鼻组织。因此，我们的方法将有利于呼吸，嗅觉，鼻腔损伤和疾病的形态学，免疫组织化学和生理研究。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Dissection&lt;/strong&gt;</td></tr><tr><td>Rongeur, 1.0 mm Jaw width</td><td>World Precision Instruments (WPI)</td><td>501333</td><td></td></tr><tr><td>Fine forceps, Dumont 3 </td><td>WPI</td><td>503235</td><td></td></tr><tr><td>Fine forceps, Dumont 55</td><td>WPI</td><td>14099</td><td></td></tr><tr><td>Fine forceps, Dumont AA</td><td>Fine Science Tools (FST)</td><td>11210-20</td><td></td></tr><tr><td>Specimen forceps, Serrated</td><td>VWR</td><td>82027-440</td><td></td></tr><tr><td>Operating scissors</td><td>WPI</td><td>501753</td><td></td></tr><tr><td>Iris scissors, Straight</td><td>Miltex</td><td>V95-304</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZ40</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Tissue embedding&lt;/strong&gt;</td></tr><tr><td>Optimum cutting temperature (OCT) compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Plastic embedding mold</td><td>VWR</td><td>15160-215</td><td></td></tr><tr><td>Aspirator vacuum pump</td><td>Fisher Scientific</td><td>09-960-2</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Section staining &lt;/strong&gt;</td></tr><tr><td>Neutral red</td><td>ACROS Organic</td><td>CAS 553-24-2</td><td>Nuclei staining</td></tr></tbody></table>

an effective manual deboning method to prepare intact mouse nasal tissue with preserved anatomical organization

哺乳动物的鼻子是一个多功能的器官，具有复杂的内部结构。鼻腔内衬各种上皮细胞，如嗅觉，呼吸，和鳞状上皮细胞明显不同的解剖位置，形态和功能。在成年小鼠中，与各种颅骨覆盖鼻子，限制了实验获得的内部结构，尤其是在后等为主体的嗅觉上皮细胞（MOE）。在这里，我们描述了一个有效的方法，获得几乎整个完整的鼻腔组织与腌制解剖组织。使用手术工具，在解剖显微镜下，我们依次取出头骨周围鼻腔组织。这个程序可以进行多聚甲醛固定，解剖新鲜，皮肤的鼠标头。去骨整个过程约需20-30分钟，这是明显短于传统化工为主的实验所需时间钙化。此外，我们提出了一个简单的方法来去除气泡被困鼻甲之间，这是获得完整的细水平线或者冠状或矢状切面​​鼻组织准备的关键。鼻组织用我们的方法制备可用于整个装载整个上皮细胞的观察，以及形态，免疫细胞化学，RNA 原位杂交，和生理的研究，尤其是在特定区域的检查和比较，还研究。

使用这种方法，可以可靠地获得几乎完全完整的鼻组织图2A示出的图像的成年人鼻试样从多聚甲醛固定磁头。在这个标本，所有四个子嗅觉感官器官，包括教育部，室间隔器官中，Gruneberg节，VNO，都完好无损。此外，呼吸道上皮细胞和上皮下的组织，如腺体和血管，将被保留。我们已经成功地使用此方法，在一些研究中，我们调查形态，分布，神经的神经支配，细胞标记的表达在各专...

Watch this Scientific Journal Video about An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization at JoVE.com

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

有效的人工剔骨方法编写完整的鼠标鼻组织与保存的解剖组织

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

an-effective-manual-deboning-method-to-prepare-intact-mouse-nasal

Research

JoVE Journal

Biology

35.3K 次观看.  University of Maryland Baltimore County. 

视频: An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

科研

神经科学

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

鼻咽癌相关淋巴组织（NALT）在鼠标的响应中的作用研究疫苗

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

免疫与感染

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

发育生物学

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.