This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

注：所有动物的程序都在查理特大学或诊所耶拿符合德国动物保护的法律，避免动物的任何不必要的痛苦处理。 1.准备解决方案和解剖职场<ol><li>解决方案 注：在开始上皮剥离之前，请准备以下解决方案。 <ol><li>在解剖过程的解决方案： <ol><li>制备林格氏液（pH7.4）中为140 mM氯化钠，5mM的氯化钾，10mM的HEPES，2mM的氯化钙 ，1mM的MgCl 2的，和10mM葡萄糖的浓度。 </li><li>制备的PBS - / -溶液（pH 7.4）以2.68毫米氯化钾，1.47毫KH 2 PO 4，136 mM氯化钠，和8.1毫摩尔的Na 2 HPO 4的浓度。 </li><li>制备固定剂溶液（pH 7.2）与浓度的1×PBS的- / - ，0.2mM的氯化钙 ，4％蔗糖，4％的多聚甲醛。蔗糖在固定液提高低温切片和回升冰冻切片的。存储在-20℃。 </li></ol></li><li>用于染色过程的解决方案<ol><li>准备PBS + / +解决方案与1X PBS的浓度- / - ， - 0.48毫米氯化镁2，0.9毫米氯化钙2。 </li><li>准备PBST + / +解决方案与1X PBS + / + 0.1％的Triton X-100的浓度。 </li><li>制备用1×PBST + / +和1％明胶的浓度封闭溶液。 </li></ol></li></ol></li><li>职场<ol><li>用于夹层的工作场所中，使用解剖显微镜用明亮的照明，以及一个液体阻滞剂笔。 </li><li>准备一个培养皿，充满林格氏溶液，和粘合剂载玻片。 </li><li>得到如下手术器械：一对手术剪一个尖锐的和一个钝头，一双弹簧剪刀直笔尖形状，双钳细尖弧形形状和剃刀刀片（ <strong&gt;图1A）。 </li></ol></li></ol> 2.准备鼻中隔<ol><li>房子的动物在笼子里批准定期获得食物和水，适当的昼/夜循环。 </li><li>在一个封闭的容器含有纱布通过测试后足部反射浸泡100％，异氟醚和显示器镇痛麻醉进行。由于颈椎脱位可引起血鼻腔，直接斩首麻醉小鼠。 </li><li>取下皮肤，露出整个颅骨和鼻子的骨，并彻底用纸巾擦去剩余的血液和组织。取出下颌前牙。 </li><li>切开鼻子的背侧骨双边在1-2毫米的距离平行于缝合线的隔垫（内侧）的一侧，从上颌骨（横向）分开。分裂的鼻子跟单切。如果残存骨骼和鼻甲仍附着于隔，去除这些精心完全暴露隔而不触及它。 </li><li>由滑动的钳子沿着该隔膜的背侧细弯曲尖，隔膜和鼻骨之间移除背鼻骨。适用于轻微的压力，骨头，推起来，将其删除。 </li><li>以提供从每个空腔访问两个隔组织，1，小心地取出头的另一侧的上颌骨。当准备年长的动物（P&gt; 28），除去额骨覆盖嗅球的一角。 </li><li>由它的微黄色的颜色（ 图1B）确定嗅觉上皮。接壤呼吸道上皮是白色的，表明运动纤毛可在解剖显微镜下可见的移动。沿嗅和呼吸道上皮的交界带双鸟语花香剪刀剪。 </li><li>延长削减和到犁骨腹连接分开隔。然后沿隔和OS筛的筛板之间的边界切即隔膜现在已经完全从所有连接到头部隔离。使用图1C中所示的切割位置。 </li></ol> 3.隔离嗅觉上皮细胞的免疫染色<ol><li>放置在培养皿中充满林格氏溶液的粘合剂载玻片。将其放置到陪替氏培养皿的边缘上，以使一个半玻璃在于林格氏溶液（ 图1A）。 </li><li>使用镊子的嗅觉上皮仔细转移筛骨到陪替氏培养皿中。抬起不与镊子尖端抓住它。 </li><li>抢筛骨垂直板有一个镊子，并使用第二个要小心地从隔的一侧取出上皮。滑动垂直板和上皮细胞之间的弧形尖剥离脱落上皮。 </li><li>总是一定要识别睫状侧，万一上皮翻转在林格氏soluti上。如果上皮翻转，这是几乎不可能事后识别睫状侧。大多数情况下，上皮细胞卷起里面的纤毛表面。 </li><li>与图1C中所示的切割位置进行比较的登记组织的形状。抢在上皮在边境的位置，并将其拉至载玻片。请勿触摸纤毛侧镊子，避免组织损伤。 </li><li>转动隔膜并与来自另一侧的上皮重复该过程。 </li><li>干燥围绕两件的嗅觉上皮用纸巾载玻片并环绕与液体阻滞剂笔的组织。 </li><li>固定带在室温10分钟150μl的固定液的组织。关于纤毛的稳定性和完整性，使用短的固定时间用于各种测试抗体。在这10分钟纤毛是固定的，但可能不是全部的上皮组织。因此，随着在S之后的显微镜直接可视泰宁程序。然而，固定液时间可能有所不同个体抗体。 </li></ol> 4.染色协议注：处理组织非常认真地保存嗅感觉神经元的纤毛结构。吹打删除解决方案。不要直接将任何解决方案到上皮细胞，机械力可能会破坏优良的纤毛。完成所有步骤在室温如果没有说明。 <ol><li>除去固定液和洗涤2次，用PBS + / +。 </li><li>孵育上皮至少1小时，以封闭液。 </li><li>离心5分钟以全速以除去任何沉淀物：通过在封闭液（200为在该研究中使用的抗体1）溶​​解该抗体制备第一抗体溶液。 </li><li>孵育在潮湿室中在4℃CO / N抗体溶液的上皮。 </li><li>洗涤上皮用PBS + / + 5分钟，在至少3次。 </li><li>离心5分钟，在全速：通过在封闭溶液（500 1）将所述抗体制备二级抗体溶液。 </li><li>孵育在黑暗腔室1小时的第二抗体溶液中的上皮。 </li><li>洗涤上皮用PBS + / + 5分钟，在至少3次。 </li><li>用刀片或纸巾除去液体阻滞剂。洗用蒸馏水上皮为5秒。 </li><li>保护组织抗淬灭安装试剂。 注：在几天内背景荧光迅速增大;因此显微分析中安装嵌入后不迟于2日内建议。特别注意有寻找正确的焦平面时，挤压组织，因为它会严重损坏准备采取没有。由于离焦的荧光，进一步调查，共焦显微镜，推荐。 </li></ol>

<ol>
	<li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">How the olfactory system makes sense of scents.</a> Nature. 413 (6852), 211-218 (2001).</li><li>Buck, L., 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>Wong, S. 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=Disruption+of+the+type+III+adenylyl+cyclase+gene+leads+to+peripheral+and+behavioral+anosmia+in+transgenic+mice.">Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice.</a> Neuron. 27 (3), 487-497 (2000).</li><li>Belluscio, L., Gold, G. H., Nemes, A., 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=Mice+deficient+in+G(olf)+are+anosmic.">Mice deficient in G(olf) are anosmic.</a> Neuron. 20 (1), 69-81 (1998).</li><li>Brunet, L. J., Gold, G. H., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel.">General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.</a> Neuron. 17 (4), 681-693 (1996).</li><li>Reisert, J., Lai, J., Yau, K. W., Bradley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+excitatory+Cl-+response+in+mouse+olfactory+receptor+neurons.">Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons.</a> Neuron. 45 (4), 553-561 (2005).</li><li>Hengl, 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=Molecular+components+of+signal+amplification+in+olfactory+sensory+cilia.">Molecular components of signal amplification in olfactory sensory cilia.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (13), 6052-6057 (2010).</li><li>Smith, D. W., Thach, S., Marshall, E. L., Mendoza, M. G., Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+NKCC1+have+normal+olfactory+sensitivity.">Mice lacking NKCC1 have normal olfactory sensitivity.</a> Physiolog., &amp; Behavior. 93 (1-2), 44-49 (2008).</li><li>Menco, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+aspects+of+olfactory+signaling.">Ultrastructural aspects of olfactory signaling.</a> Chemical Senses. 22 (3), 295-311 (1997).</li><li>Serizawa, S., 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=A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting.">A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.</a> Cell. 127 (5), 1057-1069 (2006).</li><li>Baumgart, S., 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=Scaffolding+by+MUPP1+regulates+odorant-mediated+signaling+in+olfactory+sensory+neurons.">Scaffolding by MUPP1 regulates odorant-mediated signaling in olfactory sensory neurons.</a> Journal Of Cell Science. 127 (11), 2518-2527 (2014).</li><li>Strotmann, J., Wanner, I., Krieger, J., Raming, K., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+odorant+receptors+in+spatially+restricted+subsets+of+chemosensory+neurones.">Expression of odorant receptors in spatially restricted subsets of chemosensory neurones.</a> Neuroreport. 3 (12), 1053-1056 (1992).</li><li>Jenkins, P. M., McEwen, D. P., Martens, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+cilia:+linking+sensory+cilia+function+and+human+disease.">Olfactory cilia: linking sensory cilia function and human disease.</a> Chemical Senses. 34 (5), 451-464 (2009).</li><li>Tadenev, A. 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=Loss+of+Bardet-Biedl+syndrome+protein-8+(BBS8)+perturbs+olfactory+function,+protein+localization,+and+axon+targeting.">Loss of Bardet-Biedl syndrome protein-8 (BBS8) perturbs olfactory function, protein localization, and axon targeting.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (25), 10320-10325 (2011).</li></ol>

The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer...

鼻腔鼠标嗅上皮包括6-10万双极嗅感觉神经元1。每个嗅觉神经元选择的1200气味受体基因的表达之一。检测加臭剂的开始由气味结合到嗅觉受体2，其然后激活腺苷酸环化酶III型（ACIII）3经由嗅觉特异性G蛋白GαOLF 4。由此而造成的环磷酸腺苷（cAMP）打开一个环核苷酸门控（CNG），非选择性阳离子通道，导致钙离子和钠离子，并随后钙涌入2+内流导致开口的Ca 2+活化的Cl -通道5,6。所得向外Cl -的磁通变得容易通过高细胞内氯-吸收，有可能通过的Na + / K + / Cl -的-协同转运NKCC1中，通过稳定氯浓度保持CL - / HCO3 -换热器SLC4A1，也许更多的尚未确定运输6-8。 双极嗅觉神经元具有单一的，无支链的轴突直接投射到嗅球和树枝状延伸到上皮的表面和端部作为专门隔室，树枝状旋钮。从这个旋钮，10-30纤毛，其可以达到50-60微米的长度，发出入粘液覆盖的上皮表面9。规范化的信号转导级联的蛋白质主要定位于这些纤毛的膜。上皮的增加感官表面放大，以检测气味物质的能力。由于感觉神经元的密度，纤毛从相邻的树突旋钮延伸弄乱。这种交织的结果是从不同的神经元，表达不同类型的嗅觉受体，上皮的表面上的随机混合物纤毛。的检测和细胞睫状蛋白，只存在于感觉神经元的一个子集的ular分配，因此在冷冻切片困难。另外，这种蛋白质沿纤毛的精确定位是几乎不可能的，因为冷冻切片通常比纤毛的平均长度薄。 为了使在嗅觉神经元至今未鉴定的膜蛋白的纤毛本土化的调查中，我们优化的恩面对制剂技术，它允许蛋白定位于纤毛的详细分析。简言之，将小鼠处死并在头分割中线附近。鼻甲，鼻，和额叶骨骼被去除以暴露隔膜。用的衬里上皮嗅觉部隔膜通过切割所有连接到鼻腔松开。把隔膜变成培养皿充满林格氏液后，将上皮剥离UND转移到涂覆的玻璃载片。经过简短fixat离子一步，免疫组化方法可以如果操作尽可能轻柔，以免脆弱的组织损伤进行。我们证明在古典冷冻切片和中所描述的连接面制备比较两种不同的膜蛋白的染色在嗅觉纤毛可达到的分辨率。

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">straight tip, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Surgical scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fine forceps</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">curved tips, Dumont #7, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Razor blade</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">extra thin, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Binocular with illumination</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers, Stemi 2000-C, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid-blocker pen</td>
			<td style="width:127px;">Science Services</td>
			<td style="width:119px;">N71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:119px;">J2800AMNZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal microscope</td>
			<td style="width:127px;">Leica Microsystems</td>
			<td style="width:119px;">TCS SPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">A21206, A11057</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">P36930</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">toxic, work under fume hood</td>
		</tr>
	</tbody>
</table>

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.

嗅觉上皮连接面制剂可以用于研究蛋白质的定位中的感觉神经元的纤毛，使蛋白质的详细调查，其定位是冷冻切片的分析后不清楚。这个问题可以例举在染色IRRE样蛋白2（Kirrel2）的健的情况下。 Kirrel2（也称为Neph3）是膜蛋白和功能的免疫球蛋白（Ig）超家族的同嗜粘附蛋白中的一员。它显示出在嗅觉系统中轴突引导的作用，并表达在嗅觉神经元的活性依赖性方式10的子集。 <p clas...

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

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

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 ...

whole-mount-labeling-of-cilia-in-the-main-olfactory-system-of-mice

Research

JoVE Journal

Neuroscience

11.0K 次观看.  Universitaetsklinikum Jena. 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.

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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...