Name: whole mount labeling of cilia in the main olfactory system of mice
Uploaded: 2014-12-27T14:00:00
Description: Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

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

NOTE: All animal procedures were handled at the Charit&#233; or University Clinic Jena in accord with German Animal Care laws avoiding any undue suffering of animals.
1. Preparing Solutions and Dissection Workplace
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	<li>Solutions 
	NOTE: Prepare the following solutions before starting dissection of the epithelium.
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		<li>Solutions for the dissection procedure:
		<ol>
			<li>Prepare Ringer&#8217;s solution (pH 7.4) with concentrations of 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM...

<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. 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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...

The mouse olfactory epithelium in the nasal cavity comprises 6-10 million bipolar olfactory sensory neurons1. Each olfactory neuron chooses one of 1,200 odorant receptor genes for expression. Detection of odorants starts by odorant binding to an olfactory receptor2, which then activates adenylyl cyclase type-III (ACIII)3 via the olfactory specific G protein G&#945;olf4. The resulting rise in cyclic adenosine monophosphate (cAMP) opens a cyclic nucleotide-gated (CNG), nonselective cation channel leading to influx of Ca2+ and Na+, and subsequently Ca2+ influx leads to opening of a Ca2+ activated Cl- channel5,6. The resulting outward Cl- flux is facilitated by a high intracellular Cl- concentration maintained by steady Cl- uptake, likely via the Na+/K+/Cl- cotransporter NKCC1, the Cl-/HCO3- exchanger SLC4A1, and maybe additional yet to be identified transporters6-8.
Bipolar olfactory neurons have single, unbranched axons that project directly to the olfactory bulb, and a dendrite that extends to the surface of the epithelium and ends as a specialized compartment, the dendritic knob. From this knob, 10-30 cilia, which can reach a length of up to 50-60 &#181;m, emanate into the mucus covering the epithelial surface9. Proteins of the canonical signal transduction cascade are mainly localized in the membrane of these cilia. The increased sensory surface of the epithelium amplifies the ability to detect odorants. Due to the density of sensory neurons, cilia extending from neighboring dendritic knobs intermingle. This intermingling results in a random mixture of cilia from different neurons, expressing different types of olfactory receptors, on the surface of the epithelium. The detection and cellular allocation of ciliary proteins which are only present in a subset of sensory neurons is therefore difficult in cryosections. In addition, the precise localization of such proteins along the cilia is barely possible, since cryosections are typically thinner than the average length of the cilia.
To enable investigation of ciliary localization of so far uncharacterized membrane proteins in olfactory neurons, we optimized an en face preparation technique which allows the detailed analysis of protein localization in cilia. Briefly, the mouse is sacrificed and the head split near the midline. Turbinates, nasal, and frontal bones are removed to expose the septum. The septum with the olfactory part of the lining epithelium is loosened by cutting all connections to the nasal cavity. After putting the septum into a petri dish filled with Ringer&#8217;s solution, the epithelium is peeled off und transferred to a coated glass slide. Following a short fixation step, immunostaining procedures can be performed if handling is as gentle as possible to avoid damage of the fragile tissue. We demonstrate the achievable resolution by comparing the staining of two different membrane proteins in olfactory cilia in classical cryosections and in the en face preparation described.

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			<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.

Olfactory epithelium en face preparations can be used to investigate localization of proteins in the cilia of sensory neurons, allowing the detailed investigation of proteins whose localization is unclear after analysis of cryosections. This problem can be exemplified in the case of the staining for Kin of IRRE-like protein 2 (Kirrel2). Kirrel2 (also called Neph3) is a member of the immunoglobulin (Ig) superfamily of membrane proteins and functions as a homophilic adhesion protein. It was shown to play a role in...

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.

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

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