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
<ol>
	<li>Solutions 
	NOTE: Prepare the following solutions before starting dissection of the epithelium.
	<ol>
		<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 CaCl2, 1 mM MgCl2, and 10 mM glucose.</li>
			<li>Prepare a PBS-/- solution (pH 7.4) with concentrations of 2.68 mM KCl, 1.47 mM KH2PO4, 136 mM NaCl, and 8.1 mM Na2HPO4.</li>
			<li>Prepare a fixative solution (pH 7.2) with concentrations of 1x PBS-/-, 0.2 mM CaCl2, 4% sucrose, and 4% paraformaldehyde. Sucrose in the fixative solution improves cryosectioning and pick-up of cryosections. Store at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Solutions for the staining procedure
		<ol>
			<li>Prepare a PBS+/+ solution with concentrations of 1x PBS-/-, 0.48 mM MgCl2, 0.9 mM CaCl2.</li>
			<li>Prepare a PBST+/+ solution with concentrations of 1x PBS+/+ and 0.1% Triton X-100.</li>
			<li>Prepare a blocking solution with concentrations of 1x PBST+/+ and 1% gelatine.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Workplace
	<ol>
		<li>For the dissection workplace, use a dissecting microscope with bright illumination, as well as a liquid-blocker pen.</li>
		<li>Prepare a petri dish, filled with Ringer&#8217;s solution, and adhesive glass slides.</li>
		<li>Obtain the following surgical instruments: a pair of surgical scissors with one sharp and one blunt tip, a pair of spring scissors with straight tip shape, two forceps with fine curved tip shape and a razor blade (Figure 1A).</li>
	</ol>
	</li>
</ol>
2. Preparation of the Nasal Septum
<ol>
	<li>House animals in approved cages with regular access to food and water and appropriate day/night cycle.</li>
	<li>Perform anesthesia in a closed receptacle containing gauze soaked with 100% isoflurane and monitor analgesia by testing rear foot reflexes. Since cervical dislocation can cause blood in nasal cavities, directly decapitate anesthetized mice.</li>
	<li>Remove the skin to expose the bone of the entire skull and nose, and wipe away the remaining blood and tissue thoroughly using a paper towel. Remove the lower jaw and the front teeth.</li>
	<li>Incise the dorsal bone of the nose bilaterally in 1-2 mm distance parallel to the suture line to separate one side of the septum (medially) from the maxilla (laterally). Split the nose with a single cut. If bone remnants and turbinates are still attached to the septum, remove these carefully to expose the septum completely without touching it.</li>
	<li>Remove the dorsal nasal bone by sliding the fine curved tip of a forceps along the dorsal side of the septum, between septum and nasal bone. Apply slight pressure on the bone, push it up and remove it.</li>
	<li>To provide access to the two septal tissues, one from each cavity, carefully remove the maxilla of the other side of the head. When preparing older animals (P &#62; 28), remove the tip of the frontal bone covering the olfactory bulbs.</li>
	<li>Identify the olfactory epithelium by its slightly yellow color (Figure 1B). The bordering respiratory epithelium is white and shows movement of motile cilia that can be seen under the dissecting microscope. Cut along the border between olfactory and respiratory epithelium with a pair of fine spring scissors.</li>
	<li>Extend the cut and separate the septum from the ventral connection to the vomer bone. Then cut along the border between the septum and the lamina cribrosa of the Os ethmoidale. The septum is now completely isolated from all connections to the head. Use the cutting positions shown in Figure 1C.</li>
</ol>
3. Isolation of the Olfactory Epithelium for Immunostaining
<ol>
	<li>Place an adhesive glass slide in the petri dish filled with Ringer&#8217;s solution. Place it onto the edge of the petri dish, so that one half of the glass lies in the Ringer&#8217;s solution (Figure 1A).</li>
	<li>Use a forceps to carefully transfer the ethmoid bone with the olfactory epithelium to the petri dish. Lift it without grabbing it with the forceps tips.</li>
	<li>Grab the perpendicular plate of the ethmoid bone with one forceps and use a second one to carefully remove the epithelium from one side of the septum. Slide the curved tip between the perpendicular plate and epithelium to peel the epithelium off.</li>
	<li>Be always sure to identify the ciliary side, in case the epithelium flips in the Ringer&#8217;s solution. If the epithelium flips, it is hardly possible to identify the ciliary side afterwards. Mostly, the epithelium rolls up with the ciliary surface inside.</li>
	<li>Compare the enrolled tissue shape with the cutting positions shown in Figure 1C. Grab the epithelium at a position at the border and pull it onto the glass slide. Do not touch the ciliary side with the forceps to avoid lesions of the tissue.</li>
	<li>Turn the septum and repeat the procedure with the epithelium from the other side.</li>
	<li>Dry the glass slide around both pieces of olfactory epithelium with a paper towel and encircle the tissue with a liquid-blocker pen.</li>
	<li>Fix the tissue with 150&#160;&#181;l fixative solution for 10 min at RT. Concerning cilia stability and integrity, use short fixation times for a variety of tested antibodies. Within these 10 min&#160;cilia are fixed, but probably not the whole epithelium tissue. Thus, immediate visualize with the microscope subsequent to the staining procedure. However, fixative times might vary for individual antibodies.</li>
</ol>
4. Staining Protocol
NOTE: Handle the tissue very carefully to preserve ciliary structures of the olfactory sensory neurons. Remove solutions by pipetting. Do not drop any solutions directly onto the epithelium, as mechanical forces could disrupt the fine cilia. Perform all steps at RT if not stated otherwise.
<ol>
	<li>Remove the fixative solution and wash 2 times with PBS+/+.</li>
	<li>Incubate the epithelium for at least 1 hr with blocking solution.</li>
	<li>Prepare primary antibody solution by dissolving the antibody in blocking solution (1:200 for the antibodies used in this study) and centrifuge for 5 min at full speed to remove any precipitates.</li>
	<li>Incubate the epithelium with antibody solution in a humid chamber at 4 &#176;C O/N.</li>
	<li>Wash epithelium with PBS+/+ for 5 min at least 3 times.</li>
	<li>Prepare secondary antibody solution by dissolving the antibody in blocking solution (1:500) and centrifuge for 5 min at full speed.</li>
	<li>Incubate the epithelium with secondary antibody solution for 1 hr in a dark chamber.</li>
	<li>Wash epithelium with PBS+/+ for 5 min at least 3 times.</li>
	<li>Remove the liquid-blocker with a razor blade or paper towel. Wash the epithelium with distilled water for 5 s.</li>
	<li>Preserve the tissue in antifade mounting reagent. 
	NOTE: Background fluorescence increases rapidly within days; microscopical analysis not later than 2 days after embedding in mounting medium is therefore recommended. Special care has to be taken not to squeeze the tissue when searching the correct focal plane, since it can severely damage the preparation. Due to out-of-focus fluorescence, further investigation with confocal microscopy is recommended.</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...

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.

<table><tbody><tr><td>Spring scissors</td><td></td><td></td><td>straight tip, multiple suppliers</td></tr><tr><td>Surgical scissors</td><td></td><td></td><td>sharp and blunt end, multiple suppliers</td></tr><tr><td>Fine forceps</td><td></td><td></td><td>curved tips, Dumont #7, multiple suppliers</td></tr><tr><td>Razor blade</td><td></td><td></td><td>extra thin, multiple suppliers</td></tr><tr><td>Binocular with illumination</td><td></td><td></td><td>multiple suppliers, Stemi 2000-C, Zeiss</td></tr><tr><td>Petri dish</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Liquid-blocker pen</td><td>Science Services</td><td>N71310</td><td></td></tr><tr><td>Polysine coated slides</td><td>Thermo Scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>primary antibody Goat anti-Kirrel2</td><td>R&amp;amp;D Systems</td><td>AF2930</td><td>1:200</td></tr><tr><td>primary antibody Rabbit anti-mOR-EG</td><td>Baumgart et al., 2014</td><td></td><td>1:200</td></tr><tr><td>secondary antibodies</td><td>Life Technologies</td><td>A21206, A11057</td><td>1:500</td></tr><tr><td>Mounting medium, ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>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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11.1K Views.  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.

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

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Behavior

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.

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. ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

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.

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

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

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

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

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.

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 ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

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

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

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

Biology

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

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

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