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

Summary

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.

Abstract

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.

Introduction

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 Gα_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^- channel^5,6. 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 transporters^6-8.

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 µm, 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’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.

Protocol

NOTE: All animal procedures were handled at the Charité or University Clinic Jena in accord with German Animal Care laws avoiding any undue suffering of animals.

1. Preparing Solutions and Dissection Workplace

1. Solutions
   NOTE: Prepare the following solutions before starting dissection of the epithelium.
   1. Solutions for the dissection procedure:
      1. Prepare Ringer’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.
      2. 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.
      3. 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 °C.
   2. Solutions for the staining procedure
      1. Prepare a PBS^+/+ solution with concentrations of 1x PBS^-/-, 0.48 mM MgCl₂, 0.9 mM CaCl₂.
      2. Prepare a PBST^+/+ solution with concentrations of 1x PBS^+/+ and 0.1% Triton X-100.
      3. Prepare a blocking solution with concentrations of 1x PBST^+/+ and 1% gelatine.
2. Workplace
   1. For the dissection workplace, use a dissecting microscope with bright illumination, as well as a liquid-blocker pen.
   2. Prepare a petri dish, filled with Ringer’s solution, and adhesive glass slides.
   3. 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).

2. Preparation of the Nasal Septum

1. House animals in approved cages with regular access to food and water and appropriate day/night cycle.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 > 28), remove the tip of the frontal bone covering the olfactory bulbs.
7. 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.
8. 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.

3. Isolation of the Olfactory Epithelium for Immunostaining

1. Place an adhesive glass slide in the petri dish filled with Ringer’s solution. Place it onto the edge of the petri dish, so that one half of the glass lies in the Ringer’s solution (Figure 1A).
2. 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.
3. 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.
4. Be always sure to identify the ciliary side, in case the epithelium flips in the Ringer’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.
5. 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.
6. Turn the septum and repeat the procedure with the epithelium from the other side.
7. Dry the glass slide around both pieces of olfactory epithelium with a paper towel and encircle the tissue with a liquid-blocker pen.
8. Fix the tissue with 150 µl 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 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.

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.

1. Remove the fixative solution and wash 2 times with PBS^+/+.
2. Incubate the epithelium for at least 1 hr with blocking solution.
3. 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.
4. Incubate the epithelium with antibody solution in a humid chamber at 4 °C O/N.
5. Wash epithelium with PBS^+/+ for 5 min at least 3 times.
6. Prepare secondary antibody solution by dissolving the antibody in blocking solution (1:500) and centrifuge for 5 min at full speed.
7. Incubate the epithelium with secondary antibody solution for 1 hr in a dark chamber.
8. Wash epithelium with PBS^+/+ for 5 min at least 3 times.
9. Remove the liquid-blocker with a razor blade or paper towel. Wash the epithelium with distilled water for 5 s.
10. 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.

Results

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...

Discussion

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...

Materials

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