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 Ca....

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 ....

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

List of materials used in this article

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