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

Summary

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.

Abstract

The mouse olfactory system is often used to study mechanisms of neural circuit formation　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.

Introduction

During development, neurons are precisely connected with each other to form proper neural circuits, which is critical for the normal brain function. Since aberrant neural circuits in the brain are thought to be the cause of mental disorders such as autism and schizophrenia, understanding the mechanisms of neural circuit formation is one of the major challenges in the field of neuroscience.

In the mouse olfactory system, each Olfactory Sensory Neuron (OSN) in the Olfactory Epithelium (OE) expresses only one functional Olfactory Receptor (OR) gene and OSNs expressing the same OR converge their axons to a specific pair of glomeruli at stereoty....

Protocol

All experimental procedures were performed with the approval of the animal experiment ethics committee at the University of Tokyo and according to the University of Tokyo guidelines for the care and use of laboratory animals.

1. Preparation of Solutions

1. Prepare 0.01 M Phosphate-Buffered Saline (PBS): add a PBS tablet (0.14 M NaCl, 0.0027 M KCl, 0.010 M PO₄^3-, pH 7.4) to 1 L distilled water and stir at RT until it is completely dissolved.
2. Prepare 4% paraformaldehyde (PFA) in PBS: add 4 g of PFA to 100 mL PBS. Stir the solution at 60 °C until it is completely dissolved.
   ....

Results

The olfactory glomerular map is formed by initial global targeting and subsequent glomerular segregation of OSN axons^1,2. Glomerular segregation is regulated by the adhesive/repulsive axonal interactions mediated by axon-sorting molecules whose expression levels are determined by expressed OR molecules⁷. The axon-sorting molecules involved in glomerular segregation are expressed in a position-independent mo.......

Discussion

Quadruple immunostaining of parasagittal OB sections enabled the visualization and quantification of the expression levels of as many as four axon-sorting molecules simultaneously in a larger number of glomeruli. By analyzing these multivariable data with PCA, the characteristics for the expression of those molecules can be speculated.

For successful staining, the tissue sample preparation is critically important. Some protocols suggest that tissues should be post-fixed with 4% PFA and treated.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate-Buffered Saline (PBS) Tablets, pH7.4	TAKARA BIO	T9181
Skim Milk	nacalai tesque	31149-75
goat anti-Sema7A antibody	R&D Systems	AF2068
rat anti-OLPC antibody	Merck Millipore	MABT20
mouse anti-VGLUT2 antibody	Merck Millipore	MAB5504
goat anti-BIG-2 antibody	R&D Systems	AF2205
gunea pig anti-Kirrel2 antibody	Operon Biotechnologies		Anti-Kirrel2 antibodies were generated by immunizing guinea pigs with KLH-conjugated synthetic peptides (644-673aa): CRLYRARAGYLTTPHPRAFTSYMKPTSFGP
donkey anti-mouse Alexa Fluor 405	Abcam	ab175658
donkey anti-goat Alexa Fluor 488	Jackson ImmunoResearch	705-545-003
donkey anti-guinea pig Alexa Fluor 555	Thermo Fisher Scientific	A21432
donkey anti-rat Alexa Fluor 647	Jackson ImmunoResearch	712-605-153
Paraformaldehyde (PFA)	Wako	162-16065
MAS coated slide glasses	MATSUNAMI	MAS-01
forceps	Fine Science Tools	11253-27
Vannas Spring Scissors	Fine Science Tools	15000-00
dissecting scissors	Fine Science Tools	14090-09
fluorescent microscope	KEYENCE	BZ-X700
DAPI filter cube	KEYENCE	OP-87762
GFP filter cube	KEYENCE	OP-87763
TRITC filter cube	KEYENCE	OP-87764
Cy5 filter cube	KEYENCE	OP-87766
filter paper	ADVANTEC	00011185
O.C.T compound	Sakura Finetek	M71484