Culture of Mouse Olfactory Sensory Neurons

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Olfactory sensory neurons, located in the nasal epithelium, detect and transmit odorant information to the central nervous system. This requires that these neurons form specific neuronal connections within the olfactory bulb and express receptors and signaling molecules specific for these functions. This protocol describes a primary olfactory sensory neuron culture technique that allows in vitro investigation of olfactory sensory neuron differentiation, axon outgrowth, odorant receptor expression, and function. Olfactory epithelium is obtained from the nasal cavity and is enzymatically treated to reduce stroma tissue. Dissociated olfactory sensory neurons are cultured directly on a layer of cortical astrocytes to support their survival. Using this method, cultured olfactory sensory neurons maintain their bipolar morphology and express odorant signal transduction molecules, which are specific for olfactory sensory neurons. Curr. Protoc. Neurosci. 58:3.24.1?3.24.14. © 2012 by John Wiley & Sons, Inc.

Keywords: olfactory epithelium; bipolar; astrocytes; lentivirus; odorant receptor

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Introduction
Basic Protocol 1: Culture of Primary Mouse Olfactory Sensory Neurons
Support Protocol 1: Preparation of Glass Coverslips
Support Protocol 2: Purification and Culturing Mouse Cortical Astrocytes
Support Protocol 3: Immunochemistry of OSN Culture
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Culture of Primary Mouse Olfactory Sensory Neurons

Materials

Cortical astrocytes (see protocol 3 )
Glia medium (see recipe )
70% ethanol
CMF‐HBSS/HEPES (see recipe )
OSN medium (see recipe )
C57BL/6 embryos at the desired age range (E15‐E20)
2.5% avertin (see recipe ) or alternative anesthetics (e.g., sodium pentobarbital)
Ice
Dispase (Life Technologies, cat. no. 17105041)
DNase I
Mild trypsinization solution (see recipe )
EDTA
Fetal bovine serum

12‐mm glass coverslips, culture prepared (see protocol 2 )
24‐well plates
37°C, 5% CO₂ incubator
Dissection tools:
- One 5 ¹ /₂ ‐ in. scissors
- One forceps
- One 4 ¹ /₂ ‐ in. fine scissors
- Two No. 5 forceps
- One microscissors
Horizontal laminar flow culture hood
1‐ml syringe equipped with 28‐G needles
10‐cm petri dishes, sterile
Stereo dissecting microscope
35‐mm culture dishes
Sterile glass Pasteur pipets (both flame polished and not polished)
15‐ml Falcon tubes
Benchtop centrifuge
37°C water bath
Microscope and hemacytometer

Additional reagents and equipment for counting cells using a hemacytomer (Phelan, )

Support Protocol 1: Preparation of Glass Coverslips

Materials

10 M NaOH
70% ethanol
1 mg/ml laminin stock (BD Biosciences; dissolved in ddH₂ O)
10 mg/ml poly‐D‐lysine stock (BD Biosciences; dissolved in ddH₂ O)
Appropriate culture medium (e.g., DMEM)

No. 1 12‐mm circular glass coverslip
Coverslip staining racks
Laminar flow culture hood
24‐well plates

Support Protocol 2: Purification and Culturing Mouse Cortical Astrocytes

Materials

70% ethanol
Five to ten neonatal C57 BL/6 mouse pups (P0 to P3)
Crushed ice
CMF‐HBSS/HEPES (see recipe )
Trypsinization solution (see recipe )
Fetal bovine serum
Glia medium (see recipe )
1 mM AraC (Sigma‐Aldrich)
Dimethyl sulfoxide (DMSO)
Liquid nitrogen

Dissection tools:
- One 5 ¹ /₂ ‐in. scissors
- One forceps
- One 4 ¹ /₂ ‐in. fine scissors
- Two no. 5 forceps
- One micro scissors
- One micro spatula (Ted Pella, cat. no. 13510)
Laminar flow culture hood
Latex sleeves fashioned from the fingers of latex gloves
35‐mm dishes
Stereomicroscope
50‐ml Falcon tubes
Pasteur pipets
Benchtop centrifuge
37°C water bath
5‐ml pipets
70‐µm Falcon cell strainer
Microscope and hemacytometer
75‐cm² flasks
37°C, 5% CO₂ incubator
Parafilm
Rotary platform
Cryovials
12‐mm glass coverslips, culture prepared (see protocol 2 )
24‐well plates

Additional reagents and equipment for counting cells using a hemacytometer (Phelan, )

Support Protocol 3: Immunochemistry of OSN Culture

Materials

OSN culture (see protocol 1 )
4% paraformaldehyde
Phosphate‐buffered saline (PBS; see recipe ) with 0.1% Triton X‐100 (PBS+TX)
Normal horse serum
Phosphate‐buffered saline (PBS; see recipe )
Primary antibodies (see step 4 annotation for details)
Secondary antibodies (see the annotation to step 6 for details)
Fluoromount G (Southern Biotech, cat. no. 0100‐01)

Petri dishes
Parafilm
Coverslip rack
Glass slides

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Figures

Figure 3.24.1 Dissection and isolation of olfactory neuroepithelium. (A ) Dorsal view of a neonatal mouse head. A line through the midsagittal plan indicates where the cut is. (B ) Sagittal view of the opened nasal cavity. Nasal epithelium (OE) is circled. Olfactory bulb (OB) is located superior to the nasal cavity. (C ‐D ) Different views of the dissected nasal epithelia with cartilage tissue attached. Arrowhead in C points to the nasal septum. (E ) Olfactory epithelium with underlining stroma. Arrowhead in E points to the separated stroma, which appear opaque under the stereomicroscope. (F ) Isolated olfactory neuroepithelial appear transparent under the stereomicroscope. Bar = 1 mm in A and B; 1 mm in C and D; 2 mm in E and F.

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Figure 3.24.2 Morphology and gene expression of cultured OSNs. (A ) OSNs are identified by expression of type III β‐tubulin. Sensory neurons are cultured at low density with each individual neuron extending processes without contacting other neurons. Majority of cultured neurons are bipolar in morphology. (B ‐D ) At 3 DIV, cultured OSNs exhibit bipolar morphology with a short, thick dendritic process and a long, thin axonal process. These neurons express neuronal markers, GAP43 (B), type III b‐tubulin (C) and NCAM (D). (E ‐F ) At 6 DIV, cultured OSNs express odorant signaling molecules, Golf (E) and AC3 (F). Bar, 60 µm in A; 20 µm in B‐F.

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Literature Cited

Literature Cited
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