Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons

Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616, USA.
Neural Development (Impact Factor: 3.45). 10/2008; 3(1):22. DOI: 10.1186/1749-8104-3-22
Source: PubMed


Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.
We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro.
This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.

Download full-text


Available from: Alla F Fomina
  • Source
    • "To produce functional ORs, some groups have used various OR expression systems, either mammalian cells (Chen, Dadsetan, Fomina, & Gong, 2008; Dahoun, Grasso, Vogel, & Pick, 2011; Ko & Park, 2006; Krautwurst, Yau, & Reed, 1998; Levasseur et al., 2003; Matsunami, Mainland, & Dey, 2009; Saito, Kubota, Roberts, Chi, & Matsunami, 2004; Zhuang & Matsunami, 2007), Saccharomyces cerevisiae-based cells (Fukutani et al., 2012; Minic et al., 2005; Pajot-Augy, Crowe, Levasseur, Salesse, & Connerton, 2003; Radhika et al., 2007) or Escherichia coli cells (Song, Lee, Oh, & Park, 2009; Sung, Ko, & Park, 2006) also regarded as most promising systems. Here, ORs are produced in S. cerevisiae, which constitutes an efficient heterologous expression system. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We report a dose-dependent detection of androstenone in solution, as one of the boar taint compounds, based on related OR7D4 olfactory receptors immobilized on a gold electrode through their 6-His tag and NTA-copper complex, as visualized through fluorescence microscopy. Square wave voltammetry (SWV) is for the first time, the method used to monitor the olfactory receptor/odorant recognition process. The relative variation of the Cu(I)-Cu(II) current peak increases linearly versus log (concentration of androstenone) from 10(-14)M to 10(-4)M, in buffer solution. Negative tests were performed, using an unrelated odorant, helional, itself a ligand of OR 1740. Cross-selectivity was also tested after immobilization of OR 1740. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Oct 2015 · Food Chemistry
  • Source
    • "Neurons in olfactory mucosal cells should differentiate into OSNs because we did not induce specific neurons or neural stem cells in the system, and some neurons in the gel were olfactory marker protein-positive, which is specific antigen for OSNs (data not shown). It was difficult to distinguish between neurons from olfactory mucosal cells and from cortical slices because olfactory marker protein is the marker for mature OSNs, and cultured neurons in olfactory mucosal cells are not always positive for olfactory marker protein (Chen et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Among the possible sources of autologous cells and tissues for use in spinal cord injury grafts, one promising source is the olfactory mucosa containing olfactory ensheathing cells and neural progenitor cells. Olfactory mucosa transplantation for spinal cord injury has been effective in animal models and in pilot clinical trials. However, the contributions of olfactory ensheathing cells and neurons in olfactory mucosa are unclear. For the present study, we prepared primary olfactory mucosal cells and used a cortex-Matrigel coculture assay system to examine the axonal outgrowth of olfactory mucosa. Axonal outgrowth from cortical slices was significantly enhanced in olfactory mucosal cells compared with noncell controls and respiratory mucosal cells, which have few olfactory ensheathing cells and neurons. Axonal outgrowth was severely reduced after treatment with an antineurotrophin cocktail. A conditioned medium in the olfactory mucosa-derived cell group contained neurotrophin-3. Some olfactory ensheathing cells and almost all neurons were immunopositive for neurotrophin-3. Axons originating from cortical slices targeted mainly the astrocyte-like olfactory ensheathing cells. Our findings demonstrate that the axonal outgrowth effect of olfactory mucosa is supported by both olfactory ensheathing cells and neurons in olfactory mucosa. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Jul 2014 · Journal of Neuroscience Research
  • Source
    • "Olfactory sensory neuron culture and axon outgrowth assay OSN culture was performed as described previously (Chen et al., 2008). Briefly, OE tissue was dissected from the newborn mouse nasal cavity and treated with 2 mg/mL dispase for 40 min at room temperature. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Olfactory sensory neurons (OSNs) extend their axons from the nasal epithelium to their odorant receptor-dependent locations in the olfactory bulb. Previous studies have identified several membrane proteins along the projection pathway, and on OSN axons themselves, which regulate this process; however, little is known about the signaling mechanisms through which these factors act. We have identified and characterized Rap1gap2, a novel small GTPase regulator, in OSNs during early postnatal mouse development. Rap1gap2 overexpression limits neurite outgrowth and branching in Neuro-2a cells, and counteracts Rap1-induced augmentation of neurite outgrowth. Rap1gap2 expression is developmentally regulated within OSNs, with high expression in early postnatal stages that ultimately drops to undetectable levels by adulthood. This temporal pattern coincides with an early postnatal plastic period of OSN innervation refinement at the OB glomerular layer. Rap1gap2 stunts OSN axon outgrowth when overexpressed in vitro, while knock-down of Rap1gap2 transcript results in a significant increase in axon length. These results indicate an important role of Rap1gap2 in OSN axon growth dynamics during early postnatal development.
    Full-text · Article · Jun 2012 · Molecular and Cellular Neuroscience
Show more