Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells

Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250, USA.
Journal of Neurophysiology (Impact Factor: 2.89). 06/2011; 106(3):1274-87. DOI: 10.1152/jn.00186.2011
Source: PubMed


The mammalian olfactory epithelium is made up of ciliated olfactory sensory neurons (OSNs), supporting cells, basal cells, and microvillous cells. Previously, we reported that a population of nonneuronal microvillous cells expresses transient receptor potential channel M5 (TRPM5). Using transgenic mice and immunocytochemical labeling, we identify that these cells are cholinergic, expressing the signature markers of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. This result suggests that acetylcholine (ACh) can be synthesized and released locally to modulate activities of neighboring supporting cells and OSNs. In Ca(2+) imaging experiments, ACh induced increases in intracellular Ca(2+) levels in 78% of isolated supporting cells tested in a concentration-dependent manner. Atropine, a muscarinic ACh receptor (mAChR) antagonist suppressed the ACh responses. In contrast, ACh did not induce or potentiate Ca(2+) increases in OSNs. Instead ACh suppressed the Ca(2+) increases induced by the adenylyl cyclase activator forskolin in some OSNs. Supporting these results, we found differential expression of mAChR subtypes in supporting cells and OSNs using subtype-specific antibodies against M(1) through M(5) mAChRs. Furthermore, we found that various chemicals, bacterial lysate, and cold saline induced Ca(2+) increases in TRPM5/ChAT-expressing microvillous cells. Taken together, our data suggest that TRPM5/ChAT-expressing microvillous cells react to certain chemical or thermal stimuli and release ACh to modulate activities of neighboring supporting cells and OSNs via mAChRs. Our studies reveal an intrinsic and potentially potent mechanism linking external stimulation to cholinergic modulation of activities in the olfactory epithelium.

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    • "These cells may represent a population of stem cells [46,47]. Non-neuronal ACh released in their vicinity might modulate their activity as it was shown for cells of the olfactory epithelium [48]. Meyrick et al. [31] "
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    ABSTRACT: Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect "bitter" substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel=TRPM5, phospholipase Cβ2), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways. Copyright © 2015. Published by Elsevier B.V.
    International immunopharmacology 05/2015; 29(1). DOI:10.1016/j.intimp.2015.05.028 · 2.47 Impact Factor
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    • "The method of OSN isolation was adapted from our previous study (Ogura et al., 2011). Briefly, mice were euthanized by CO 2 asphyxiation followed by cervical dislocation and exsanguination through an open heart. "
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    ABSTRACT: Phospholipase C (PLC) and internal Ca(2+) stores are involved in a variety of cellular functions. However, our understanding of PLC in mammalian olfactory sensory neurons (OSNs) is generally limited to its controversial role in odor transduction. Here we employed single-cell Ca(2+) imaging and molecular approaches to investigate PLC-mediated Ca(2+) responses and its isozyme gene transcript expression. We found that the pan-PLC activator m-3M3FBS (25 μM) induces intracellular Ca(2+) increases in vast majority of isolated mouse OSNs tested. Both the response amplitude and percent responding cells depend on m-3M3FBS concentrations. In contrast, the inactive analog o-3M3FBS fails to induce Ca(2+) responses. The m-3M3FBS-induced Ca(2+) increase is blocked by the PLC inhibitor U73122, while its inactive analog U73433 has no effect. Removal of extracellular Ca(2+) does not change significantly the m-3M3FBS-induced Ca(2+) response amplitude. Additionally, in the absence of external Ca(2+), we found that a subset of OSNs respond to an odorant mixture with small Ca(2+) increases, which are significantly suppressed by U73122. Furthermore, using reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction, we found that multiple PLC isozyme gene transcripts are expressed in olfactory turbinate tissue in various levels. Using RNA in situ hybridization analysis, we further show expression of β4, γ1, γ2 gene transcripts in OSNs. Taken together, our results establish that PLC isozymes are potent enzymes for mobilizing intracellular Ca(2+) in mouse OSNs and provide molecular insight for PLC isozymes-mediated complex cell signaling and regulation in the peripheral olfactory epithelium.
    Frontiers in Cellular Neuroscience 10/2014; 8:336. DOI:10.3389/fncel.2014.00336 · 4.29 Impact Factor
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    • "The reduction is statistically significant (Student’s t-test, p < 0.001). Since we did not observe apparent zonal variation in the density of ChAT/Trpm5-expressing microvillous cells in our previous studies [5,12], or in the present study, we believe these results are representative and that Skn-1a knockout diminishes the population of ChAT/Trpm5-expressing microvillous cells in the MOE. Thus, combined with the lack of Trpm5, villin, and ChAT expression in the Skn-1a-/- mice, our results strongly indicate that Skn-1a functions as a critical regulator for the generation of Trpm5-microvillous cells in the MOE. "
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    ABSTRACT: The main olfactory epithelium (MOE) in mammals is a specialized organ to detect odorous molecules in the external environment. The MOE consists of four types of cells: olfactory sensory neurons, supporting cells, basal cells, and microvillous cells. Among these, development and function of microvillous cells remain largely unknown. Recent studies have shown that a population of microvillous cells expresses the monovalent cation channel Trpm5 (transient receptor potential channel M5). To examine functional differentiation of Trpm5-expressing microvillous cells in the MOE, we investigated the expression and function of Skn-1a, a POU (Pit-Oct-Unc) transcription factor required for functional differentiation of Trpm5-expressing sweet, umami, and bitter taste bud cells in oropharyngeal epithelium and solitary chemosensory cells in nasal respiratory epithelium. Skn-1a is expressed in a subset of basal cells and apical non-neuronal cells in the MOE of embryonic and adult mice. Two-color in situ hybridization revealed that a small population of Skn-1a-expressing cells was co-labeled with Mash1/Ascl1 and that most Skn-1a-expressing cells coexpress Trpm5. To investigate whether Skn-1a has an irreplaceable role in the MOE, we analyzed Skn-1a-deficient mice. In the absence of Skn-1a, olfactory sensory neurons differentiate normally except for a limited defect in terminal differentiation in ectoturbinate 2 of some of MOEs examined. In contrast, the impact of Skn-1a deficiency on Trpm5-expressing microvillous cells is much more striking: Trpm5, villin, and choline acetyltransferase, cell markers previously shown to identify Trpm5-expressing microvillous cells, were no longer detectable in Skn-1a-deficient mice. In addition, quantitative analysis demonstrated that the density of superficial microvillous cells was significantly decreased in Skn-1a-deficient mice. Skn-1a is expressed in a minority of Mash1-positive olfactory progenitor cells and a majority of Trpm5-expressing microvillous cells in the main olfactory epithelium. Loss-of-function mutation of Skn-1a resulted in complete loss of Trpm5-expressing microvillous cells, whereas most of olfactory sensory neurons differentiated normally. Thus, Skn-1a is a critical regulator for the generation of Trpm5-expressing microvillous cells in the main olfactory epithelium in mice.
    BMC Neuroscience 01/2014; 15(1):13. DOI:10.1186/1471-2202-15-13 · 2.67 Impact Factor
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