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Gap junctions in olfactory neurons modulate olfactory sensitivity.

Zhang C - BMC Neurosci (2010)

Bottom Line: Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT.Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subsets of ORNs.These data suggest that gap junctional communication or hemichannel activity plays a critical role in maintaining olfactory sensitivity and odor perception.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA. zhangc@iit.edu

ABSTRACT

Background: One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs) behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43) dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT) support my hypothesis.

Results: I generated OlfDNCX mice that express a dominant negative Cx43 protein, Cx43/β-gal, in mature ORNs to inactivate gap junctions and hemichannels composed of Cx43 or other structurally related connexins. Characterization of OlfDNCX revealed that Cx43/β-gal was exclusively expressed in areas where mature ORNs resided. Real time quantitative PCR indicated that cellular machineries of OlfDNCX were normal in comparison to WT. Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT. Octaldehyde-elicited glomerular activity in the olfactory bulb, measured according to odor-elicited c-fos mRNA upregulation in juxtaglomerular cells, was confined to smaller areas of the glomerular layer in OlfDNCX compared to WT. In WT mice, octaldehyde sensitive neurons exhibited reduced response magnitudes after application of gap junction uncoupling reagents and the effects were specific to subsets of neurons.

Conclusions: My study has demonstrated that altered assembly of Cx43 or structurally related connexins in ORNs modulates olfactory responses and changes olfactory activation maps in the olfactory bulb. Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subsets of ORNs. These data suggest that gap junctional communication or hemichannel activity plays a critical role in maintaining olfactory sensitivity and odor perception.

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Characterization of OlfDNCX. A. The PCR results from reverse transcription. Templates used for PCR reaction were the plasmid of the construct (plasmid), cDNA of olfactory turbinates in wild type (WT) and OlfDNCX mice. In OlfDNCX, OE- is the control that was processed identically as OE+ except missing SuperScript II in reverse transcription. B. Western analysis of protein homogenates of olfactory turbinates revealed a protein band around 120 kD in OlfDNCX, but not in WT, that was immunoreactive to antibodies against connexin 43 and β-galactosidase. C. A cartoon indicates the arrangement of epithelial cells in the olfactory epithelium. D and E. In situ hybridization in the olfactory epithelium. In situ hybridization using the antisense β-galactosidase riboprobe showed that the signal was localized to a band in the middle of the olfactory epithelial layer (D). The control (E) was processed identically as (D) except that sense β-galactosidase riboprobe was used. F and G. Confocal images displaying immunoreactivity for β-galactosidase (red) overlaid on Nomarski images. Immunostaining was observed in OlfDNCX (F), but not in WT (G). Arrowheads point to a, apical surface; and b, basal lamina. Bar = 20 μm.
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Figure 2: Characterization of OlfDNCX. A. The PCR results from reverse transcription. Templates used for PCR reaction were the plasmid of the construct (plasmid), cDNA of olfactory turbinates in wild type (WT) and OlfDNCX mice. In OlfDNCX, OE- is the control that was processed identically as OE+ except missing SuperScript II in reverse transcription. B. Western analysis of protein homogenates of olfactory turbinates revealed a protein band around 120 kD in OlfDNCX, but not in WT, that was immunoreactive to antibodies against connexin 43 and β-galactosidase. C. A cartoon indicates the arrangement of epithelial cells in the olfactory epithelium. D and E. In situ hybridization in the olfactory epithelium. In situ hybridization using the antisense β-galactosidase riboprobe showed that the signal was localized to a band in the middle of the olfactory epithelial layer (D). The control (E) was processed identically as (D) except that sense β-galactosidase riboprobe was used. F and G. Confocal images displaying immunoreactivity for β-galactosidase (red) overlaid on Nomarski images. Immunostaining was observed in OlfDNCX (F), but not in WT (G). Arrowheads point to a, apical surface; and b, basal lamina. Bar = 20 μm.

Mentions: Our studies have demonstrated expression of multiple connexins (36, 43 and 45, 57) in ORNs in addition to their expression in the olfactory bulb [23-26]. These connexins are heterogeneously distributed within the olfactory epithelium in regional and partially overlapping patterns. We postulate that gap junctional coupling between ORNs, or between ORNs and sustentacular cells, plays a role in modulating olfactory neuronal activity. However, in a follow-up freeze-fracture immunocytochemical study to visualize gap junctions in the olfactory epithelium and olfactory bulb, we did not identify gap junction plaques in ORNs [27]. This result diverges from our earlier studies where we used a combination of approaches including utilizing transgenic mice [23,24]. Even though the negative results from freeze-facture studies do not establish an absence of gap junctions in ORNs due to its technical nature, it certainly casts doubts on presence of gap junctions in ORNs. One probable explanation for this discrepancy is that the number of gap junctional channels in ORNs is sparse and cannot be identified by freeze-fracture studies since non-clustered gap junctional channels would not form typical gap junction plaques. However, a few sparsely distributed gap junctions, if present in ORNs, could profoundly modulate olfactory coding. To further address the possible involvement of gap junctions in olfactory coding, I used a dominant negative transgenic approach to specifically disrupt gap junctions in mature ORNs while gap junctions in sustentacular cells and basal cells in the olfactory epithelium and gap junctions in other tissues remain intact. The dominant negative transgenic mouse OlfDNCX expresses an olfactory marker protein (OMP) promoter driven dominant negative variant Cx43/β-galactosidase fusion protein (Cx43/β-gal) in mature ORNs with minimal expression in sustentacular cells, basal cells and immature ORNs (Figure 1 and 2). This fusion protein has a β-gal reporter protein directly fused to the C-terminus of Cx43 [28,29]. Cx43/β-gal (inactive) interferes with endogenous Cx43 during protein trafficking and thus decreases transport of Cx43 to the plasma membrane [29,30], limiting the formation of functional gap junctions [31]. Transgenic mice that express Cx43/β-gal driven by the human elongation factor-1α promoter die shortly after birth and display phenotypes typical of Cx43 knock out mice, including reduced dye coupling and heart malformation [28]. This indicates that in vivo expression of Cx43/β-gal can powerfully inhibit gap junctions. Using OlfDNCX mice, I demonstrate that gap junctional communication in the olfactory epithelium modulates olfactory activity at the peripheral level and alters glomerular activation patterns (odor maps) in the olfactory bulb. Topological changes in odor maps due to gap junctional modulation could affect perception of odor quality or quantity.


Gap junctions in olfactory neurons modulate olfactory sensitivity.

Zhang C - BMC Neurosci (2010)

Characterization of OlfDNCX. A. The PCR results from reverse transcription. Templates used for PCR reaction were the plasmid of the construct (plasmid), cDNA of olfactory turbinates in wild type (WT) and OlfDNCX mice. In OlfDNCX, OE- is the control that was processed identically as OE+ except missing SuperScript II in reverse transcription. B. Western analysis of protein homogenates of olfactory turbinates revealed a protein band around 120 kD in OlfDNCX, but not in WT, that was immunoreactive to antibodies against connexin 43 and β-galactosidase. C. A cartoon indicates the arrangement of epithelial cells in the olfactory epithelium. D and E. In situ hybridization in the olfactory epithelium. In situ hybridization using the antisense β-galactosidase riboprobe showed that the signal was localized to a band in the middle of the olfactory epithelial layer (D). The control (E) was processed identically as (D) except that sense β-galactosidase riboprobe was used. F and G. Confocal images displaying immunoreactivity for β-galactosidase (red) overlaid on Nomarski images. Immunostaining was observed in OlfDNCX (F), but not in WT (G). Arrowheads point to a, apical surface; and b, basal lamina. Bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Characterization of OlfDNCX. A. The PCR results from reverse transcription. Templates used for PCR reaction were the plasmid of the construct (plasmid), cDNA of olfactory turbinates in wild type (WT) and OlfDNCX mice. In OlfDNCX, OE- is the control that was processed identically as OE+ except missing SuperScript II in reverse transcription. B. Western analysis of protein homogenates of olfactory turbinates revealed a protein band around 120 kD in OlfDNCX, but not in WT, that was immunoreactive to antibodies against connexin 43 and β-galactosidase. C. A cartoon indicates the arrangement of epithelial cells in the olfactory epithelium. D and E. In situ hybridization in the olfactory epithelium. In situ hybridization using the antisense β-galactosidase riboprobe showed that the signal was localized to a band in the middle of the olfactory epithelial layer (D). The control (E) was processed identically as (D) except that sense β-galactosidase riboprobe was used. F and G. Confocal images displaying immunoreactivity for β-galactosidase (red) overlaid on Nomarski images. Immunostaining was observed in OlfDNCX (F), but not in WT (G). Arrowheads point to a, apical surface; and b, basal lamina. Bar = 20 μm.
Mentions: Our studies have demonstrated expression of multiple connexins (36, 43 and 45, 57) in ORNs in addition to their expression in the olfactory bulb [23-26]. These connexins are heterogeneously distributed within the olfactory epithelium in regional and partially overlapping patterns. We postulate that gap junctional coupling between ORNs, or between ORNs and sustentacular cells, plays a role in modulating olfactory neuronal activity. However, in a follow-up freeze-fracture immunocytochemical study to visualize gap junctions in the olfactory epithelium and olfactory bulb, we did not identify gap junction plaques in ORNs [27]. This result diverges from our earlier studies where we used a combination of approaches including utilizing transgenic mice [23,24]. Even though the negative results from freeze-facture studies do not establish an absence of gap junctions in ORNs due to its technical nature, it certainly casts doubts on presence of gap junctions in ORNs. One probable explanation for this discrepancy is that the number of gap junctional channels in ORNs is sparse and cannot be identified by freeze-fracture studies since non-clustered gap junctional channels would not form typical gap junction plaques. However, a few sparsely distributed gap junctions, if present in ORNs, could profoundly modulate olfactory coding. To further address the possible involvement of gap junctions in olfactory coding, I used a dominant negative transgenic approach to specifically disrupt gap junctions in mature ORNs while gap junctions in sustentacular cells and basal cells in the olfactory epithelium and gap junctions in other tissues remain intact. The dominant negative transgenic mouse OlfDNCX expresses an olfactory marker protein (OMP) promoter driven dominant negative variant Cx43/β-galactosidase fusion protein (Cx43/β-gal) in mature ORNs with minimal expression in sustentacular cells, basal cells and immature ORNs (Figure 1 and 2). This fusion protein has a β-gal reporter protein directly fused to the C-terminus of Cx43 [28,29]. Cx43/β-gal (inactive) interferes with endogenous Cx43 during protein trafficking and thus decreases transport of Cx43 to the plasma membrane [29,30], limiting the formation of functional gap junctions [31]. Transgenic mice that express Cx43/β-gal driven by the human elongation factor-1α promoter die shortly after birth and display phenotypes typical of Cx43 knock out mice, including reduced dye coupling and heart malformation [28]. This indicates that in vivo expression of Cx43/β-gal can powerfully inhibit gap junctions. Using OlfDNCX mice, I demonstrate that gap junctional communication in the olfactory epithelium modulates olfactory activity at the peripheral level and alters glomerular activation patterns (odor maps) in the olfactory bulb. Topological changes in odor maps due to gap junctional modulation could affect perception of odor quality or quantity.

Bottom Line: Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT.Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subsets of ORNs.These data suggest that gap junctional communication or hemichannel activity plays a critical role in maintaining olfactory sensitivity and odor perception.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA. zhangc@iit.edu

ABSTRACT

Background: One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs) behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43) dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT) support my hypothesis.

Results: I generated OlfDNCX mice that express a dominant negative Cx43 protein, Cx43/β-gal, in mature ORNs to inactivate gap junctions and hemichannels composed of Cx43 or other structurally related connexins. Characterization of OlfDNCX revealed that Cx43/β-gal was exclusively expressed in areas where mature ORNs resided. Real time quantitative PCR indicated that cellular machineries of OlfDNCX were normal in comparison to WT. Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT. Octaldehyde-elicited glomerular activity in the olfactory bulb, measured according to odor-elicited c-fos mRNA upregulation in juxtaglomerular cells, was confined to smaller areas of the glomerular layer in OlfDNCX compared to WT. In WT mice, octaldehyde sensitive neurons exhibited reduced response magnitudes after application of gap junction uncoupling reagents and the effects were specific to subsets of neurons.

Conclusions: My study has demonstrated that altered assembly of Cx43 or structurally related connexins in ORNs modulates olfactory responses and changes olfactory activation maps in the olfactory bulb. Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subsets of ORNs. These data suggest that gap junctional communication or hemichannel activity plays a critical role in maintaining olfactory sensitivity and odor perception.

Show MeSH
Related in: MedlinePlus