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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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Gap junction coupling is neuron specific. Raw data of calcium imaging showing representative neuronal responses to stimuli in a period over 30 min. Data were binned every four frames after recordings using the Excel program and presented in the ratio of F340/F380. For the convenience of description, data are grouped into 5 phases. Duration of stimulation is indicated by black (for odorants) or red (for BGA) bars on the top. BEN, 100 μM benzaldehyde; BGA, 1 μM 18β-glycyrrhetinic acid, a gap junction uncoupling reagent; IBMX, 500 μM 3-isobutyl-1-methylxanthine; OCT, 100 μM octaldehyde.
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Figure 4: Gap junction coupling is neuron specific. Raw data of calcium imaging showing representative neuronal responses to stimuli in a period over 30 min. Data were binned every four frames after recordings using the Excel program and presented in the ratio of F340/F380. For the convenience of description, data are grouped into 5 phases. Duration of stimulation is indicated by black (for odorants) or red (for BGA) bars on the top. BEN, 100 μM benzaldehyde; BGA, 1 μM 18β-glycyrrhetinic acid, a gap junction uncoupling reagent; IBMX, 500 μM 3-isobutyl-1-methylxanthine; OCT, 100 μM octaldehyde.

Mentions: I used calcium imaging to monitor individual neuronal responses with or without the influence of gap junction uncoupling reagents in WT mice. Calcium imaging was conducted on the surface of the epithelium in intact turbinates as shown in Figure 3A. Because the recording ORNs were situated within an intact olfactory epithelium, they were under optimal biological conditions they could possibly have in an in situ preparation. This in situ preparation allowed me to study cell-to-cell communication under physiological conditions. Figure 4 shows examples of individual ORNs responding to odorants before and after application of the gap junction uncoupling reagent 18β-glycyrrhetinic acid (BGA) at 1 μM. At this concentration, BGA itself induced negligible intracellular calcium changes (Phase 2A, 2B, 4A and 4B in Figure 4). Interestingly, at this concentration the effects of BGA to odor-evoked responses were neuron specific. Neuron A, an octaldehyde-responsive neuron, was responsive to 500 μM IBMX as well (Phase 1A). After application of 1 μM BGA, responsiveness of Neuron A to octaldehyde was reduced to threshold levels (Phase 2A). Since the effect of BGA is reversible [46,47], Neuron A regained its response magnitude to octaldehyde minutes later (Phase 3A). Phase 4A in Figure 4 shows that responsiveness of Neuron A to IBMX was not influenced by application of BGA. The same applied to its responsiveness to 76 mM KCl (high K) (data not shown). These results indicate that BGA does not interfere with normal physiological capability of Neuron A because it did not interfere with IBMX- or high K-evoked responses. However, the responses to 100 μM octaldehyde observed in Phase 1A were significantly modulated by the BGA treatment. I speculate that the olfactory receptor of Neuron A is only moderately sensitive to octaldehyde and gap junctional coupling with other neurons is necessary to sustain the response magnitude observed in Phase 1A.


Gap junctions in olfactory neurons modulate olfactory sensitivity.

Zhang C - BMC Neurosci (2010)

Gap junction coupling is neuron specific. Raw data of calcium imaging showing representative neuronal responses to stimuli in a period over 30 min. Data were binned every four frames after recordings using the Excel program and presented in the ratio of F340/F380. For the convenience of description, data are grouped into 5 phases. Duration of stimulation is indicated by black (for odorants) or red (for BGA) bars on the top. BEN, 100 μM benzaldehyde; BGA, 1 μM 18β-glycyrrhetinic acid, a gap junction uncoupling reagent; IBMX, 500 μM 3-isobutyl-1-methylxanthine; OCT, 100 μM octaldehyde.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944353&req=5

Figure 4: Gap junction coupling is neuron specific. Raw data of calcium imaging showing representative neuronal responses to stimuli in a period over 30 min. Data were binned every four frames after recordings using the Excel program and presented in the ratio of F340/F380. For the convenience of description, data are grouped into 5 phases. Duration of stimulation is indicated by black (for odorants) or red (for BGA) bars on the top. BEN, 100 μM benzaldehyde; BGA, 1 μM 18β-glycyrrhetinic acid, a gap junction uncoupling reagent; IBMX, 500 μM 3-isobutyl-1-methylxanthine; OCT, 100 μM octaldehyde.
Mentions: I used calcium imaging to monitor individual neuronal responses with or without the influence of gap junction uncoupling reagents in WT mice. Calcium imaging was conducted on the surface of the epithelium in intact turbinates as shown in Figure 3A. Because the recording ORNs were situated within an intact olfactory epithelium, they were under optimal biological conditions they could possibly have in an in situ preparation. This in situ preparation allowed me to study cell-to-cell communication under physiological conditions. Figure 4 shows examples of individual ORNs responding to odorants before and after application of the gap junction uncoupling reagent 18β-glycyrrhetinic acid (BGA) at 1 μM. At this concentration, BGA itself induced negligible intracellular calcium changes (Phase 2A, 2B, 4A and 4B in Figure 4). Interestingly, at this concentration the effects of BGA to odor-evoked responses were neuron specific. Neuron A, an octaldehyde-responsive neuron, was responsive to 500 μM IBMX as well (Phase 1A). After application of 1 μM BGA, responsiveness of Neuron A to octaldehyde was reduced to threshold levels (Phase 2A). Since the effect of BGA is reversible [46,47], Neuron A regained its response magnitude to octaldehyde minutes later (Phase 3A). Phase 4A in Figure 4 shows that responsiveness of Neuron A to IBMX was not influenced by application of BGA. The same applied to its responsiveness to 76 mM KCl (high K) (data not shown). These results indicate that BGA does not interfere with normal physiological capability of Neuron A because it did not interfere with IBMX- or high K-evoked responses. However, the responses to 100 μM octaldehyde observed in Phase 1A were significantly modulated by the BGA treatment. I speculate that the olfactory receptor of Neuron A is only moderately sensitive to octaldehyde and gap junctional coupling with other neurons is necessary to sustain the response magnitude observed in Phase 1A.

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