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In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta.

Nolte A, Funk NW, Mukunda L, Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M - PLoS ONE (2013)

Bottom Line: Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms.We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta.Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Physiology, University of Kassel, Kassel, Germany.

ABSTRACT
The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cβ. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca(2+)-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

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VUAA1- dependent MsexOrco activation increases spontaneous as well as background activity of olfactory receptor neurons (ORNs).Original recordings show spontaneous activity without previous BAL-stimulation (A) and background activity (C) after BAL-stimulation of bombykal- (BAL) sensitive ORNs (larger amplitude). Action potentials of smaller amplitude were generated by the second BAL-insensitive ORN. (B) Spontaneous activity was dose-dependently increased by VUAA1 stimulation, with lower VUAA1 concentrations required for saturation in the activity phase. (D) Lower VUAA1 concentrations increased the background activity already maximally during the first 20 min (beginning) of the recordings and more strongly than the spontaneous activity. Furthermore, MsexOrco appeared to express Zeitgebertime-dependent changes. Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).
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pone-0062648-g004: VUAA1- dependent MsexOrco activation increases spontaneous as well as background activity of olfactory receptor neurons (ORNs).Original recordings show spontaneous activity without previous BAL-stimulation (A) and background activity (C) after BAL-stimulation of bombykal- (BAL) sensitive ORNs (larger amplitude). Action potentials of smaller amplitude were generated by the second BAL-insensitive ORN. (B) Spontaneous activity was dose-dependently increased by VUAA1 stimulation, with lower VUAA1 concentrations required for saturation in the activity phase. (D) Lower VUAA1 concentrations increased the background activity already maximally during the first 20 min (beginning) of the recordings and more strongly than the spontaneous activity. Furthermore, MsexOrco appeared to express Zeitgebertime-dependent changes. Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).

Mentions: Next we examined whether Orco forms an ion channel involved in modulating spontaneous activity in the absence of pheromone. A significant VUAA1 dose-dependent increase in spontaneous activity without previous pheromone stimulation could be observed during activity and resting phase, with higher sensitivity to VUAA1 during activity phase (Fig. 4B;Tab. S3,S4). Also, the background activity between two pheromone stimulations was examined. Application of VUAA1 significantly increased background activity (Fig. 4C,D;Tab. S2,S4). However, no dose-dependent effect was found. While long-term control recordings showed a continuous decline over the time course, the decline was counteracted in 100 µM VUAA1 recordings during activity and resting phase (Fig. S4). Background activity was significantly higher than spontaneous activity in control and 1 µM VUAA1 recordings (P<0.001 for all). However, during the activity phase spontaneous activity was significantly higher than background activity under the influence of 100 µM VUAA1 (P = 0.006).


In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta.

Nolte A, Funk NW, Mukunda L, Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M - PLoS ONE (2013)

VUAA1- dependent MsexOrco activation increases spontaneous as well as background activity of olfactory receptor neurons (ORNs).Original recordings show spontaneous activity without previous BAL-stimulation (A) and background activity (C) after BAL-stimulation of bombykal- (BAL) sensitive ORNs (larger amplitude). Action potentials of smaller amplitude were generated by the second BAL-insensitive ORN. (B) Spontaneous activity was dose-dependently increased by VUAA1 stimulation, with lower VUAA1 concentrations required for saturation in the activity phase. (D) Lower VUAA1 concentrations increased the background activity already maximally during the first 20 min (beginning) of the recordings and more strongly than the spontaneous activity. Furthermore, MsexOrco appeared to express Zeitgebertime-dependent changes. Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643954&req=5

pone-0062648-g004: VUAA1- dependent MsexOrco activation increases spontaneous as well as background activity of olfactory receptor neurons (ORNs).Original recordings show spontaneous activity without previous BAL-stimulation (A) and background activity (C) after BAL-stimulation of bombykal- (BAL) sensitive ORNs (larger amplitude). Action potentials of smaller amplitude were generated by the second BAL-insensitive ORN. (B) Spontaneous activity was dose-dependently increased by VUAA1 stimulation, with lower VUAA1 concentrations required for saturation in the activity phase. (D) Lower VUAA1 concentrations increased the background activity already maximally during the first 20 min (beginning) of the recordings and more strongly than the spontaneous activity. Furthermore, MsexOrco appeared to express Zeitgebertime-dependent changes. Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).
Mentions: Next we examined whether Orco forms an ion channel involved in modulating spontaneous activity in the absence of pheromone. A significant VUAA1 dose-dependent increase in spontaneous activity without previous pheromone stimulation could be observed during activity and resting phase, with higher sensitivity to VUAA1 during activity phase (Fig. 4B;Tab. S3,S4). Also, the background activity between two pheromone stimulations was examined. Application of VUAA1 significantly increased background activity (Fig. 4C,D;Tab. S2,S4). However, no dose-dependent effect was found. While long-term control recordings showed a continuous decline over the time course, the decline was counteracted in 100 µM VUAA1 recordings during activity and resting phase (Fig. S4). Background activity was significantly higher than spontaneous activity in control and 1 µM VUAA1 recordings (P<0.001 for all). However, during the activity phase spontaneous activity was significantly higher than background activity under the influence of 100 µM VUAA1 (P = 0.006).

Bottom Line: Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms.We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta.Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Physiology, University of Kassel, Kassel, Germany.

ABSTRACT
The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cβ. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca(2+)-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

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