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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 does not increase the pheromone-dependent sensillum potential amplitude (SPA) nor the bombykal-dependent action potential (AP) frequency as it would be expected for an Orco-based ionotropic mechanism of pheromone transduction.(A–C) Box plots represent pheromone responses during the first 20 minutes (beginning) of the tip-recordings. (A) The pheromone-dependent SPA was never affected by VUAA1. (B) The pheromone-dependent AP response (first 5 APs) was not affected by VUAA1 during the activity phase, but was decreased at high agonist concentrations at rest. (C) Only at rest the first pheromone-dependent AP was delayed by VUAA1. Significant differences are indicated by asterisks (n.s. = not significant; **P<0.01, ***P<0.001; Mann-Whitney test).
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pone-0062648-g002: VUAA1-dependent MsexOrco activation does not increase the pheromone-dependent sensillum potential amplitude (SPA) nor the bombykal-dependent action potential (AP) frequency as it would be expected for an Orco-based ionotropic mechanism of pheromone transduction.(A–C) Box plots represent pheromone responses during the first 20 minutes (beginning) of the tip-recordings. (A) The pheromone-dependent SPA was never affected by VUAA1. (B) The pheromone-dependent AP response (first 5 APs) was not affected by VUAA1 during the activity phase, but was decreased at high agonist concentrations at rest. (C) Only at rest the first pheromone-dependent AP was delayed by VUAA1. Significant differences are indicated by asterisks (n.s. = not significant; **P<0.01, ***P<0.001; Mann-Whitney test).

Mentions: In long-term tip-recordings infusion of VUAA1 did not affect pheromone-dependent sensillum potential amplitudes (SPA), neither during activity nor during resting phase (Fig. 2A,S1; Tab. S2,S4). Thus, Orco appears not to contribute to the rise of the BAL-dependent receptor potential. Also, analysis of the phasic action potential (AP) response did not provide evidence for a BAL-gated OR-Orco-dependent ion channel opening during the first ∼25 ms of the pheromone response. In the beginning of the long-term tip-recordings the BAL-dependent AP frequency was not affected by VUAA1 during the activity phase (Fig. 2B). A significant decline only occurred with perfusion of 100 µM VUAA1 at rest. Both control and VUAA1 recordings showed a significant decline in AP frequency over the time course (Fig. S2A–D; Tab. S2,S4). This decline was significantly stronger in the presence of 100 µM VUAA1.


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 does not increase the pheromone-dependent sensillum potential amplitude (SPA) nor the bombykal-dependent action potential (AP) frequency as it would be expected for an Orco-based ionotropic mechanism of pheromone transduction.(A–C) Box plots represent pheromone responses during the first 20 minutes (beginning) of the tip-recordings. (A) The pheromone-dependent SPA was never affected by VUAA1. (B) The pheromone-dependent AP response (first 5 APs) was not affected by VUAA1 during the activity phase, but was decreased at high agonist concentrations at rest. (C) Only at rest the first pheromone-dependent AP was delayed by VUAA1. Significant differences are indicated by asterisks (n.s. = not significant; **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-g002: VUAA1-dependent MsexOrco activation does not increase the pheromone-dependent sensillum potential amplitude (SPA) nor the bombykal-dependent action potential (AP) frequency as it would be expected for an Orco-based ionotropic mechanism of pheromone transduction.(A–C) Box plots represent pheromone responses during the first 20 minutes (beginning) of the tip-recordings. (A) The pheromone-dependent SPA was never affected by VUAA1. (B) The pheromone-dependent AP response (first 5 APs) was not affected by VUAA1 during the activity phase, but was decreased at high agonist concentrations at rest. (C) Only at rest the first pheromone-dependent AP was delayed by VUAA1. Significant differences are indicated by asterisks (n.s. = not significant; **P<0.01, ***P<0.001; Mann-Whitney test).
Mentions: In long-term tip-recordings infusion of VUAA1 did not affect pheromone-dependent sensillum potential amplitudes (SPA), neither during activity nor during resting phase (Fig. 2A,S1; Tab. S2,S4). Thus, Orco appears not to contribute to the rise of the BAL-dependent receptor potential. Also, analysis of the phasic action potential (AP) response did not provide evidence for a BAL-gated OR-Orco-dependent ion channel opening during the first ∼25 ms of the pheromone response. In the beginning of the long-term tip-recordings the BAL-dependent AP frequency was not affected by VUAA1 during the activity phase (Fig. 2B). A significant decline only occurred with perfusion of 100 µM VUAA1 at rest. Both control and VUAA1 recordings showed a significant decline in AP frequency over the time course (Fig. S2A–D; Tab. S2,S4). This decline was significantly stronger in the presence of 100 µM VUAA1.

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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