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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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During the first 20 min of each recording VUAA1-dependent MsexOrco activation does not affect the first 150 ms or first 1000 ms of the pheromone response.Rather, MsexOrco-ion channel opening affects bombykal (BAL)-response kinetics at the time scale of minutes, at the last 20 min of the 2 h recording. (A,B) Post stimulus time histograms show the mean number of APs generated within the first 1000 ms after BAL stimulation (binsize = 10 ms). The number of APs within the first 150 ms (shaded area A,B) and the first 1000 ms did not change VUAA1-dependently during the first 20 minutes (beginning) of the recording. At the end of the tip-recordings (last 20 minutes) the pheromone responses shifted to a more tonic response pattern in the presence of 100 µM VUAA1 as compared to the beginning (see also Fig. S3).
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pone-0062648-g003: During the first 20 min of each recording VUAA1-dependent MsexOrco activation does not affect the first 150 ms or first 1000 ms of the pheromone response.Rather, MsexOrco-ion channel opening affects bombykal (BAL)-response kinetics at the time scale of minutes, at the last 20 min of the 2 h recording. (A,B) Post stimulus time histograms show the mean number of APs generated within the first 1000 ms after BAL stimulation (binsize = 10 ms). The number of APs within the first 150 ms (shaded area A,B) and the first 1000 ms did not change VUAA1-dependently during the first 20 minutes (beginning) of the recording. At the end of the tip-recordings (last 20 minutes) the pheromone responses shifted to a more tonic response pattern in the presence of 100 µM VUAA1 as compared to the beginning (see also Fig. S3).

Mentions: To determine further ion channel activation by VUAA1 within the first second of the pheromone response, post stimulus time histograms (PSTHs) were prepared (Fig. 3). The number of BAL-dependent APs was analyzed during the first 150 ms and 1000 ms after onset of the BAL-dependent sensillum potential (Fig. S3, Tab. S2, S4). In the beginning of the recordings neither VUAA1 concentration had any effect on the number of APs generated in the first 150 ms (Fig. 3A,B; Tab S2,S4). Neither was the number of APs during the first 1000 ms of the activity phase affected by VUAA1 (Fig. S3C,D;Tab. S2,S4), while at rest only 1 µM VUAA1 caused a significant decline (Tab. S2,S4). Comparison of the distribution (Fig. 3) and number of APs (Fig. S3) in controls over the course of the recordings indicates that the kinetics of the pheromone response shifted to a more tonic response pattern during rest. In addition, the decrease of the number of APs in the first 150 ms in control experiments indicated an increase in threshold at rest (Fig. S3, Tab. S2,S4). Application of the Orco agonist further enhanced this shift in kinetics and BAL-sensitivity during the course of the recording. This suggests that Orco activation affected the kinetics as well as the sensitivity of the BAL response on the time scale of minutes rather than milliseconds.


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)

During the first 20 min of each recording VUAA1-dependent MsexOrco activation does not affect the first 150 ms or first 1000 ms of the pheromone response.Rather, MsexOrco-ion channel opening affects bombykal (BAL)-response kinetics at the time scale of minutes, at the last 20 min of the 2 h recording. (A,B) Post stimulus time histograms show the mean number of APs generated within the first 1000 ms after BAL stimulation (binsize = 10 ms). The number of APs within the first 150 ms (shaded area A,B) and the first 1000 ms did not change VUAA1-dependently during the first 20 minutes (beginning) of the recording. At the end of the tip-recordings (last 20 minutes) the pheromone responses shifted to a more tonic response pattern in the presence of 100 µM VUAA1 as compared to the beginning (see also Fig. S3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643954&req=5

pone-0062648-g003: During the first 20 min of each recording VUAA1-dependent MsexOrco activation does not affect the first 150 ms or first 1000 ms of the pheromone response.Rather, MsexOrco-ion channel opening affects bombykal (BAL)-response kinetics at the time scale of minutes, at the last 20 min of the 2 h recording. (A,B) Post stimulus time histograms show the mean number of APs generated within the first 1000 ms after BAL stimulation (binsize = 10 ms). The number of APs within the first 150 ms (shaded area A,B) and the first 1000 ms did not change VUAA1-dependently during the first 20 minutes (beginning) of the recording. At the end of the tip-recordings (last 20 minutes) the pheromone responses shifted to a more tonic response pattern in the presence of 100 µM VUAA1 as compared to the beginning (see also Fig. S3).
Mentions: To determine further ion channel activation by VUAA1 within the first second of the pheromone response, post stimulus time histograms (PSTHs) were prepared (Fig. 3). The number of BAL-dependent APs was analyzed during the first 150 ms and 1000 ms after onset of the BAL-dependent sensillum potential (Fig. S3, Tab. S2, S4). In the beginning of the recordings neither VUAA1 concentration had any effect on the number of APs generated in the first 150 ms (Fig. 3A,B; Tab S2,S4). Neither was the number of APs during the first 1000 ms of the activity phase affected by VUAA1 (Fig. S3C,D;Tab. S2,S4), while at rest only 1 µM VUAA1 caused a significant decline (Tab. S2,S4). Comparison of the distribution (Fig. 3) and number of APs (Fig. S3) in controls over the course of the recordings indicates that the kinetics of the pheromone response shifted to a more tonic response pattern during rest. In addition, the decrease of the number of APs in the first 150 ms in control experiments indicated an increase in threshold at rest (Fig. S3, Tab. S2,S4). Application of the Orco agonist further enhanced this shift in kinetics and BAL-sensitivity during the course of the recording. This suggests that Orco activation affected the kinetics as well as the sensitivity of the BAL response on the time scale of minutes rather than milliseconds.

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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