Four Irritating Odorants Target the Trigeminal Chemoreceptor TRPA1

Abstract

The trigeminal nerve responds to a variety of nociceptive stimuli, including many chemicals that activate the olfactory system at lower concentrations. However, the mechanisms by which specific odorants activate the trigeminal nerve are largely undetermined. We used an integrative approach to determine whether TRPA1 channels were the target of eight olfactory stimuli known to activate the trigeminal nerve. In a mammalian cell line stably expressing the human TRPA1 channel, we observed significant increases in intracellular calcium levels upon application of α-terpineol, amyl acetate, benzaldehyde, and toluene. Notably, these responses were greatly reduced when these chemicals were applied in conjunction with the TRPA1 inhibitor HC-030031. To determine whether TRPA1 is required for detecting these odorants as irritants in vivo, we evaluated physiological and behavioral responses to these stimuli in mice lacking the TRPA1 channel. A decline in respiration was seen in wild-type but not TRPA1 knockout mice upon exposure to α-terpineol, benzaldehyde, and toluene. Furthermore, we observed either attenuated or absent avoidance behaviors in TRPA1 knockout mice compared to wild-type mice when exposed to α-terpineol, amyl acetate, and benzaldehyde. Our results show that TRPA1 is a molecular target for four different odorants-α-terpineol, amyl acetate, benzaldehyde, and toluene. Additionally, this study suggests that TRPA1 is required for detection of α-terpineol, benzaldehyde, and toluene as trigeminal irritants. However, TRPA1 is likely only one of multiple trigeminal receptors detecting amyl acetate.

Dissecting the role of TRPV1 in detecting multiple trigeminal irritants in three behavioral assays for sensory irritation

“…It is also produced synthetically for use as an artificial flavoring because of its distinctly fruity odor 39 . In addition to stimulating the olfactory system, at high concentrations amyl acetate also stimulates the trigeminal system 4, 25 . Previous work has demonstrated that while amyl acetate activates TRPA1 it is not the only mechanism by which amyl acetate is detected 25 .…”

“…The sap of the tolu balsam tree contains toluene and has been used as an ingredient in cough syrups 41 . Both benzaldehyde and toluene stimulate the trigeminal nerve 4, 24 , fail to activate TRPV1 in vitro 4 , activate TRPA1 in vivo and fail to induce the expiratory pause reflex in TRPA1 knockouts 25 . However, TRPA1 knockouts find benzaldehyde less aversive in the cotton swab paradigm but respond to toluene normally 25 .…”

“…The cotton swab test has been used previously to test the aversion of TRPA1 knockout mice to a number of chemicals 25 . For liquid stimuli, cotton applicators (Puritan Medical Products, Guilford, ME, USA) were saturated with the pure compound before presentation to the animal.…”

“…We have previously used this assay to assess the response of TRPA1 knockouts 25 and capsaicin-desensitized rats 26 to trigeminal irritants. For an individual mouse, a mean behavioral score was calculated for each substance and the scores from each animal were then averaged to ensure normally distributed data.…”

“…In another assay, a modified two-bottle preference test, which avoids mixing irritants with the animal’s drinking water to prevent post-ingestive effects, was used to assess aversion. In the final assay, a custom-built air-dilution olfactometer was used to administer volatile compounds to mice restrained in a double-chambered plethysmograph where respiratory reflexes were monitored, this representing a modified “Alarie Test” 24, 25 . Our data suggest that, although many compounds may activate TRPV1, only a few, such as cyclohexanone, are exclusively detected by the channel, a finding that underlies the biological redundancy typified by the trigeminal system.…”