Fig 1.
PEA mitigated behavioural aversion to the volatile irritant cyclohexanone in mice.
A: Depiction of the aluminum annular housing (h = 11 mm; w = 30 mm; D = 8.5 mm and d = 4 mm) that were filled with pieces of felt soaked in irritant and odorant solutions and placd around water sipping tubes in home cages. B, lower: Mice had acces to water ad libitum from two water sources throughout the experiment. B, upper: After seven-days acclimatization to two water sources, housing were filled with an irritant, odorant or vehicle randomized for the side (left/right) over 3 consecutive days. C: Water consumption from each of two bottles. In the absence of irritant or odorant there was no bottle preference (paired t-test, n = 10, p = 0.60). D: Similarly, there was no drinking preference bottles with pure odorant PEA and vehicle (paired t-test, n = 10, p = 0.26). However, absolute water consumption was diminished by the introduction of PEA (panel D, upper right corner: paired t-test, n = 10, p < 0.01). E: Mice showed aversion to the volatile irritant cyclohexanone (panel E, left: 2-way ANOVA, factor cyclo, p < 0.05, post hoc Tukey HSD, water vs cyclo, p = 0.01) and this aversion was mitigated with the addition of PEA to both housings (panel E, right: 2-way ANOVA, factor cyclo, p < 0.05, post hoc Tukey HSD, PEA vs cyclo, p = 0.94) although absolute water consumption did not change (panel F, inset upper right corner: paired t-test, n = 12, p = 0.16). F: Mice showed aversion to the volatile irritant AITC (panel F, left: 2-way ANOVA, factor AITC, p < 0.01, post hoc Tukey HSD, water vs AITC, p < 0.01) and aversion persisted with the addition of PEA to both housings (panel F, right: 2-way ANOVA, factor AITC, p < 0.01, post hoc Tukey HSD, PEA vs PEA + AITC, p < 0.01). Absolute water consumption did not change (panel F, inset upper right corner: paired t-test, n = 10, p = 0.40).
Fig 2.
Ethmoid innervation of the rodent nasal cavity.
A: Spatial distribution of ethmoid nerve revealed by anterograde tracing with the dextran amine DiI (red tracing) overlaid on a sketch of the rat skull in mid-sagittal section (scale bar: 2 mm, background adapted from barrios et al. 2014 [69]).B-D. B-D: Representative immunohistological sections of anterior ethmoid nerve (left images, scale bar: 10 μm) and trigeminal ganglion (right images, scale bar: 50 μm) from TRPM8eGFP mice (B, green), TRPA1-immuno-stainings in WT mice (C, green) and NaV1.8::tdTomato mice (D, NaV1.8-red, TRPA1-green). E, F: Images from isolated mouse olfactory epithelium showing nerve terminals expressing the TRPM8eGFP reporter (E, green) and TRPA1-immuno-label (F, green). DAPI staining is shown in blue in panels B-F.
Table 1.
Primary antibodies.
Fig 3.
Functional assessment of individual sensory axons in the ethmoid nerve.
A: Extracellular signals were recorded from axons in the ethmoid nerve with receptive fields in the respiratory and olfactory epithelia identified by electrical (A; white markers) and mechanical (A; black markers) stimuli. (Scale bar: 2 mm;, background adapted from barrios et al. 2014 [69]) B: Distribution of axonal velocities for 82 individual trigeminal afferents in the ethmoid nerve. Mechano-sensitive axons are displayed as filled bars (grey; 0.2–5.2 m/s) and electrically-evoked axons are shown as white bars (0.2–2 m/s) C: Receptive field sites for thermally sensitive trigeminal axons identified by mechanical (black markers, n = 12) and electrical (white marker, n = 9) search stimuli (scale bar: 2 mm; background image adapted from Barrios et al., 2014). D&E: Pooled temperature threshold of trigeminal afferents to heating (D) and cooling (E) and initially identified by mechanical (M, black markers and shading) and electrical (E, white markers and shading) search stimuli. F: Falling leaf display of a trigeminal afferent responding to repeat electrical stimulation during a heating and cooling stimulus (red trace). G-J: An example of trigeminal afferents as part of a multi-unit recording responding with an increase in firing during cooling (I). Response patterns for two individual units (G&H), identified by spike shape (G&H insets), are shown during cooling. A box and whisker plot representing pooled data for the threshold temperature at which an increase in firing rate was identified in 11 non-tracked units observed in multi-unit recordings (K).
Fig 4.
Characterization of responses to chemical stimuli in individual ethmoid afferents.
A: Receptive field locations within the respiratory and olfactory epithelia for individual trigeminal afferents responding to chemical stimuli and identified using electrical (A; white markers) and mechanical (A, black markers) search stimuli (scale bar: 2 mm;, background adapted from barrios et al. 2014 [69]). B: Illustrative example of an individual trigeminal afferent responding at fixed latency to repeat electrical stimulation, shown as a falling leaf display. Application of ammonia (NH3, blue trace) activates the axon leaving it refractory to electrical stimulation before it is once again activated and re-establishes a stable response latency to electrical stimulation. C: Normalized latency of single afferent response to electrical stimulation across time before, during and after application of phenylethyl alcohol PEA (dashed line) D: Pooled data for latency changes in response to pure odorant phenylethyl alcohol (PEA; n = 6, paired t-test, p = 0.77). E-P: Representative examples of changes in electrical response latency of individual sensory axons during application of chemical stimuli to the nose (upper panels) and corresponding pooled data for latency changes in response to capsaicin (250 nM; E&F, n = 2), (-)-menthol (10μM; G&H, n = 1), allyl isothiocyanate (AITC, 20μM; I&J, n = 2), cyclohexanone (1%; K&L, n = 5), icilin (10μM; M&N, n = 2) and ammonia (NH3; O&P, n = 4).
Fig 5.
Assessment of functional interactions between olfactory and trigeminal sensory afferents in the nasal cavity.
A: Electrical receptive field locations for seven trigeminal afferents (A, blue markers) recorded in OMP/ChR2-YFP mice (scale bar: 2 mm, background adapted from barrios et al. 2014 [69]). B: Panel B shows the fusion protein ChR2-YFP (green) and nuclear DAPI (blue) staining in the olfactory epithelium in transverse section. C: Photoactivation of olfactory sensory neurons was verified by recording extracellular electro-olfactogram (EOG) signals with an electrode positioned on the second turbinate (A, II) in response to sinusoidal light pulses varying in duration from 2-100ms. D: The absolute amplitude of the EOG signal was maximal in response to a 14 ms sinusoidal light pulse (D, black trace), while the positive going EOG was maximal for stimuli of 9 ms duration (D; red trace) and the negative-going EOG signal had a maximum amplitude at stimulus widths of 18 ms (D; blue trace). E: The response latency of action potentials in single trigeminal axons evoked by electrical stimulation (E, lower black trace) was monitored during electrical stimulation alone every 4s (E, grey traces) and combined with photoactivation (E, lower blue trace) of olfactory sensory neurons every 12s (E, blue traces). F-G:The average response latency to electrical stimulation alone (F, open grey markers) was compared to the electrical response latency when applied with light stimulation (F, blue markers) and the ratio of these two latencies determined (G). H: Pooled latency ratios for electrically-evoked responses in trigeminal afferents without light stimulation (control) and in combination with photostimulation (LED) are shown for seven fibres.
Fig 6.
Functional assessment of trigeminal axonal branching to the olfactory epithelium and the olfactory dura mater.
A-C: Recording from the distal cut end of the ethmoid nerve at its entry into the anterior cranial fossa (B) it was possible to verify electrical (A&C) receptive fields for single trigeminal axons both in the nasal cavity (C) and in the olfactory dura mater (A). D-F:Using the same recording configuration, it was possible to verify action potential activity (D & F) in response to mechanical von Frey probing (1.47 mN, D & F, vertical black markers) in the nasal cavity (E, dashed blue circle) and in the olfactory dura mater (E, dashed green circle). Responses to von Frey (1.47 mN) stimulation were assessed by determining the variance of recorded signal over consecutive 1 s bins (D&F, middle traces and see Methods). G-I: In the same half-skull preparation as shown in panels D through F, the position of the recording electrode was changed to a more distal site, specifically the proximal cut end of the anterior ethmoid nerve upon its entry into the nasal cavity through the cribroethmoid foramen (H). At this site, it is only possible principally to record anti-dromic activity in the trigeminal axons. We verified this using electrical stimulation to evoke a multi-fibre compound C-fibre action potential response (I) when stimulating at the original site of recording on the ethmoid nerve (H, blue dot). Functional assessment of whether axon reflex signals could propagate from sites in the anterior cranial fossa anti-dromically into the nasal cavity were determined by stimulating with a von Frey filament (G; black vertical markers) at sites within the olfactory dura mater (G, dashed green circle). Mechanical stimulation with a von Frey filament (1.47 mN) did not increase activity as determined by analysis of signal variance (G, centre trace). J: Action potential activity was seen in response to random mechanical probing of the nasal cavity but not during von Frey stimulation of the olfactory dura (paired t-test; n = 6, p < 0.01). Scale bar: 2mm, background adapted from barrios et al. 2014 [69].