Olfactory Adaptation is Dependent on Route of Delivery

Olfactory Adaptation is Dependent on Route of Delivery Abstract Odorants are perceived orthonasally (nostrils) or retronasally (oral cavity). Prior research indicates route of delivery impacts odorant perception, pleasantness, and directed behaviors thus suggesting differential processing of olfactory information. Adaptation is a form of neural processing resulting in decreased perceived intensity of a stimulus following prolonged and continuous exposure. The present study objective was to determine whether route of delivery differentially impacts olfactory adaptation and whether cross-adaptation occurs between orthonasal and retronasal pathways. Linalool (12%) or vanillin (25%) were delivered orthonasally [6 L/min (LPM)] and retronasally (8 LPM) in air phase through a custom-built olfactometer. Perceived odorant intensity was collected every 5 min over 10-min exposure. Immediately following the exposure period, cross-adaptation was assessed by shunting the delivery of the odorant from the nostrils to the oral cavity, or vice versa. A control study was also completed in which subjects underwent the orthonasal adaptation protocol using stimulus concentrations matched to the intensity of restronasal stimuli (e.g., 1.5% linalool and 6.25% vanillin). Following orthonasal delivery of both high and low vanillin concentrations, results showed perceived intensity decreased significantly at 5 and 10 min. High concentrations of orthonasal linalool similarly decreased significantly whereas lower concentrations decreased but did not reach statistical significance. Linalool and vanillin delivered retronasally did not adapt as perceived intensity actually increased significantly following a 10-min exposure. In addition, evidence of cross-adaptation was not obvious following extended odorant exposure from either delivery pathway. This study suggests that olfactory processing may be affected by the route of odorant delivery. Introduction Odorants are delivered to the olfactory epithelium via retronasal or orthonasal routes. Retronasal delivery occurs when odorants reach the olfactory epithelium through the mouth. Studies have used the ascent of odorants through the posterior nares of the nasopharynx as a defining feature of retronasal olfaction (DeWeese and Saunders 1968; Voirol and Daget 1986; Roberts and Acree 1995), where odor identification was more likely for odorants when subjects exhaled solely through their nose after oral cavity inhalation (Chen and Halpern 2008). Additionally, because retronasal delivery occurs through the oral cavity, associated sensations are referred to the mouth and contribute to our perceptions of flavor (Murphy et al. 1977; Rozin 1982; Small et al. 2005). Orthonasal olfaction occurs when odorants are inhaled through the nostrils and these stimuli are typically associated with the external environment. Although both routes deliver the same odorants to the same receptor fields in the olfactory epithelium, sensation and perception have reportedly differed (Rozin 1982; Pierce and Halpern 1996; Espinosa Diaz 2004; Small et al. 2005; Hummel et al. 2006; Pickering et al. 2007; Hummel and Heilmann 2008; Bender et al. 2009; Welge-Lüssen et al. 2009; Lee and Halpern 2013). Indeed, when delivered orthonasally and retronasally, the same odorants can evoke different physiological responses (Heilmann and Hummel 2004; Small et al. 2005; Hummel et al. 2006), thresholds (Pierce and Halpern 1996; Hummel et al. 2006; Visschers et al. 2006), perceptual qualities (Pierce and Halpern 1996; Hummel et al. 2006), hedonic responses (Small et al. 2005), and behaviors (Rozin 1982; Burdach and Doty 1987; Heilmann and Hummel 2004; although see Sakai et al. 2001 for contradictory results). These differences support the Duality of Smell hypothesis (Rozin 1982) that suggests olfaction operates as a dual system in which orthonasal and retronasal stimuli are processed differently although the specific mechanism enabling this distinction is unknown. Adaptation may provide insight into how orthonasal and retronasal sensations are processed. Adaptation, defined as the diminution in sensitivity to a stimulus following prolonged and constant exposure (Dalton 2000), occurs among all human sensory systems (Koshland et al. 1982). It is thought that adaptation provides adaptive value by limiting over-stimulation (Ferguson and Caron 1998), and filtering insignificant sensory cues to highlight those distinct from the background (Kadohisa and Wilson 2006). Adaption to an odorant stimulus has been proposed to occur at the peripheral level following the phosphorylation of G-protein coupled receptors in olfactory receptor cells (Ferguson and Caron 1998; Mashukova et al. 2006) and at the cortical level through homosynaptic depression of afferents to the pirform cortex (Kadohisa and Wilson 2006). Olfactory adaptation has been reported in multiple studies (e.g. Cain 1974; Berglund et al. 1986; Dalton and Wysocki 1996; Best and Wilson 2004), and manifests as an increase of threshold odorant concentration (Berglund et al. 1986). Olfactory adaptation appears to be concentration dependent (Stone et al. 1972; Wuttke and Tompkins 2000) as higher concentrations make for more efficient adaptation (Stone et al. 1972; Wuttke 2000; Jacob et al. 2003). Historically, adaptation to orthonasal stimuli is well described, whereas retronasal adaptation is not. Similarly, cross-adaptation, whereby extended exposure to a stimulus delivered via one pathway (i.e., ortho- or retronasal) induces adaptation of the same stimulus when delivered via the alternative pathway (retro- or orthonasal), has not been investigated. One study, however, found salivary flow rates to decline with prolonged delivery of retronasal food (but not non-food) odors that rebounded when the same odor was subsequently presented orthonasally (Bender et al. 2009). The results are consistent with a lack of cross-adaptation between odorant pathways. The objective of the present study was to assess olfactory adaptation following extended exposure to odorant stimuli delivered orthonasally and retronasally. Additionally, we aim to assess the potential for cross-adaptation by evaluating orthonasal odorant intensity after extended exposure to the same stimulus delivered retronasally, and vice versa. Finally, as orthonasal and retronasal pathways are closely associated with environmental and flavor sensations, respectively, we further sought to determine whether any observed adaptation or cross-adaptation effects were dependent on the congruency of the odorant (food or non-food) with the specific pathway. Materials and methods Subjects Sixty subjects were recruited via the Ohio State University’s sensory testing database and enrolled in the study under informed consent (approved IRB protocol 2014B0597). Thirty panelists (13 female, 17 male; ages 23–76) performed this procedure for linalool and 30 (19 female, 11 male; ages 21–72) panelists performed this procedure for vanillin. Recruits were tested if they were over 18 years of age, had no known smell deficits, no history of heart disease, pulmonary issues (e.g., asthma, fragrance allergies), or dry mouth (xerostomia). They could not smoke and not currently be using any psychoactive (e.g., antidepressants or antipsychotics), or cardiopulmonary (e.g., hypertensive) drugs, statins, beta-blockers, neurologic medications, motion sickness, or smoking/alcohol cessation medications. Participants were also excluded if they were undergoing cancer therapy (chemotherapy, radiation therapy). They were told in the recruitment email they would receive $20 for approximately 40 min of testing and to refrain from wearing perfume or cologne. The recruitment email indicated they would be evaluating aromas and/or flavors delivered to their nose or oral cavity, respectively, by a customized aroma delivery device and that they would be asked to assess various aspects of the aromas/flavors. Olfactory stimulation Linalool (12% w/w; Sigma-Aldrich, St. Louis, MO) or vanillin (25% w/w; Sigma-Aldrich, St. Louis, MO) were dissolved in miglyol (Nature’s Oil, Streetsboro, OH) or propylene glycol (Fischer Scientific, Fair Lawn, NJ), respectively, and delivered in the air phase at 8 L/min (retronasal) or 6 L/min (orthonasal) with deodorized, humidified breathing air through a customized aroma delivery device. Initial testing indicated the 8 LPM flow evoked a sensation for each odorant that was perceived as considerably more intense than the 6 LPM flow. The combination of odorant concentration and flow rate was therefore optimized in preliminary experiments to deliver odorant concentrations that elicited approximately equally intense sensations when delivered orthonasally and retronasally. For retronasal delivery, subjects were fitted with a silicone mouthpiece (Figure 1A) attached to a glass manifold that was connected via silicone tubing to the aroma source. Subjects were instructed to inhale through their mouth and exhale through their nose. Accumulation of saliva was common during retronasal evaluations. To minimize this, saliva was suctioned out using a dental suction device (Henry Schein, Melville, NY) inserted around the mouthpiece and placed bilaterally between the cheek and gum. For orthonasal evaluations, subjects were fitted with a glass conical nose piece (Figure 1B) and instructed to inhale through their nose and exhale through their mouth. For cross-adaptation studies, subjects were fitted with both retro- and orthonasal delivery devices. Figure 1. View largeDownload slide Retronasal (A) and orthonasal (B) aroma delivery systems. Note, the retronasal system was disassembled to show component parts (mouthpiece, glass manifold, and connecting tube). Each system was connected to an aroma source (12 or 1.5% linalool and 25 or 6.25% vanillin) that delivered the odorant at 8 LPM (retronasal) or 6 LPM (orthonasal) using deodorized, humidified breathing air. The depicted individual has provided written consent (available upon request) for her image to be published in print and online in perpetuity. Figure 1. View largeDownload slide Retronasal (A) and orthonasal (B) aroma delivery systems. Note, the retronasal system was disassembled to show component parts (mouthpiece, glass manifold, and connecting tube). Each system was connected to an aroma source (12 or 1.5% linalool and 25 or 6.25% vanillin) that delivered the odorant at 8 LPM (retronasal) or 6 LPM (orthonasal) using deodorized, humidified breathing air. The depicted individual has provided written consent (available upon request) for her image to be published in print and online in perpetuity. Panelists were told the goal of the study was to understand intensity perception of certain odors better and that researchers needed them to rate perceived intensity at different time points and in different smelling conditions. They were then prompted to practice the breathing patterns without being fitted to the device to familiarize themselves with the study conditions. After practicing breathing patterns, panelists smelled a reference solution of the same concentration they would be assessing of either vanillin or linalool to provide familiarity with the odorant character and intensity. Subjects were then fitted with the aroma delivery device and rated perceived odorant intensity during 10 min of continuous delivery at 0, 5, and 10 min using the generalized labeled magnitude scale (Bartoshuk et al. 2004). Retronasal exposure occurred first, followed after a short recovery period (~5 min), by orthonasal delivery. This order of exposure was intentional as we sought to avoid potential context effects (Schifferstein 1996) resulting in the retronasal stimuli receiving “artificially” lower ratings and leading to a potential floor effect, due to prior exposure to the higher intensity orthonasal stimuli. During the extended exposure, they performed arithmetic problems as a distracter, except when prompted to provide their present perceived intensity ratings. The intensity ratings were collected on an iPad tablet device (Apple Inc., Cupertino, CA) using Compusense software (Guelph, Ontario, CAN). For cross-adaptation evaluations, subjects were fitted with both orthonasal and retronasal delivery devices. Panelists first provided an initial (pre-adaptation) rating to the stimulus. The route of delivery was then switched after which the subject continued to inhale the odorant for 10 min. Following the 10-min exposure period, the route of odorant delivery was switched back to its original setting and subjects immediately inhaled the stimulus again to provide a cross-adaptation rating. Next, subjects inhaled clean deodorized air through the nose and oral cavity for a period of 2 min to cleanse any residual effects from the persistent exposure and the odorant was delivered again to assess recovery from cross-adaptation. A second experiment was conducted to control for the initial perceived intensity differences observed between orthonasal and retronasal stimuli in the first experiment. Preliminary testing was conducted to identify orthonasal concentrations of stimuli that better matched the perceived intensity of the retronasal odorants in the previous experiment. An additional 53 subjects were recruited and underwent the orthonasal adaptation protocol using 1.5% (w/w) linalool (n = 26; ages 23–69) or 6.25% (w/w) vanillin (n = 27; ages 22–70). Statistical analysis For each treatment, intensity ratings were subjected to a repeated measures, within subjects, Analysis of Variance (ANOVA). Post hoc Tukey’s test was used to assess significant differences in time. As the retronasal stimuli were perceived to be rather weak, we sought to determine whether the lack of adaptation may be the result of a floor effect. To this end, we divided subjects into groups that perceived the retronasal odors as “more intense” or “less intense” to determine whether there was a difference in the proportion of subject’s displaying an adapting response. Each subject cohort (linalool subjects or vanillin subjects) was divided at the median perceived intensity and the proportion in each group that adapted to the stimulus (or not) was determined. The proportions of non-adapting responses from each group were then compared by calculating the z-score. Finally, we compared the responses of intensity matched orthonasal stimuli from the second experiment and retronasal stimuli from the first experiment by subjecting intensity ratings to a mixed-model repeated measures ANOVA with time as a within-subjects factor and route of delivery as a between-subjects factor. Greenhouse–Geisser correction was used when the test of sphericity was violated. Post hoc least significant difference (LSD) tests were performed to determine which time points differed between the breathing conditions. All data are reported as means ± SE. Results Adaptation High-intensity orthonasal delivery When delivered orthonasally, prolonged exposure to the odorants linalool and vanillin resulted in progressively weaker intensity ratings consistent with adaptation (Figure 2A). For linalool, the aroma intensity was perceived as significantly (P = 0.010) lower following the 10-min exposure period compared to the initial, pre-exposure rating (16.5 ± 1.2 vs. 23.7 ± 1.1, respectively). Subjects perceived the linalool intensity to be weaker at 5-min post exposure (19.1 ± 1.1), although it did not reach statistical significance (P = 0.169). A similar pattern of response was noted following prolonged vanillin delivery, however, compared to the pre-exposure rating (26.5 ± 1.1), the perceived intensity was significantly lower at both 5 min (19.2 ± 1.2; P = 0.005) and 10 min (17.7 ± 1.2; P < 0.001). Figure 2. View largeDownload slide Orthonasal (A) and retronasal (B) perceived intensity before, during, and after 10 min of continuous linalool (12%; open circles) or vanillin (25%; closed circles) delivery. Orthonasal aroma delivery resulted in adaptation whereas the perceived intensity of retronasal stimuli increased with continued exposure. For each panel, letters above or below the white and black symbols denote significant differences in perceived linalool or vanillin intensities, respectively. Figure 2. View largeDownload slide Orthonasal (A) and retronasal (B) perceived intensity before, during, and after 10 min of continuous linalool (12%; open circles) or vanillin (25%; closed circles) delivery. Orthonasal aroma delivery resulted in adaptation whereas the perceived intensity of retronasal stimuli increased with continued exposure. For each panel, letters above or below the white and black symbols denote significant differences in perceived linalool or vanillin intensities, respectively. Retronasal delivery When delivered retronasally, neither linalool nor vanillin intensities adapted following a 10-min exposure period (Figure 2B). This contrasts what was observed following orthonasal delivery of the same aroma compounds. In fact, following 5-min continuous exposure to the retronasal odorants, the perceived intensities of both linalool (11.0 ± 1.2) and vanillin (9.4 ± 1.2) were significantly (P = 0.011 and P = 0.032, respectively) higher compared to the pre-adaptation intensities (linalool: 6.4 ± 1.2; vanillin: 6.4 ± 1.1). At 10 min, the perceived vanillin intensity continued to increase (10.7 ± 1.2), whereas linalool intensity plateaued or slightly decreased (8.1 ± 1.2). This lack of an adapting response was consistent regardless of whether subjects perceived the retronasal stimuli as more or less intense. For linalool, 10/15 subjects in the “more intense” group and 14/15 subjects in the “less intense” group produced intensity ratings reflecting a lack of adaptation. These proportions are not significantly different (z = −1.83; P > 0.05). Similarly, for vanillin, 12/15 in the “more intense” and 13/15 in the “less intense” groups produced patterns over time reflecting a lack of adaptation. These proportions are not significantly different (z = −0.49; P > 0.05). Low-intensity orthonasal delivery To determine if the lack of adaptation observed following retronasal delivery was a consequence of initial perceived intensity, a control experiment was conducted in which the concentrations of the linalool and vanillin stimuli were adjusted so that when delivered orthonasally, they more closely matched the retronasal intensities obtained previously. When comparing the responses obtained retronasally to those obtained orthonasally with lowered odorant concentrations, a significant time*condition effect was observed for both linalool (P = 0.006) and vanillin (P < 0.001) indicating that the pattern of responses obtained were not consistent across delivery routes. Indeed, for linalool, orthonasal delivery resulted in a slight decrease in perceived intensity over the 10-min exposure period consistent with adaptation (although the decrease was not significant; P = 0.179) compared to the significant (P = 0.015) increase following retronasal delivery (Figure 3A). At 5 min, the perceived linalool intensity was significantly greater when delivered retronasally compared to orthonasally (P < 0.001; Figure 3A). Similarly, for vanillin, orthonasal delivery resulted in a significant (P = 0.016) decrease in perceived intensity after 5 min of constant exposure that subsequently plateaued (Figure 3B). This is in contrast to retronasal delivery that showed a progressive increase in perceived intensity. For vanillin, there was also a significant (P= 0.006) difference in the mean intensity observed for the initial orthonasal and retronasal rating indicating that the orthonasal vanillin was perceived as more intense even though it was 1/8th the concentration of the retronasal stimulus (Figure 3B). Figure 3. View largeDownload slide Perceived intensity of a weak (A) linalool (1.5%) or (B) vanillin (6.25%) odorant stimulus before, during, and after 10 min of continuous retronasal (open circles) or orthonasal (closed circles) delivery. Note for each odorant, orthonasal delivery elicits a response that tends to adapt over time whereas perceived intensity of the same stimulus increases when delivered retronasally. For each panel, letters above or below the white and black symbols denote significant increases or decreases in perceived retronasal or orthonasal intensities, respectively. The asterisk (*) above a pair of symbols indicates a significant difference between the ortho- and retronasal stimuli at a given time point. Figure 3. View largeDownload slide Perceived intensity of a weak (A) linalool (1.5%) or (B) vanillin (6.25%) odorant stimulus before, during, and after 10 min of continuous retronasal (open circles) or orthonasal (closed circles) delivery. Note for each odorant, orthonasal delivery elicits a response that tends to adapt over time whereas perceived intensity of the same stimulus increases when delivered retronasally. For each panel, letters above or below the white and black symbols denote significant increases or decreases in perceived retronasal or orthonasal intensities, respectively. The asterisk (*) above a pair of symbols indicates a significant difference between the ortho- and retronasal stimuli at a given time point. Cross adaptation Orthonasal to retronasal Evidence of a retronasal stimulus adapting following 10 min of continuous orthonasal exposure was not obvious. For linalool, the perceived intensity of the retronasal stimulus immediately following (4.1 ± 1.2) prolonged orthonasal exposure was lower compared to when the stimulus preceded orthonasal exposure (6.4 ± 1.2) although this difference was not significant (P = 0.057; Figure 4A). After inhaling clean air, the perceived intensity of retronasal linalool rebounded to a significantly (P = 0.009) higher level (7.3 ± 1.2). For vanillin, no evidence of orthonasal to retronasal cross-adaptation was observed. The perceived intensities of the retronasal vanillin stimuli immediately preceding (6.4 ± 1.2) and following (5.3 ± 1.2) the 10-min orthonasal stimulus did not significantly (P = 0.259) differ (Figure 4A). Figure 4. View largeDownload slide Cross-adaptation. (A) Orthonasal to retronasal cross-adaptation. Evidence of a retronasal stimulus adapting following 10 min of continuous orthonasal exposure was not observed for linalool (white circles) or vanillin (black circles). (B) Retronasal to orthonasal cross-adaptation. Evidence of an orthonasal stimulus adapting following 10-min of continuous retronasal exposure was not obvious for linalool (white circles) or vanillin (black circles). For each panel, letters above or below the white and black symbols denote significant differences in perceived vanillin or linalool intensities, respectively. Figure 4. View largeDownload slide Cross-adaptation. (A) Orthonasal to retronasal cross-adaptation. Evidence of a retronasal stimulus adapting following 10 min of continuous orthonasal exposure was not observed for linalool (white circles) or vanillin (black circles). (B) Retronasal to orthonasal cross-adaptation. Evidence of an orthonasal stimulus adapting following 10-min of continuous retronasal exposure was not obvious for linalool (white circles) or vanillin (black circles). For each panel, letters above or below the white and black symbols denote significant differences in perceived vanillin or linalool intensities, respectively. Retronasal to orthonasal Evidence of an orthonasal stimulus adapting following 10 min of continuous retronasal exposure to the same odorant was not observed. Rather, instead of cross-adaptation, the orthonasal stimulus intensity tended to be higher following prolonged retronasal delivery (Figure 4B). This was true for both linalool and vanillin. For linalool, the intensity of the orthonasal stimulus following retronasal exposure (18.9 ± 1.2) was greater than that observed before retronasal exposure (14.9 ± 1.1), although this difference was not significant (P = 0.211). For vanillin, the intensity of the orthonasal stimulus following retronasal exposure (22.5 ± 1.1) was greater than that observed before retronasal exposure (19.8 ± 1.2); this difference was not significant (P = 0.463). Following the inhalation of clean air, the perceived intensity of both linalool and vanillin increased further and was found to be significantly (P = 0.004 and P = 0.024, respectively) greater than pre-adapting levels (linalool: 23.7 ± 1.0 and vanillin: 26.5 ± 1.1). Discussion Presently we show that olfactory adaptation is dependent upon whether the odorant is delivered orthonasally or retronasally. Whereas orthonasal stimuli adapt readily, retronasally delivered odorants do not. This is the first report that we know of specifically addressing retronasal adaptation and showing a lack thereof. In addition to the lack of retronasal adaptation, we saw no evidence of cross-adaptation whereby extended exposure to a stimulus delivered via one pathway (i.e., ortho- or retronasal) induces adaptation of the same stimulus when delivered via the alternative pathway (retro- or orthonasal). These findings were consistent across 2 odorants—linalool, a non-food odorant, and vanillin, a food-related odorant—and suggest this lack of adaptation may be a specific property of retronasal olfaction. One reason contributing to the dearth of retronasal adaptation studies is the logistical complications associated with the continuous delivery of a retronasal stimulus. In most prior studies investigating retronasal olfaction, the odorant stimulus has been dissolved in water and delivered in solution where subjects are either asked to swallow the stimulus bolus (Cerf-Ducastel and Murphy 2001; Espinosa Diaz 2004) or it was placed in a container on the back of the tongue where panelists were expected to use throat contractions to pump the aroma past the velum (Pierce and Halpern 1996; Sun and Halpern 2005; Lee and Halpern 2013) and into the nasal sinus. In either case, prolonged stimulus delivery is hindered by the complications associated with consuming large volumes of stimulus or placing an aroma bolus on the back of the tongue. We overcame these issues by delivering volatilized aroma compounds in the air phase directly into the oral cavity through a mouthpiece and training subjects to inhale through the mouth and exhale through the nose. Such a procedure enables the study of purely retronasal stimuli without the confounding issues associated with other delivery methods. Despite these improvements to stimulus control, a purely retronasal stimulus of this kind is rarely experienced in real life. As such, caution should be taken when interpreting these results and additional investigations, using more naturalistic delivery methods, should seek to replicate the present findings. Using this methodology, we presently observed higher concentrations of linalool and vanillin, when delivered retronasally, elicited perceived intensity ratings that were significantly lower than when the compounds were delivered at lower concentrations orthonasally. This finding is consistent with previous reports that retronasal olfaction is less sensitive than orthonasal olfaction (Espinosa Diaz 2004; Heilmann and Hummel 2004; Hummel et al. 2006; Furudono et al. 2013). This differential sensitivity may reflect differences in air-flow patterns (Zhao et al. 2006) as well as non-uniform receptor distributions across the olfactory epithelium (Schoenfeld and Cleland 2006). In addition, recent evidence suggests that odorant molecules can be retained in the lung leading to the possible reduction of retronasal odor concentrations and alterations to the odor mixture makeup (Verhagen 2015). In the present experimental design, subjects were instructed to inhale through their mouth and exhale through their nose for retronasal stimulus delivery. As such, odorants necessarily entered the lung prior to being exhaled through the nasal cavity which may have resulted in lower retronasal odorant concentrations. Nevertheless, higher detection thresholds and decreased perceived intensities have also been observed when the same odorant concentration was delivered orthonasally or retronasally through a nasal cannula inserted through the nares to the rostral and caudal aspects of the nasal sinus, respectively (Heilmann and Hummel 2004). In the present experiment, we also observed adaptation to high and low (intensity matched to a retronasal stimulus) concentrations of linalool and vanillin—both pleasant aromas—when delivered orthonasally. Surprisingly, when even higher concentrations of linalool or vanillin were delivered retronasally, no evidence of adaptation was seen. Moreover, given the intensity matched orthonasal stimuli adapted whereas the same stimuli delivered retronasally did not suggests that perceived intensity per se is not responsible for the observed effect. In humans, adaptation to orthonasal aroma stimuli has been reported previously (Cain 1974; Berglund et al. 1986; Dalton and Wysocki 1996; Best and Wilson 2004) and the extent to which it occurs depends on the odorant concentration and duration of exposure (Jacob et al. 2003) as well as the stimulus relevance (Dalton 2000; Kobyashi et al. 2007) and pleasantness (Jacob et al. 2003). These same stimulus parameters have not been evaluated during retronasal olfaction and the degree to which they may impact the processing of retronasal stimuli is unknown. Indeed, given the differences between the 2 olfactory pathways it is not inconceivable that such stimulus effects have no effect. This lack of adaptation to a retronasal olfactory stimulus was unexpected. Prior studies have noted adaptation across all sensory systems, with the possible exception of pain that exhibits sensitization, hyperalgesia, and allodynia (Sessle 2006). Mechanistically, it is unclear why adaptation following prolonged retronasal stimulation was not observed. We do not think this finding is due to a floor effect in which the retronasal stimuli evoke a sensation that is perceived as too weak to further adapt. Indeed, this lack of adaptation was observed across subjects regardless of whether they perceived the initial retronasal stimulus as relatively more intense or less intense. In addition, when orthonasal stimuli were reduced to levels that evoked initial sensations of equivalent intensity to retronasal stimuli, adaptation was still observed. Taken together, these results suggest that although perceived as weak, subjects could still have perceived and appropriately scaled an adapting pattern of sensation had it occurred in the retronasal delivery condition. Alternative mechanisms may also contribute to the lack of adaptation and/or sensitizing pattern of sensation following prolonged retronasal exposure. One possibility is that adaptation to a retronasal stimulus requires a longer duration of exposure. In the present experiment, subjects were exposed to the stimulus for 10 min. Exposure for periods longer than this may be required to observe retronasal adaptation. Alternatively, an odorant’s physicochemical properties may contribute to the sensitization effect seen presently. Sensitization could be the result of the unknown accumulation of odor compounds absorbed and desorbed from the oral mucosa over time. Linforth et al. (2002) found that hydrophobic molecules, such as those assessed presently, were better absorbed into the oral mucosa when compared to the nasal mucosa. With time, absorption to the oral mucosa would saturate and as molecules began to desorb, concentrations reaching the olfactory mucosa could increase. Further studies are needed to delineate the likely contribution of this mechanism. Finally, these results are consistent with the Duality of Smell hypothesis (Rozin 1982) and suggest that the same stimulus is processed differently whether it is delivered orthonasally or retronasally. Differential processing of orthonasal and retronasal odors has been suggested to underpin the intensity and quality differences observed between the orthonasal and retronasal delivery of the same odorants (Heilmann and Hummel 2004; Small et al. 2005; Hummel et al. 2006; Verhagen 2015). This differential processing may also impart decreased susceptibility to adaptation. The lack of retronasal adaptation could have adaptive value in that the continued presence of a pleasant retronasal sensation could motivate continued feeding behavior. Additional support for the Duality of Smell hypothesis comes from the lack of cross-adaptation observed in our present experiments. As retronasal stimuli did not adapt, a lack of cross-adaption to orthonasal stimuli may be expected. However, for both linalool and vanillin, orthonasal adaptation was observed. Interestingly, during the period of orthonasal adaptation, panelists were still rather sensitive to the same odorant when delivered retronasally. This lack of cross-adaptation suggests that orthonasal and retronasal information is processed differently and, like differential sensitivity, could result from differential air flow (Zhao et al. 2006) or non-uniform receptor distribution in the olfactory epithelium (Schoenfeld and Cleland 2006). Funding Research support provided by state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center (Award # 2017007). Acknowledgements This study served in partial fulfillment of the dissertation requirements for AMP. References Bartoshuk LM, Duffy VB, Green BG, Hoffman HJ, Ko CW, Lucchina LA, Marks LE, Snyder DJ, Weiffenbach JM. 2004. 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Olfactory Adaptation is Dependent on Route of Delivery

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Abstract

Abstract Odorants are perceived orthonasally (nostrils) or retronasally (oral cavity). Prior research indicates route of delivery impacts odorant perception, pleasantness, and directed behaviors thus suggesting differential processing of olfactory information. Adaptation is a form of neural processing resulting in decreased perceived intensity of a stimulus following prolonged and continuous exposure. The present study objective was to determine whether route of delivery differentially impacts olfactory adaptation and whether cross-adaptation occurs between orthonasal and retronasal pathways. Linalool (12%) or vanillin (25%) were delivered orthonasally [6 L/min (LPM)] and retronasally (8 LPM) in air phase through a custom-built olfactometer. Perceived odorant intensity was collected every 5 min over 10-min exposure. Immediately following the exposure period, cross-adaptation was assessed by shunting the delivery of the odorant from the nostrils to the oral cavity, or vice versa. A control study was also completed in which subjects underwent the orthonasal adaptation protocol using stimulus concentrations matched to the intensity of restronasal stimuli (e.g., 1.5% linalool and 6.25% vanillin). Following orthonasal delivery of both high and low vanillin concentrations, results showed perceived intensity decreased significantly at 5 and 10 min. High concentrations of orthonasal linalool similarly decreased significantly whereas lower concentrations decreased but did not reach statistical significance. Linalool and vanillin delivered retronasally did not adapt as perceived intensity actually increased significantly following a 10-min exposure. In addition, evidence of cross-adaptation was not obvious following extended odorant exposure from either delivery pathway. This study suggests that olfactory processing may be affected by the route of odorant delivery. Introduction Odorants are delivered to the olfactory epithelium via retronasal or orthonasal routes. Retronasal delivery occurs when odorants reach the olfactory epithelium through the mouth. Studies have used the ascent of odorants through the posterior nares of the nasopharynx as a defining feature of retronasal olfaction (DeWeese and Saunders 1968; Voirol and Daget 1986; Roberts and Acree 1995), where odor identification was more likely for odorants when subjects exhaled solely through their nose after oral cavity inhalation (Chen and Halpern 2008). Additionally, because retronasal delivery occurs through the oral cavity, associated sensations are referred to the mouth and contribute to our perceptions of flavor (Murphy et al. 1977; Rozin 1982; Small et al. 2005). Orthonasal olfaction occurs when odorants are inhaled through the nostrils and these stimuli are typically associated with the external environment. Although both routes deliver the same odorants to the same receptor fields in the olfactory epithelium, sensation and perception have reportedly differed (Rozin 1982; Pierce and Halpern 1996; Espinosa Diaz 2004; Small et al. 2005; Hummel et al. 2006; Pickering et al. 2007; Hummel and Heilmann 2008; Bender et al. 2009; Welge-Lüssen et al. 2009; Lee and Halpern 2013). Indeed, when delivered orthonasally and retronasally, the same odorants can evoke different physiological responses (Heilmann and Hummel 2004; Small et al. 2005; Hummel et al. 2006), thresholds (Pierce and Halpern 1996; Hummel et al. 2006; Visschers et al. 2006), perceptual qualities (Pierce and Halpern 1996; Hummel et al. 2006), hedonic responses (Small et al. 2005), and behaviors (Rozin 1982; Burdach and Doty 1987; Heilmann and Hummel 2004; although see Sakai et al. 2001 for contradictory results). These differences support the Duality of Smell hypothesis (Rozin 1982) that suggests olfaction operates as a dual system in which orthonasal and retronasal stimuli are processed differently although the specific mechanism enabling this distinction is unknown. Adaptation may provide insight into how orthonasal and retronasal sensations are processed. Adaptation, defined as the diminution in sensitivity to a stimulus following prolonged and constant exposure (Dalton 2000), occurs among all human sensory systems (Koshland et al. 1982). It is thought that adaptation provides adaptive value by limiting over-stimulation (Ferguson and Caron 1998), and filtering insignificant sensory cues to highlight those distinct from the background (Kadohisa and Wilson 2006). Adaption to an odorant stimulus has been proposed to occur at the peripheral level following the phosphorylation of G-protein coupled receptors in olfactory receptor cells (Ferguson and Caron 1998; Mashukova et al. 2006) and at the cortical level through homosynaptic depression of afferents to the pirform cortex (Kadohisa and Wilson 2006). Olfactory adaptation has been reported in multiple studies (e.g. Cain 1974; Berglund et al. 1986; Dalton and Wysocki 1996; Best and Wilson 2004), and manifests as an increase of threshold odorant concentration (Berglund et al. 1986). Olfactory adaptation appears to be concentration dependent (Stone et al. 1972; Wuttke and Tompkins 2000) as higher concentrations make for more efficient adaptation (Stone et al. 1972; Wuttke 2000; Jacob et al. 2003). Historically, adaptation to orthonasal stimuli is well described, whereas retronasal adaptation is not. Similarly, cross-adaptation, whereby extended exposure to a stimulus delivered via one pathway (i.e., ortho- or retronasal) induces adaptation of the same stimulus when delivered via the alternative pathway (retro- or orthonasal), has not been investigated. One study, however, found salivary flow rates to decline with prolonged delivery of retronasal food (but not non-food) odors that rebounded when the same odor was subsequently presented orthonasally (Bender et al. 2009). The results are consistent with a lack of cross-adaptation between odorant pathways. The objective of the present study was to assess olfactory adaptation following extended exposure to odorant stimuli delivered orthonasally and retronasally. Additionally, we aim to assess the potential for cross-adaptation by evaluating orthonasal odorant intensity after extended exposure to the same stimulus delivered retronasally, and vice versa. Finally, as orthonasal and retronasal pathways are closely associated with environmental and flavor sensations, respectively, we further sought to determine whether any observed adaptation or cross-adaptation effects were dependent on the congruency of the odorant (food or non-food) with the specific pathway. Materials and methods Subjects Sixty subjects were recruited via the Ohio State University’s sensory testing database and enrolled in the study under informed consent (approved IRB protocol 2014B0597). Thirty panelists (13 female, 17 male; ages 23–76) performed this procedure for linalool and 30 (19 female, 11 male; ages 21–72) panelists performed this procedure for vanillin. Recruits were tested if they were over 18 years of age, had no known smell deficits, no history of heart disease, pulmonary issues (e.g., asthma, fragrance allergies), or dry mouth (xerostomia). They could not smoke and not currently be using any psychoactive (e.g., antidepressants or antipsychotics), or cardiopulmonary (e.g., hypertensive) drugs, statins, beta-blockers, neurologic medications, motion sickness, or smoking/alcohol cessation medications. Participants were also excluded if they were undergoing cancer therapy (chemotherapy, radiation therapy). They were told in the recruitment email they would receive $20 for approximately 40 min of testing and to refrain from wearing perfume or cologne. The recruitment email indicated they would be evaluating aromas and/or flavors delivered to their nose or oral cavity, respectively, by a customized aroma delivery device and that they would be asked to assess various aspects of the aromas/flavors. Olfactory stimulation Linalool (12% w/w; Sigma-Aldrich, St. Louis, MO) or vanillin (25% w/w; Sigma-Aldrich, St. Louis, MO) were dissolved in miglyol (Nature’s Oil, Streetsboro, OH) or propylene glycol (Fischer Scientific, Fair Lawn, NJ), respectively, and delivered in the air phase at 8 L/min (retronasal) or 6 L/min (orthonasal) with deodorized, humidified breathing air through a customized aroma delivery device. Initial testing indicated the 8 LPM flow evoked a sensation for each odorant that was perceived as considerably more intense than the 6 LPM flow. The combination of odorant concentration and flow rate was therefore optimized in preliminary experiments to deliver odorant concentrations that elicited approximately equally intense sensations when delivered orthonasally and retronasally. For retronasal delivery, subjects were fitted with a silicone mouthpiece (Figure 1A) attached to a glass manifold that was connected via silicone tubing to the aroma source. Subjects were instructed to inhale through their mouth and exhale through their nose. Accumulation of saliva was common during retronasal evaluations. To minimize this, saliva was suctioned out using a dental suction device (Henry Schein, Melville, NY) inserted around the mouthpiece and placed bilaterally between the cheek and gum. For orthonasal evaluations, subjects were fitted with a glass conical nose piece (Figure 1B) and instructed to inhale through their nose and exhale through their mouth. For cross-adaptation studies, subjects were fitted with both retro- and orthonasal delivery devices. Figure 1. View largeDownload slide Retronasal (A) and orthonasal (B) aroma delivery systems. Note, the retronasal system was disassembled to show component parts (mouthpiece, glass manifold, and connecting tube). Each system was connected to an aroma source (12 or 1.5% linalool and 25 or 6.25% vanillin) that delivered the odorant at 8 LPM (retronasal) or 6 LPM (orthonasal) using deodorized, humidified breathing air. The depicted individual has provided written consent (available upon request) for her image to be published in print and online in perpetuity. Figure 1. View largeDownload slide Retronasal (A) and orthonasal (B) aroma delivery systems. Note, the retronasal system was disassembled to show component parts (mouthpiece, glass manifold, and connecting tube). Each system was connected to an aroma source (12 or 1.5% linalool and 25 or 6.25% vanillin) that delivered the odorant at 8 LPM (retronasal) or 6 LPM (orthonasal) using deodorized, humidified breathing air. The depicted individual has provided written consent (available upon request) for her image to be published in print and online in perpetuity. Panelists were told the goal of the study was to understand intensity perception of certain odors better and that researchers needed them to rate perceived intensity at different time points and in different smelling conditions. They were then prompted to practice the breathing patterns without being fitted to the device to familiarize themselves with the study conditions. After practicing breathing patterns, panelists smelled a reference solution of the same concentration they would be assessing of either vanillin or linalool to provide familiarity with the odorant character and intensity. Subjects were then fitted with the aroma delivery device and rated perceived odorant intensity during 10 min of continuous delivery at 0, 5, and 10 min using the generalized labeled magnitude scale (Bartoshuk et al. 2004). Retronasal exposure occurred first, followed after a short recovery period (~5 min), by orthonasal delivery. This order of exposure was intentional as we sought to avoid potential context effects (Schifferstein 1996) resulting in the retronasal stimuli receiving “artificially” lower ratings and leading to a potential floor effect, due to prior exposure to the higher intensity orthonasal stimuli. During the extended exposure, they performed arithmetic problems as a distracter, except when prompted to provide their present perceived intensity ratings. The intensity ratings were collected on an iPad tablet device (Apple Inc., Cupertino, CA) using Compusense software (Guelph, Ontario, CAN). For cross-adaptation evaluations, subjects were fitted with both orthonasal and retronasal delivery devices. Panelists first provided an initial (pre-adaptation) rating to the stimulus. The route of delivery was then switched after which the subject continued to inhale the odorant for 10 min. Following the 10-min exposure period, the route of odorant delivery was switched back to its original setting and subjects immediately inhaled the stimulus again to provide a cross-adaptation rating. Next, subjects inhaled clean deodorized air through the nose and oral cavity for a period of 2 min to cleanse any residual effects from the persistent exposure and the odorant was delivered again to assess recovery from cross-adaptation. A second experiment was conducted to control for the initial perceived intensity differences observed between orthonasal and retronasal stimuli in the first experiment. Preliminary testing was conducted to identify orthonasal concentrations of stimuli that better matched the perceived intensity of the retronasal odorants in the previous experiment. An additional 53 subjects were recruited and underwent the orthonasal adaptation protocol using 1.5% (w/w) linalool (n = 26; ages 23–69) or 6.25% (w/w) vanillin (n = 27; ages 22–70). Statistical analysis For each treatment, intensity ratings were subjected to a repeated measures, within subjects, Analysis of Variance (ANOVA). Post hoc Tukey’s test was used to assess significant differences in time. As the retronasal stimuli were perceived to be rather weak, we sought to determine whether the lack of adaptation may be the result of a floor effect. To this end, we divided subjects into groups that perceived the retronasal odors as “more intense” or “less intense” to determine whether there was a difference in the proportion of subject’s displaying an adapting response. Each subject cohort (linalool subjects or vanillin subjects) was divided at the median perceived intensity and the proportion in each group that adapted to the stimulus (or not) was determined. The proportions of non-adapting responses from each group were then compared by calculating the z-score. Finally, we compared the responses of intensity matched orthonasal stimuli from the second experiment and retronasal stimuli from the first experiment by subjecting intensity ratings to a mixed-model repeated measures ANOVA with time as a within-subjects factor and route of delivery as a between-subjects factor. Greenhouse–Geisser correction was used when the test of sphericity was violated. Post hoc least significant difference (LSD) tests were performed to determine which time points differed between the breathing conditions. All data are reported as means ± SE. Results Adaptation High-intensity orthonasal delivery When delivered orthonasally, prolonged exposure to the odorants linalool and vanillin resulted in progressively weaker intensity ratings consistent with adaptation (Figure 2A). For linalool, the aroma intensity was perceived as significantly (P = 0.010) lower following the 10-min exposure period compared to the initial, pre-exposure rating (16.5 ± 1.2 vs. 23.7 ± 1.1, respectively). Subjects perceived the linalool intensity to be weaker at 5-min post exposure (19.1 ± 1.1), although it did not reach statistical significance (P = 0.169). A similar pattern of response was noted following prolonged vanillin delivery, however, compared to the pre-exposure rating (26.5 ± 1.1), the perceived intensity was significantly lower at both 5 min (19.2 ± 1.2; P = 0.005) and 10 min (17.7 ± 1.2; P < 0.001). Figure 2. View largeDownload slide Orthonasal (A) and retronasal (B) perceived intensity before, during, and after 10 min of continuous linalool (12%; open circles) or vanillin (25%; closed circles) delivery. Orthonasal aroma delivery resulted in adaptation whereas the perceived intensity of retronasal stimuli increased with continued exposure. For each panel, letters above or below the white and black symbols denote significant differences in perceived linalool or vanillin intensities, respectively. Figure 2. View largeDownload slide Orthonasal (A) and retronasal (B) perceived intensity before, during, and after 10 min of continuous linalool (12%; open circles) or vanillin (25%; closed circles) delivery. Orthonasal aroma delivery resulted in adaptation whereas the perceived intensity of retronasal stimuli increased with continued exposure. For each panel, letters above or below the white and black symbols denote significant differences in perceived linalool or vanillin intensities, respectively. Retronasal delivery When delivered retronasally, neither linalool nor vanillin intensities adapted following a 10-min exposure period (Figure 2B). This contrasts what was observed following orthonasal delivery of the same aroma compounds. In fact, following 5-min continuous exposure to the retronasal odorants, the perceived intensities of both linalool (11.0 ± 1.2) and vanillin (9.4 ± 1.2) were significantly (P = 0.011 and P = 0.032, respectively) higher compared to the pre-adaptation intensities (linalool: 6.4 ± 1.2; vanillin: 6.4 ± 1.1). At 10 min, the perceived vanillin intensity continued to increase (10.7 ± 1.2), whereas linalool intensity plateaued or slightly decreased (8.1 ± 1.2). This lack of an adapting response was consistent regardless of whether subjects perceived the retronasal stimuli as more or less intense. For linalool, 10/15 subjects in the “more intense” group and 14/15 subjects in the “less intense” group produced intensity ratings reflecting a lack of adaptation. These proportions are not significantly different (z = −1.83; P > 0.05). Similarly, for vanillin, 12/15 in the “more intense” and 13/15 in the “less intense” groups produced patterns over time reflecting a lack of adaptation. These proportions are not significantly different (z = −0.49; P > 0.05). Low-intensity orthonasal delivery To determine if the lack of adaptation observed following retronasal delivery was a consequence of initial perceived intensity, a control experiment was conducted in which the concentrations of the linalool and vanillin stimuli were adjusted so that when delivered orthonasally, they more closely matched the retronasal intensities obtained previously. When comparing the responses obtained retronasally to those obtained orthonasally with lowered odorant concentrations, a significant time*condition effect was observed for both linalool (P = 0.006) and vanillin (P < 0.001) indicating that the pattern of responses obtained were not consistent across delivery routes. Indeed, for linalool, orthonasal delivery resulted in a slight decrease in perceived intensity over the 10-min exposure period consistent with adaptation (although the decrease was not significant; P = 0.179) compared to the significant (P = 0.015) increase following retronasal delivery (Figure 3A). At 5 min, the perceived linalool intensity was significantly greater when delivered retronasally compared to orthonasally (P < 0.001; Figure 3A). Similarly, for vanillin, orthonasal delivery resulted in a significant (P = 0.016) decrease in perceived intensity after 5 min of constant exposure that subsequently plateaued (Figure 3B). This is in contrast to retronasal delivery that showed a progressive increase in perceived intensity. For vanillin, there was also a significant (P= 0.006) difference in the mean intensity observed for the initial orthonasal and retronasal rating indicating that the orthonasal vanillin was perceived as more intense even though it was 1/8th the concentration of the retronasal stimulus (Figure 3B). Figure 3. View largeDownload slide Perceived intensity of a weak (A) linalool (1.5%) or (B) vanillin (6.25%) odorant stimulus before, during, and after 10 min of continuous retronasal (open circles) or orthonasal (closed circles) delivery. Note for each odorant, orthonasal delivery elicits a response that tends to adapt over time whereas perceived intensity of the same stimulus increases when delivered retronasally. For each panel, letters above or below the white and black symbols denote significant increases or decreases in perceived retronasal or orthonasal intensities, respectively. The asterisk (*) above a pair of symbols indicates a significant difference between the ortho- and retronasal stimuli at a given time point. Figure 3. View largeDownload slide Perceived intensity of a weak (A) linalool (1.5%) or (B) vanillin (6.25%) odorant stimulus before, during, and after 10 min of continuous retronasal (open circles) or orthonasal (closed circles) delivery. Note for each odorant, orthonasal delivery elicits a response that tends to adapt over time whereas perceived intensity of the same stimulus increases when delivered retronasally. For each panel, letters above or below the white and black symbols denote significant increases or decreases in perceived retronasal or orthonasal intensities, respectively. The asterisk (*) above a pair of symbols indicates a significant difference between the ortho- and retronasal stimuli at a given time point. Cross adaptation Orthonasal to retronasal Evidence of a retronasal stimulus adapting following 10 min of continuous orthonasal exposure was not obvious. For linalool, the perceived intensity of the retronasal stimulus immediately following (4.1 ± 1.2) prolonged orthonasal exposure was lower compared to when the stimulus preceded orthonasal exposure (6.4 ± 1.2) although this difference was not significant (P = 0.057; Figure 4A). After inhaling clean air, the perceived intensity of retronasal linalool rebounded to a significantly (P = 0.009) higher level (7.3 ± 1.2). For vanillin, no evidence of orthonasal to retronasal cross-adaptation was observed. The perceived intensities of the retronasal vanillin stimuli immediately preceding (6.4 ± 1.2) and following (5.3 ± 1.2) the 10-min orthonasal stimulus did not significantly (P = 0.259) differ (Figure 4A). Figure 4. View largeDownload slide Cross-adaptation. (A) Orthonasal to retronasal cross-adaptation. Evidence of a retronasal stimulus adapting following 10 min of continuous orthonasal exposure was not observed for linalool (white circles) or vanillin (black circles). (B) Retronasal to orthonasal cross-adaptation. Evidence of an orthonasal stimulus adapting following 10-min of continuous retronasal exposure was not obvious for linalool (white circles) or vanillin (black circles). For each panel, letters above or below the white and black symbols denote significant differences in perceived vanillin or linalool intensities, respectively. Figure 4. View largeDownload slide Cross-adaptation. (A) Orthonasal to retronasal cross-adaptation. Evidence of a retronasal stimulus adapting following 10 min of continuous orthonasal exposure was not observed for linalool (white circles) or vanillin (black circles). (B) Retronasal to orthonasal cross-adaptation. Evidence of an orthonasal stimulus adapting following 10-min of continuous retronasal exposure was not obvious for linalool (white circles) or vanillin (black circles). For each panel, letters above or below the white and black symbols denote significant differences in perceived vanillin or linalool intensities, respectively. Retronasal to orthonasal Evidence of an orthonasal stimulus adapting following 10 min of continuous retronasal exposure to the same odorant was not observed. Rather, instead of cross-adaptation, the orthonasal stimulus intensity tended to be higher following prolonged retronasal delivery (Figure 4B). This was true for both linalool and vanillin. For linalool, the intensity of the orthonasal stimulus following retronasal exposure (18.9 ± 1.2) was greater than that observed before retronasal exposure (14.9 ± 1.1), although this difference was not significant (P = 0.211). For vanillin, the intensity of the orthonasal stimulus following retronasal exposure (22.5 ± 1.1) was greater than that observed before retronasal exposure (19.8 ± 1.2); this difference was not significant (P = 0.463). Following the inhalation of clean air, the perceived intensity of both linalool and vanillin increased further and was found to be significantly (P = 0.004 and P = 0.024, respectively) greater than pre-adapting levels (linalool: 23.7 ± 1.0 and vanillin: 26.5 ± 1.1). Discussion Presently we show that olfactory adaptation is dependent upon whether the odorant is delivered orthonasally or retronasally. Whereas orthonasal stimuli adapt readily, retronasally delivered odorants do not. This is the first report that we know of specifically addressing retronasal adaptation and showing a lack thereof. In addition to the lack of retronasal adaptation, we saw no evidence of cross-adaptation whereby extended exposure to a stimulus delivered via one pathway (i.e., ortho- or retronasal) induces adaptation of the same stimulus when delivered via the alternative pathway (retro- or orthonasal). These findings were consistent across 2 odorants—linalool, a non-food odorant, and vanillin, a food-related odorant—and suggest this lack of adaptation may be a specific property of retronasal olfaction. One reason contributing to the dearth of retronasal adaptation studies is the logistical complications associated with the continuous delivery of a retronasal stimulus. In most prior studies investigating retronasal olfaction, the odorant stimulus has been dissolved in water and delivered in solution where subjects are either asked to swallow the stimulus bolus (Cerf-Ducastel and Murphy 2001; Espinosa Diaz 2004) or it was placed in a container on the back of the tongue where panelists were expected to use throat contractions to pump the aroma past the velum (Pierce and Halpern 1996; Sun and Halpern 2005; Lee and Halpern 2013) and into the nasal sinus. In either case, prolonged stimulus delivery is hindered by the complications associated with consuming large volumes of stimulus or placing an aroma bolus on the back of the tongue. We overcame these issues by delivering volatilized aroma compounds in the air phase directly into the oral cavity through a mouthpiece and training subjects to inhale through the mouth and exhale through the nose. Such a procedure enables the study of purely retronasal stimuli without the confounding issues associated with other delivery methods. Despite these improvements to stimulus control, a purely retronasal stimulus of this kind is rarely experienced in real life. As such, caution should be taken when interpreting these results and additional investigations, using more naturalistic delivery methods, should seek to replicate the present findings. Using this methodology, we presently observed higher concentrations of linalool and vanillin, when delivered retronasally, elicited perceived intensity ratings that were significantly lower than when the compounds were delivered at lower concentrations orthonasally. This finding is consistent with previous reports that retronasal olfaction is less sensitive than orthonasal olfaction (Espinosa Diaz 2004; Heilmann and Hummel 2004; Hummel et al. 2006; Furudono et al. 2013). This differential sensitivity may reflect differences in air-flow patterns (Zhao et al. 2006) as well as non-uniform receptor distributions across the olfactory epithelium (Schoenfeld and Cleland 2006). In addition, recent evidence suggests that odorant molecules can be retained in the lung leading to the possible reduction of retronasal odor concentrations and alterations to the odor mixture makeup (Verhagen 2015). In the present experimental design, subjects were instructed to inhale through their mouth and exhale through their nose for retronasal stimulus delivery. As such, odorants necessarily entered the lung prior to being exhaled through the nasal cavity which may have resulted in lower retronasal odorant concentrations. Nevertheless, higher detection thresholds and decreased perceived intensities have also been observed when the same odorant concentration was delivered orthonasally or retronasally through a nasal cannula inserted through the nares to the rostral and caudal aspects of the nasal sinus, respectively (Heilmann and Hummel 2004). In the present experiment, we also observed adaptation to high and low (intensity matched to a retronasal stimulus) concentrations of linalool and vanillin—both pleasant aromas—when delivered orthonasally. Surprisingly, when even higher concentrations of linalool or vanillin were delivered retronasally, no evidence of adaptation was seen. Moreover, given the intensity matched orthonasal stimuli adapted whereas the same stimuli delivered retronasally did not suggests that perceived intensity per se is not responsible for the observed effect. In humans, adaptation to orthonasal aroma stimuli has been reported previously (Cain 1974; Berglund et al. 1986; Dalton and Wysocki 1996; Best and Wilson 2004) and the extent to which it occurs depends on the odorant concentration and duration of exposure (Jacob et al. 2003) as well as the stimulus relevance (Dalton 2000; Kobyashi et al. 2007) and pleasantness (Jacob et al. 2003). These same stimulus parameters have not been evaluated during retronasal olfaction and the degree to which they may impact the processing of retronasal stimuli is unknown. Indeed, given the differences between the 2 olfactory pathways it is not inconceivable that such stimulus effects have no effect. This lack of adaptation to a retronasal olfactory stimulus was unexpected. Prior studies have noted adaptation across all sensory systems, with the possible exception of pain that exhibits sensitization, hyperalgesia, and allodynia (Sessle 2006). Mechanistically, it is unclear why adaptation following prolonged retronasal stimulation was not observed. We do not think this finding is due to a floor effect in which the retronasal stimuli evoke a sensation that is perceived as too weak to further adapt. Indeed, this lack of adaptation was observed across subjects regardless of whether they perceived the initial retronasal stimulus as relatively more intense or less intense. In addition, when orthonasal stimuli were reduced to levels that evoked initial sensations of equivalent intensity to retronasal stimuli, adaptation was still observed. Taken together, these results suggest that although perceived as weak, subjects could still have perceived and appropriately scaled an adapting pattern of sensation had it occurred in the retronasal delivery condition. Alternative mechanisms may also contribute to the lack of adaptation and/or sensitizing pattern of sensation following prolonged retronasal exposure. One possibility is that adaptation to a retronasal stimulus requires a longer duration of exposure. In the present experiment, subjects were exposed to the stimulus for 10 min. Exposure for periods longer than this may be required to observe retronasal adaptation. Alternatively, an odorant’s physicochemical properties may contribute to the sensitization effect seen presently. Sensitization could be the result of the unknown accumulation of odor compounds absorbed and desorbed from the oral mucosa over time. Linforth et al. (2002) found that hydrophobic molecules, such as those assessed presently, were better absorbed into the oral mucosa when compared to the nasal mucosa. With time, absorption to the oral mucosa would saturate and as molecules began to desorb, concentrations reaching the olfactory mucosa could increase. Further studies are needed to delineate the likely contribution of this mechanism. Finally, these results are consistent with the Duality of Smell hypothesis (Rozin 1982) and suggest that the same stimulus is processed differently whether it is delivered orthonasally or retronasally. Differential processing of orthonasal and retronasal odors has been suggested to underpin the intensity and quality differences observed between the orthonasal and retronasal delivery of the same odorants (Heilmann and Hummel 2004; Small et al. 2005; Hummel et al. 2006; Verhagen 2015). This differential processing may also impart decreased susceptibility to adaptation. The lack of retronasal adaptation could have adaptive value in that the continued presence of a pleasant retronasal sensation could motivate continued feeding behavior. Additional support for the Duality of Smell hypothesis comes from the lack of cross-adaptation observed in our present experiments. As retronasal stimuli did not adapt, a lack of cross-adaption to orthonasal stimuli may be expected. However, for both linalool and vanillin, orthonasal adaptation was observed. Interestingly, during the period of orthonasal adaptation, panelists were still rather sensitive to the same odorant when delivered retronasally. This lack of cross-adaptation suggests that orthonasal and retronasal information is processed differently and, like differential sensitivity, could result from differential air flow (Zhao et al. 2006) or non-uniform receptor distribution in the olfactory epithelium (Schoenfeld and Cleland 2006). Funding Research support provided by state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center (Award # 2017007). Acknowledgements This study served in partial fulfillment of the dissertation requirements for AMP. References Bartoshuk LM, Duffy VB, Green BG, Hoffman HJ, Ko CW, Lucchina LA, Marks LE, Snyder DJ, Weiffenbach JM. 2004. 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Chemical SensesOxford University Press

Published: Mar 1, 2018

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