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Thermally evoked parotid salivation
Physiology & Behavior 87 (2006) 757 – 764
Thermally evoked parotid salivation Andy Lee a , Steve Guest b,⁎, Greg Essick c a
Department of Biochemistry, North Carolina State University, Raleigh, NC, United States Center for Neurosensory Disorders, School of Dentistry, University of North Carolina, Chapel Hill, NC, United States Department of Prosthodontics and Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC, United States b
c
Received 30 September 2005; received in revised form 6 December 2005; accepted 19 January 2006
Abstract Parotid salivation is known to be influenced by the temperature of liquids moved around the mouth. Here we investigated the ability of nonliquid thermal stimuli to change the rate of salivation. Unilateral parotid saliva was collected using a Lashley Cup from 12 normally hydrated subjects. Thermal stimuli were delivered through a copper tube, in which temperature-controlled water flowed, resting statically on the anterior tongue. During separate trials, the tube was 10, 22, or 44 °C, or the resting temperature of the tongue (or hypothenar of the hand, the control site). On each trial, the unstimulated salivation rate was first measured for 6 min while the subject remained seated with the mouth closed. Subsequently, salivation was measured for 6 min during application of the thermal stimulus. The tube was then removed for 1–2 min before the next trial. During the trials, subjects repeatedly rated the subjective temperature of the tongue (or hypothenar) and its perceived wetness/dryness. Stimulated salivation, expressed as a proportion of the previously measured unstimulated salivation, differed among body sites and temperatures (P b 0.03). A significant increase in salivation was seen only for the 10 °C stimulus applied to the tongue. Wetness ratings and salivation rates were positively correlated, albeit weakly. These results demonstrate that temperature-evoked changes in parotid salivation do not require the unique spatiotemporal dynamics of the tongue and jaw movements in wetting the oral mucosa. © 2006 Elsevier Inc. All rights reserved. Keywords: Parotid saliva; Temperature; Flow rate; Wetness perception
1. Introduction A variety of stimulus properties have been shown to alter saliva production in the human. For example, many basic taste solutions when swilled around the mouth lead to concomitant increases in whole or parotid salivation. This has been shown to occur for sour tastes for parotid [1–3] and whole [4] saliva, for salt solutions [2,3,5,6], for sweet stimuli [2,7] and for bitter tastes [2]. Additionally, parotid salivation has been shown to increase in response to trigeminal stimuli such as chemical irritants (e.g., capsaicin) [8] and astringents [9], although this latter effect is not universal [10]. In addition to these factors, non-stimulus-centered correlates of salivation exist, such as those associated with natural variations in individuals' state of hydration over the course of the year [11]. ⁎ Corresponding author. 2160 Old Dental Bldg., School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7450, United States. Fax: +1 919 966 3683. E-mail address: [email protected] (S. Guest). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.01.021
One aspect of salivation that has received relatively little attention is the role of thermal stimulation in altering the rate of saliva production. Indeed, some reviews of the factors affecting salivation omit thermal stimulation entirely [12]. Regardless, relatively warm or cool thermal stimuli presented to the mouth might induce salivation as a mechanism to protect the delicate oral tissues. In this respect, there is indeed some evidence that cold (0–3 °C) water presented to the mouth leads to greater production of whole saliva [13,14] than for water samples of more moderate temperature, and there is also evidence that parotid saliva can be evoked similarly by a cold (0°C) or hot (55–66 °C) thermal liquid challenge in the mouth [5,6]. Further, it has been proposed that the increase in saliva as induced by cold stimuli plays a role in the perceived pleasantness of drinks [13]. However, other research has found parotid salivation to be unaffected by prolonged presentation to the mouth of liquids with temperatures in the range 0 to 40°C [14]. In each of the experiments reported above, the thermal challenge was in the form of a liquid stimulus. As such, it is currently unknown whether the spatiotemporal dynamics of
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liquid manipulation in the mouth are necessary for significant saliva production in response to thermal stimulation. That is, if increased salivation does occur in response to a liquid thermal stimulus, it is not currently known whether the primary driver of salivation is the thermal challenge per se, or whether the thermal challenge must be combined with the types of mechanoreceptor activation that are strongly associated with saliva production, i.e., those activated by chewing-type actions of the tongue, jaw and facial tissues [15–17]. Although the role of spatiotemporal dynamics during oral manipulation has received little direct attention, the importance of periodontal mechanoreceptors to salivation was highlighted by Dawes et al. [14], who found that manipulation of an acrylic block and water at 20°C in the mouth led to considerably greater (whole mouth) salivation than water at 20 °C alone, but less salivation than a block of ice of the same dimensions as the acrylic block. Although the precise mouth movements used by the subjects of Dawes et al. remain unclear, it is likely that periodontal mechanoreceptors would have been stimulated periodically when moving the acrylic block (or ice block), even if the block was not explicitly bitten. Here we investigated the effect of a non-liquid thermal stimulus held statically on the surface of the tongue in altering the rate of parotid salivation. Although the use of such an ‘immaterial’ (i.e., non-gustatory) stimulus was suggested by Brown [12], to the best of our knowledge no such stimulus has been used in human subjects to date. Because the thermal stimulus we used was not manipulated between the teeth, the teeth were slightly separated, and no chewing movements were made, there was comparatively little stimulation to periodontal or other mechanoreceptors, and thus minimal mechanoreceptor-induced salivation [15–17]. As such, it was possible to determine to what extent prolonged thermal stimulation in the absence of periodontal reflexes affects salivation. This is of interest because if the major role of thermally invoked salivation is protective in nature, one would expect a static, non-liquid stimulus to evoke salivation in a similar way and with a similar magnitude of response to a liquid stimulus manipulated in the mouth. Conversely, if salivation does not occur in response to non-liquid thermal stimulation, then this would call into question the role of thermally evoked salivation primarily as a mechanism to protect against thermal damage. We also measured parotid salivation in response to an extraoral thermal stimulus, namely one presented to the palm of the hand. Although this was primarily designed as a control site, some previous work has found non-oral stimuli to alter salivation rates [18]. For example, in the rat, saliva production has been shown to increase in response to heating of the trunk [19]. However, saliva is used by the rat in heat-stress situations as an evaporative cooling agent, which is not the case in humans. As such, we did not hypothesize any effect of thermal stimulation of the hand on the rate of parotid salivation. Lastly, we considered the role of parotid saliva in the perception of mouth wetness. Although systematic reductions in salivary output (e.g., in Sjogren's syndrome) are associated with a dry mouth [20], it is unclear whether non-pathological between-individual variation in salivation rates leads to concomitant individual differences in perceived mouth wetness.
It is also unclear to what extent parotid salivation changes, as induced by thermal stimulation, might account for changes in mouth wetness ratings. It is not guaranteed that salivation is the only factor in perceived mouth wetness differences for liquids of different temperatures. For example, in the results of Brunstrom et al. [13] a component of the feeling of wetness in the mouth might have been induced by the combination of tactile and thermal stimulation which would exist in addition to any elicitation of saliva. That is, a cold stimulus placed in the mouth might elicit a feeling of wetness, even if the stimulus were dry. 2. Methods 2.1. Subjects Twelve subjects (10 males, 2 females, median age 28years) consented to take part in the study. All were reimbursed $10per hour for their time. The study was approved on ethical and safety grounds by the School of Dentistry Institutional Review Board (IRB) at the University of North Carolina at Chapel Hill. 2.2. Apparatus Thermal stimuli were delivered with copper tubing through which water flowed from a temperature-controlled bath (Fig. 1A). Before being placed in the mouth or on the hand of a subject, the tubing was covered with thin, flexible, plastic film. Unilateral parotid saliva was collected using a Lashley cup [6] which was positioned over Stenson's duct, and held in place using light suction, applied with a hypodermic syringe. The vacuum pressure was continually monitored by the experimenter Thermocouple readout to PC
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Water in 2.8cm
2cm Water out
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B Position of device on tongue or palm
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Palm of hand
Fig. 1. (A) Thermal stimulator, not to scale. The device was made of copper tubing, and was covered with thin, flexible film during use. (B) The body sites to which the thermal stimulator was applied.
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via a dial gauge. Saliva flowed down a capillary tube to a beaker placed on the plate of precision balance interfaced to a PC, enabling saliva weight to be recorded continually. The capillary tube between cup and balance was pre-filled with water, eliminating the need to wait for the subject's own saliva to fill the tubing before collection could begin. Priming the collection tube with water did not lead to incorrect saliva weights being recorded during initial collection because the specific gravity of parotid saliva is but trivially different from the specific gravity of the water used to prime the tube: the specific gravity of unstimulated parotid saliva lies in the range 1.0025 to 1.0040 [21], and although some aspects of parotid saliva composition vary with flow rate and duration of stimulation [22], the specific gravity of stimulated parotid saliva remains very close to that of water even during gustatory or masticatory stimulation [23]. The resting temperature of the tongue or palm was obtained using an electronic thermometer which was either held on the anterior tongue with the subject's mouth closed, or placed under the hypothenar of the hand. After approximately 45 s, the temperature reading was taken and used as the neutral temperature (NT) of the subject's tongue or palm. The resting temperature was obtained immediately before the trial that included thermal stimulation at the neutral temperature. Cues to subjects and response scales were presented on a 15″ LCD panel located approximately 60cm in front of the subject. Responses demanded of subjects were made using a cordless joystick which moved a slider on the response scale shown, with selection of a response made by the joystick trigger. 2.3. Design and procedure Thermal stimuli at 10, 22, NT (i.e., resting temperature of tongue or palm) and 44 °C were applied to either the anterior tongue or the hypothenar of the hand (Fig. 1B). Each thermal stimulus was presented for 6-min. The presentation of thermal stimuli was either in increasing (10, 22, NT and 44 °C) or decreasing (44, NT, 22, 10°C) order. Before presentation of a stimulus, the unstimulated salivation rate was measured for 6 min. During this period, there was no stimulus present in the mouth, and the mouth was closed. Thus a trial consisted of two periods, a period where no thermal stimulus was delivered, followed by a period when thermal stimulation was presented to the subject. Between periods of a trial, there was a delay of 1– 2 min. For both unstimulated and stimulated periods, the subject was allowed to swallow as and when they wished. Although the subject's jaws were not fixed in place, the subject was asked to avoid making gratuitous jaw/mouth movements. Testing took place over two days. On one day, stimuli were delivered to the hand, on the other day stimuli were delivered to the mouth. The ordering of which body site was tested on which day was pseudo randomized such that approximately equal numbers of subjects were tested on both possible orderings. Each subject returned to the lab at approximately the same time on each of the two days of testing, to minimize variation in salivation according to time of day [24]. In the hour preceding the experiment, subjects drank 500–700ml of water, to ensure adequate hydration, and the subjects did not eat during this period.
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During both unstimulated and stimulated periods of a trial, subjects rated the perceived wetness or dryness of their mouth or hand. During stimulated periods, subjects were informed that the ratings should reflect the feel of their tongue or palm under the thermal stimulator itself. During unstimulated periods, the wetness ratings reflected wetness of the mouth or hand. Ratings were also made of the perceived temperature of the hand or mouth. During stimulated periods, these ratings represented the perceived temperature of the thermal stimulus, whereas during unstimulated periods of the trials the temperature ratings represented the resting temperature of the hand or mouth. All ratings were made using bisemantic visual analogue scales (Fig. 2). The temperature scale is a linear version of the temperature wheel of Sandick et al. [25], whereas the wetness/ dryness scale is similar in form to the temperature scale. Both scales consisted of 100 discrete steps, as presented by the computerized data collection program. This number of steps was sufficient for the scale to feel continuous to the subjects. 2.4. Statistical analysis Salivary flow was analyzed separately for unstimulated and stimulated periods using mixed-model analysis of variance (ANOVA). Six estimates of salivation rate were extracted during each 6 min period, namely the rates obtained in consecutive 1 min intervals. Both analyses included repeated-measures factors of body site, temperature and time (i.e., corresponding to the six consecutive salivation estimates). Note that for the unstimulated salivation analysis, the body site and temperature factors referred to the site and temperature that were to be subsequently tested. The analysis of stimulated salivation was carried out on relative salivation rates, as has been the case in much previous research [5,13,18]. Each subject's rates were expressed as a proportion of their mean unstimulated rate. As
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Fig. 2. Response scales used to collect (A) subjective impressions of temperature, and (B) perceived wetness/dryness.
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such, the value ‘1’ indicated a stimulated salivation rate no different than the previous unstimulated rate, values greater than one denoted increased salivation, and values less than one indicated decreased salivation. Prior to analysis, all wetness and temperature ratings were converted into the range − 1 to 1. The driest and coldest labels of each scale (‘extremely dry’ and ‘very cold’) were given the value − 1, the wettest and hottest labels (‘extremely wet’ and ‘very hot’) the value 1, and each rating made along the scales assigned a value accordingly. After conversion, the value zero indicated the midpoint of the scale, signifying either a neither wet nor dry feeling, or a neither warm nor cool percept. The ratings were analyzed in similar fashion to the salivary data. Wetness and temperature ratings were analyzed separately, with separate mixed-model ANOVAs carried out for the unstimulated and stimulated data. In all cases the repeated-measures factors were the same as in the saliva analysis. Finally, relationships between salivation rate and ratings of mouth wetness were sought using both within- and betweensubjects correlations. Within-subjects correlations were calculated between the wetness ratings and the salivation rates obtained in the 45s preceding the rating. Each correlation included the wetness ratings and associated salivation rates for the mouth data only, for all combinations of stimulated or unstimulated conditions × time × temperature. Log salivation rates were used, after inspection of the data suggested better fits to linearity with this transform. The hand data was not included because the wetness ratings in this case, being the wetness of the hand, would have no known relationship with the rate of salivation. Between-subjects correlations were calculated for the mouth data, one correlation for the unstimulated data, grouped across the different temperatures, and then one for each of the four stimulated periods at the different temperatures. For the stimulated data, each subject provided four data pairs, consisting of the mean wetness rating and average (log) salivation rate over the 6min stimulus period for each temperature. Additionally, a mixed-model ANOVA was carried out on the stimulated data, with wetness ratings as the dependent variable, and (log) salivation rate, temperature, and their interaction as factors in the model to determine whether overall, salivation rate influenced wetness ratings, and whether any such influence was different for the different temperatures. Any pairwise tests reported were corrected for multiple comparisons using the Tukey–Kramer method. 3. Results 3.1. Parotid salivation Prior to stimulation, the mean unilateral parotid flow did not vary according to either the body site or the temperature of the thermal stimulus subsequently presented. The overall mean unstimulated flow rate was 0.051 g min− 1. In contrast, the stimulated (relative) salivation rates were influenced by the presence of the thermal stimulus, specifically, salivation rates varied according to body site and temperature in an interactive manner (P b 0.02). Fig. 3 illustrates the least-square mean
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Fig. 3. Stimulated relative salivation rates obtained during presentation of a static thermal stimulus to either the anterior tongue or hypothenar of the hand. Values greater than one indicate increased salivation.
relative salivation rates obtained during stimulation for the different body sites and temperatures. Pairwise comparisons of the least-square means indicated that the same relative salivation rate was elicited when the thermal stimulus was present on the hand, regardless of the temperature of the stimulus. However, in the mouth, the 10 °C stimulus led to significantly greater salivation than 22 °C or NT (P b 0.05). Although greater salivation was suggested for the 10°C stimulus than the 44 °C stimulus, the difference fell short of statistical significance (P = 0.078). Of all the relative salivation rates, only that for the 10°C stimulus presented to the mouth was greater than the baseline rate (i.e., a relative salivation rate of N 1). In addition to the effects of stimulus site and temperature, an additional effect was identified, namely that of time (P b 0.001). Inspection of the data revealed that by far the greatest salivation occurred in the first 60s interval of the stimulus trials. The five subsequent minute-long intervals showed salivation rates very similar to those gathered during resting measurements. 3.2. Ratings 3.2.1. Temperature ratings At rest, the mouth and hand were both rated as slightly warm (mean = 0.06, where zero indicates the midpoint of the scale) and did not differ in their perceived warmth. During thermal stimulation, ratings varied according to the temperature of the stimulus (P b 0.0001; Fig. 4), and were similar for both hand and mouth. 3.2.2. Wetness ratings When no thermal stimulus was applied to mouth or hand, the mouth was rated as wetter than the hand (P b 0.0001). The mean wetness ratings for hand and mouth were 0.01 and 0.12,
A. Lee et al. / Physiology & Behavior 87 (2006) 757–764 0.6
binations of stimulus site and temperature. It is clear from the figure that only in the case of the 10°C stimulus delivered to the mouth, did ratings of wetness increase greatly over time. Wetness ratings for the hand were not well differentiated for the different temperature stimuli; there was no evidence that the non-liquid, but cold stimulus led to a perception of wetness on the palm skin under the stimulator. It is also clear that the significant interaction between time and stimulus site was a consequence of certain mouth wetness ratings increasing over time while the hand wetness ratings remained relatively constant.
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3.3. Relationship between salivation and wetness ratings
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Temperature (°C) Fig. 4. Temperature ratings (averaged over hand and mouth body sites) of four different thermal stimuli. Error bars show +1 SE.
respectively. The former value indicates a neither wet nor dry feel, whereas the latter value indicates a feeling approaching ‘slightly wet’; the ‘slightly wet’ label occurs at a value of 0.2 on the wetness response scale (see Fig. 2). Additionally, there was a significant (P b 0.02) interaction between the time at which the rating was obtained and the stimulus site. However, inspection of the ratings indicated that this interaction was not meaningful: Ratings on both hand and mouth were always within 0.04units (2% of the whole scale) of their respective means, and at every time point, the mouth was rated as wetter than the hand. When the tubing was present in the mouth or on the hand, the wetness ratings varied by time (P b 0.001), the interaction between time and stimulus site (P b 0.0001) and the interaction of time, stimulus site and temperature (P b 0.02). Fig. 5 illustrates the ratings made at each of six sample times, for the com-
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Finally, the association between parotid salivation rate and ratings of mouth wetness was investigated. Correlations were calculated for each subject's mouth data, between their wetness ratings and the salivation rates obtained in the 45s preceding the rating. Of the 12 correlations, one for each subject, four were significant (P b 0.05) such that increasing wetness ratings were associated with greater salivation rates. However, these correlations ranged in magnitude from 0.27 to 0.44, suggesting relatively weak relationships. Additionally, three subjects showed an inverse relationship between their wetness ratings and salivation rates, and two of these correlations were statistically significant (r = − 0.30 and − 0.34). These results indicate that overall, for some subjects, the relationship between salivation and wetness ratings was statistically significant, but the relationship was never strong, nor visible in all subject's data.
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Time (min) Fig. 5. Ratings of the wetness of mouth or hand during the course of 6-min trials where a thermal stimulus at 10, 22, NT or 44°C was present on the hand or on the anterior tongue.
Salivation rate (g/min) Fig. 6. Wetness ratings plotted against salivation rate for four temperatures of stimulus, applied to anterior tongue. A least-squares regression line through all data points is shown. A mixed-model ANOVA indicated that the effect of salivation rate on wetness ratings was significant (P b 0.05), but there was no influence of temperature or the interaction of temperature and salivation rate on the ratings.
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Subsequently, the between-subjects relationship between salivation rate and wetness ratings was investigated. For the stimulated data, a mixed-model ANOVA indicated that wetness ratings were influenced by (log) salivation rate (P b 0.006), but not by temperature or the interaction of salivation rate and temperature, indicating that the relationship between wetness ratings and salivation rates was similar for all four stimulated temperatures. A scatterplot of wetness versus salivation rates is shown in Fig. 6. Of the correlations for each of the individual temperatures, all were indicative of higher wetness ratings being associated with greater salivation. These positive correlation values ranged from 0.45 (for the 10 °C stimulus) to 0.62 (NT stimulus). One of the correlations was statistically significant (P b 0.05), namely that for the NT stimulus. For the unstimulated periods, the correlation between salivation rates and wetness ratings was not significant (r = 0.37, P N 0.05). 4. Discussion A non-liquid (10°C) stimulus, positioned statically against the anterior tongue, increases the rate of parotid saliva production, although the magnitude of this increase, namely just more than a doubling on average, appears to be somewhat less than the increase seen with liquids at approximately the same temperature; other researchers have found approximately two- to fivefold increases in parotid salivation for cold temperatures of liquids [5]. Other non-liquid, static stimuli in the range 22–44°C, presented to the mouth, do not increase – and may even decrease – parotid salivation (Fig. 3). Thermal stimuli placed on the hypothenar of the hand do not alter salivation. These results illustrate that although movement of a liquid stimulus around the mouth is not necessary to invoke increased parotid salivation, consideration of the literature suggests that the combination of mechanical stimulation and thermal stimulation leads to greater salivation than thermal stimulation alone. It is likely that proprioceptive input and stimulation of periodontal mechanoreceptors during swishing of a liquid around the mouth [15–17] drives this difference; the ability of a non-liquid thermal stimulus to evoke salivation is clearly rather weak, especially when considering the strength of the most extreme stimuli we used: Both the coldest and warmest temperatures were such that they were the lowest (or highest) that could be reliably tolerated by all subjects, and our stimuli were applied for longer than is typically used for liquids. Yet even at these extremes, the change in evoked salivation was modest. Although the increase in salivation upon stimulation of the tongue with the 10°C challenge is assumed to be a response to the thermal nature of the stimulus per se, one of the reviewers raised the interesting possibility that the salivation may have been mediated by thermally induced taste sensations. Cruz and Green [26] found that cooling the tongue to 10 °C led to, on average, a weak sensation of sourness, with most subjects in the test population showing this thermally induced taste sensation. Given that sour stimuli are potent inducers of parotid salivation, it is possible that any thermally induced sourness could act similarly. We did not collect perceived taste sensations from subjects, and as such we cannot ascertain the magnitude of any
thermal-taste-induced salivation, or indeed even if such salivation actually occurs. The possibility is worth investigating in future studies. A further possible factor mediating the modest salivation changes we found is in the local nature of our thermal stimulus. The stimulus predominately heated or cooled the tongue, with minimal temperature change being delivered directly to many other oral surfaces, such as the labial mucosa. This differs from liquids swilled around the mouth which provide thermal stimulation to most or indeed all of the oral cavity. This wider application of thermal stimulation when using liquid stimuli could well drive greater salivation than for more focal thermal challenges. The results we found broadly agree with those of Pangborn et al. [5] and Brunstrom et al. [13], and differ in some respects from that found by Dawes et al. [14]. In particular, Dawes et al. found that liquid stimuli in the range 0 to 37°C pumped continually through the mouth did not elicit different rates of parotid salivation, whereas we found that sustained application of a 10°C stimulus to the tongue did lead to increased flow. This is perhaps unexpected given that the stimuli of Dawes et al. were at a relatively constant sustained temperature, similar to those we used, by virtue of the continual flow method used by the Dawes et al., as opposed to the sip and spit method often used with liquid stimuli. Note that Dawes et al. did find that stimulation with ice was a relatively potent producer of saliva, and this stimulus appears more similar in some respect to our (cold) thermal stimulus, although ice would presumably provide some periodontal mechanoreceptor stimulation that our thermal device minimized, and the ice would directly stimulate mucosal surfaces in addition to the tongue. Overall, ratings of mouth wetness between-subjects were positively related to salivation rates albeit not especially strongly, with at most 36% of the variance in mouth wetness ratings being accounted for. That is, subjects who salivated relatively more when the thermal tubing was in the mouth did indeed tend to rate their mouth as wetter than subjects who salivated less freely. This relationship also held for some of the within-subjects correlation analyses. Thus the wetness ratings appeared, to at least some extent, to be based on the veridical perception of wetness, although it is possible that a component of the wetness ratings for the 10 °C stimulus was illusory, induced by the pressure of a cold object against the tongue [27]. No illusory wetness on the palm was induced by a cold stimulus pressing on the hand. That the relationships between wetness and salivation rates were not stronger is perhaps unsurprising, for at least two reasons. First, unilateral parotid saliva contributes only a small portion (c. 15%) of the mouth's total salivary volume: unstimulated whole saliva typically flows at a rate of c. 0.3–0.6ml/min [20,28,29], whereas the unilateral unstimulated parotid flow rate lies in the range 0.01–0.09ml/min [3,5,30,31] as found in the current experiment. Submandibular flow rates have found to be similar to those found for parotid saliva [32], with flow from sublingual and minor glands making up the remaining components of whole saliva. Given these different salivary sources, the relationship between wetness and parotid salivation would be expected to be weak if saliva volume
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is the main determinant of perceived mouth wetness, and if the other salivary sources (e.g., sublingual, submandibular) are relatively thermally unresponsive, or if their flow rates are otherwise poorly correlated with the parotid flow rate. There is some evidence this might be so given that some authors have found thermal differences in whole saliva flow rates, but failed to find such differences in parotid flow [14]. It is also true that diverting saliva from one of the parotid glands to the collection tube prevents that saliva from being delivered to the oral mucosa. This would also reduce the potential impact of parotid saliva upon mouth wetness and may have contributed to the relatively weak relationships we found between saliva rates and perceived mouth wetness. Second, the amount of saliva in the mouth at the point of swallowing is probably a critical quantity, because this would be the point at which maximum mouth wetness would be expected. Given that we did not know when the subject swallowed, we were restricted to using ratings that would not necessarily sample these times of maximum mouth wetness. Indeed, it is possible that mouth wetness would remain relatively constant even with increased salivation, by virtue of the subject choosing to swallow more often than when their salivation was less profound. Although all but the 10 °C stimulus suggested a reduction in salivation, the ratings of mouth wetness remained relatively constant, or increased over trials (re. Fig. 5). The basis for this effect could be if the thermal tubing hindered the complete swallowing of accumulated saliva in the mouth. If suppression of salivation occurs in response to its constant presence, this could be a reason for the suggested suppression in salivation seen for the 22–44 °C stimuli. To the best of our knowledge effects of accumulated saliva on salivation has not been studied. If a suppression mechanism is indeed plausible, then the experimental paradigm may have, in fact, underestimated the saliva-stimulating effect of sustained presentation of a static, cold thermal stimulus. A further possible explanation for the presence of reduced salivation is in the relative lack of periodontal mechanoreceptive stimulation in the stimulated versus unstimulated periods. During unstimulated periods the subject's upper and lower teeth could contact each other, whereas during stimulated periods the presence of the stimulus tubing prevented any such direct contact. In conclusion, we find that cooling the tongue with a dry, static stimulus can induce increased parotid salivation, albeit to a lesser extent than is seen when cold liquids are swished around the mouth. This result is consistent with the common suggestion that thermally evoked parotid secretion is primarily a mechanism to protect the oral mucosa against heat or cold induced damage. The parotid salivation rate was but a weak predictor of perceived mouth wetness. Although this may suggest that increased salivation in response to cold fluids is unlikely to be the basis of the high perceived pleasantness of cold water taken into a dry mouth [13], the suggestion warrants caution given that we only considered one component of saliva (i.e., parotid). As expected, thermal stimulation of the hand did not alter salivation, and may be added to the list of other such non-sialagogic stimuli as odors and mental imagery [1,18].
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References [1] Drummond PD. Effect of imagining and actually tasting a sour taste on one side of the tongue. Physiol Behav 1995;57:373–6. [2] Chauncey HH, Shannon IL. Parotid gland secretion rate as a method for measuring response to gustatory stimuli in humans. Proc Soc Exp Biol Med 1960;103:459–63. [3] Froehlich DA, Pangborn RM, Whitaker JR. The effect of oral stimulation on human parotid salivary flow rate and alpha-amylase secretion. Physiol Behav 1987;41:209–17. [4] Christensen CM, Brand JG, Malmud D. Salivary changes in solution pH: a source of individual differences in taste perception. Physiol Behav 1987;40:221–7. [5] Pangborn RM, Chrisp RB, Bertolero LL. Gustatory, salivary, and oral thermal responses to solutions of sodium chloride at four temperatures. Percept Psychophys 1970;8:69–75. [6] Lashley KS. Reflex secretion of the human parotid gland. J Exp Psychol 1916;1:461–93. [7] Hyde RJ, Pangborn RM. Taste modifiers: parotid reflexes can differ from taste judgements. Chem Senses 1987;12:577–89. [8] Nasrawi CW, Pangborn RM. Temporal gustatory and salivary responses to capsaicin upon repeated stimulation. Physiol Behav 1990;47:611–5. [9] Guinard J-X, Zoumas-Morse C, Walchak C. Relation between parotid saliva flow and composition and the perception of gustatory and trigeminal stimuli in foods. Physiol Behav 1998;63:109–18. [10] Lyman BJ, Green BG. Oral astringency: effects of repeated exposure and interactions with sweeteners. Chem Senses 1990;15:151–64. [11] Shannon IL. Climatological effects on human parotid gland function. Arch Oral Biol 1966;11:451–3. [12] Brown CC. The parotid puzzle: a review of the literature on human salivation and its applications to psychophysiology. Psychophysiology 1970;7:66–85. [13] Brunstrom JM, MacRae AW, Roberts B. Mouth-state dependent changes in the judged pleasantness of water at different temperatures. Physiol Behav 1997;61:667–9. [14] Dawes C, O'Connor AM, Aspen JM. The effect on human salivary flow rate of the temperature of a gustatory stimulus. Arch Oral Biol 2000;45:957–61. [15] Winsor AL, Bayne TL. Unconditioned salivary responses in man. Am J Psychol 1929;41:271–6. [16] Anderson DJ, Hector MP. Periodontal mechanoreceptors and parotid secretion in animals and man. J Dent Res 1987;66:518–23. [17] Hector MP, Linden RWA. The possible role of periodontal mechanoreceptors in the control of parotid secretion in man. Q J Exp Psychol 1987;72:285–301. [18] Lee VM, Linden RWA. The effect of odours on stimulated parotid salivary flow in humans. Physiol Behav 1992;52:1121–5. [19] Kanosue K, Nakayama T, Tanaka H, Yanase M, Yasuda H. Modes of action of local hypothalamic and skin thermal stimulation on salivary secretion in rats. J Physiol 1990;424:459–71. [20] Wang SL, Zhao ZT, Li J, Zhu XZ, Dong H, Zhang YG. Investigation of the clinical value of total saliva flow rates. Arch Oral Biol 1998;43:39–43. [21] Shannon IL. Specific gravity and osmolarity of human parotid fluid. Caries Res 1969;3:149–58. [22] Dawes C. The effects of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human parotid saliva. Arch Oral Biol 1969;14:277–94. [23] Shannon IL. Specific gravity of oral fluids. Tex Rep Biol Med 1968;26:349–55. [24] Palmai G, Blackwell B. The diurnal pattern of salivary flow in normal and depressed patients. Br J Psychiatry 1965;111:334–8. [25] Sandick BL, Engell DB, Maller O. Perception of drinking water temperature and effects for humans after exercise. Physiol Behav 1984;32:851–5. [26] Cruz A, Green BG. Thermal stimulation of taste. Nature 2000;403:889–92. [27] Guest S, Essick GK, Young M, Lee A, Phillips N, McGlone FP. Oral hydration, parotid salivation and the perceived pleasantness of small water volumes. Physiology and Behavior submitted for publication.
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[28] Dawes C, Kubieniec K. The effects of prolonged gum chewing on salivary flow rate and composition. Arch Oral Biol 2004;49:665–9. [29] Percival RS, Challacombe SJ, Marsh PD. Flow rates of resting whole and stimulated parotid saliva in relation to age and gender. J Dent Res 1994;73:1416–20. [30] Hodson NA, Linden RWA. Is there a parotid-salivary reflex response to fat stimulation in humans? Physiol Behav 2004;82:805–13.
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Thermally evoked parotid salivation Andy Lee a , Steve Guest b,⁎, Greg Essick c a
Department of Biochemistry, North Carolina State University, Raleigh, NC, United States Center for Neurosensory Disorders, School of Dentistry, University of North Carolina, Chapel Hill, NC, United States Department of Prosthodontics and Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC, United States b
c
Received 30 September 2005; received in revised form 6 December 2005; accepted 19 January 2006
Abstract Parotid salivation is known to be influenced by the temperature of liquids moved around the mouth. Here we investigated the ability of nonliquid thermal stimuli to change the rate of salivation. Unilateral parotid saliva was collected using a Lashley Cup from 12 normally hydrated subjects. Thermal stimuli were delivered through a copper tube, in which temperature-controlled water flowed, resting statically on the anterior tongue. During separate trials, the tube was 10, 22, or 44 °C, or the resting temperature of the tongue (or hypothenar of the hand, the control site). On each trial, the unstimulated salivation rate was first measured for 6 min while the subject remained seated with the mouth closed. Subsequently, salivation was measured for 6 min during application of the thermal stimulus. The tube was then removed for 1–2 min before the next trial. During the trials, subjects repeatedly rated the subjective temperature of the tongue (or hypothenar) and its perceived wetness/dryness. Stimulated salivation, expressed as a proportion of the previously measured unstimulated salivation, differed among body sites and temperatures (P b 0.03). A significant increase in salivation was seen only for the 10 °C stimulus applied to the tongue. Wetness ratings and salivation rates were positively correlated, albeit weakly. These results demonstrate that temperature-evoked changes in parotid salivation do not require the unique spatiotemporal dynamics of the tongue and jaw movements in wetting the oral mucosa. © 2006 Elsevier Inc. All rights reserved. Keywords: Parotid saliva; Temperature; Flow rate; Wetness perception
1. Introduction A variety of stimulus properties have been shown to alter saliva production in the human. For example, many basic taste solutions when swilled around the mouth lead to concomitant increases in whole or parotid salivation. This has been shown to occur for sour tastes for parotid [1–3] and whole [4] saliva, for salt solutions [2,3,5,6], for sweet stimuli [2,7] and for bitter tastes [2]. Additionally, parotid salivation has been shown to increase in response to trigeminal stimuli such as chemical irritants (e.g., capsaicin) [8] and astringents [9], although this latter effect is not universal [10]. In addition to these factors, non-stimulus-centered correlates of salivation exist, such as those associated with natural variations in individuals' state of hydration over the course of the year [11]. ⁎ Corresponding author. 2160 Old Dental Bldg., School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7450, United States. Fax: +1 919 966 3683. E-mail address: [email protected] (S. Guest). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.01.021
One aspect of salivation that has received relatively little attention is the role of thermal stimulation in altering the rate of saliva production. Indeed, some reviews of the factors affecting salivation omit thermal stimulation entirely [12]. Regardless, relatively warm or cool thermal stimuli presented to the mouth might induce salivation as a mechanism to protect the delicate oral tissues. In this respect, there is indeed some evidence that cold (0–3 °C) water presented to the mouth leads to greater production of whole saliva [13,14] than for water samples of more moderate temperature, and there is also evidence that parotid saliva can be evoked similarly by a cold (0°C) or hot (55–66 °C) thermal liquid challenge in the mouth [5,6]. Further, it has been proposed that the increase in saliva as induced by cold stimuli plays a role in the perceived pleasantness of drinks [13]. However, other research has found parotid salivation to be unaffected by prolonged presentation to the mouth of liquids with temperatures in the range 0 to 40°C [14]. In each of the experiments reported above, the thermal challenge was in the form of a liquid stimulus. As such, it is currently unknown whether the spatiotemporal dynamics of
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liquid manipulation in the mouth are necessary for significant saliva production in response to thermal stimulation. That is, if increased salivation does occur in response to a liquid thermal stimulus, it is not currently known whether the primary driver of salivation is the thermal challenge per se, or whether the thermal challenge must be combined with the types of mechanoreceptor activation that are strongly associated with saliva production, i.e., those activated by chewing-type actions of the tongue, jaw and facial tissues [15–17]. Although the role of spatiotemporal dynamics during oral manipulation has received little direct attention, the importance of periodontal mechanoreceptors to salivation was highlighted by Dawes et al. [14], who found that manipulation of an acrylic block and water at 20°C in the mouth led to considerably greater (whole mouth) salivation than water at 20 °C alone, but less salivation than a block of ice of the same dimensions as the acrylic block. Although the precise mouth movements used by the subjects of Dawes et al. remain unclear, it is likely that periodontal mechanoreceptors would have been stimulated periodically when moving the acrylic block (or ice block), even if the block was not explicitly bitten. Here we investigated the effect of a non-liquid thermal stimulus held statically on the surface of the tongue in altering the rate of parotid salivation. Although the use of such an ‘immaterial’ (i.e., non-gustatory) stimulus was suggested by Brown [12], to the best of our knowledge no such stimulus has been used in human subjects to date. Because the thermal stimulus we used was not manipulated between the teeth, the teeth were slightly separated, and no chewing movements were made, there was comparatively little stimulation to periodontal or other mechanoreceptors, and thus minimal mechanoreceptor-induced salivation [15–17]. As such, it was possible to determine to what extent prolonged thermal stimulation in the absence of periodontal reflexes affects salivation. This is of interest because if the major role of thermally invoked salivation is protective in nature, one would expect a static, non-liquid stimulus to evoke salivation in a similar way and with a similar magnitude of response to a liquid stimulus manipulated in the mouth. Conversely, if salivation does not occur in response to non-liquid thermal stimulation, then this would call into question the role of thermally evoked salivation primarily as a mechanism to protect against thermal damage. We also measured parotid salivation in response to an extraoral thermal stimulus, namely one presented to the palm of the hand. Although this was primarily designed as a control site, some previous work has found non-oral stimuli to alter salivation rates [18]. For example, in the rat, saliva production has been shown to increase in response to heating of the trunk [19]. However, saliva is used by the rat in heat-stress situations as an evaporative cooling agent, which is not the case in humans. As such, we did not hypothesize any effect of thermal stimulation of the hand on the rate of parotid salivation. Lastly, we considered the role of parotid saliva in the perception of mouth wetness. Although systematic reductions in salivary output (e.g., in Sjogren's syndrome) are associated with a dry mouth [20], it is unclear whether non-pathological between-individual variation in salivation rates leads to concomitant individual differences in perceived mouth wetness.
It is also unclear to what extent parotid salivation changes, as induced by thermal stimulation, might account for changes in mouth wetness ratings. It is not guaranteed that salivation is the only factor in perceived mouth wetness differences for liquids of different temperatures. For example, in the results of Brunstrom et al. [13] a component of the feeling of wetness in the mouth might have been induced by the combination of tactile and thermal stimulation which would exist in addition to any elicitation of saliva. That is, a cold stimulus placed in the mouth might elicit a feeling of wetness, even if the stimulus were dry. 2. Methods 2.1. Subjects Twelve subjects (10 males, 2 females, median age 28years) consented to take part in the study. All were reimbursed $10per hour for their time. The study was approved on ethical and safety grounds by the School of Dentistry Institutional Review Board (IRB) at the University of North Carolina at Chapel Hill. 2.2. Apparatus Thermal stimuli were delivered with copper tubing through which water flowed from a temperature-controlled bath (Fig. 1A). Before being placed in the mouth or on the hand of a subject, the tubing was covered with thin, flexible, plastic film. Unilateral parotid saliva was collected using a Lashley cup [6] which was positioned over Stenson's duct, and held in place using light suction, applied with a hypodermic syringe. The vacuum pressure was continually monitored by the experimenter Thermocouple readout to PC
A
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Fig. 1. (A) Thermal stimulator, not to scale. The device was made of copper tubing, and was covered with thin, flexible film during use. (B) The body sites to which the thermal stimulator was applied.
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via a dial gauge. Saliva flowed down a capillary tube to a beaker placed on the plate of precision balance interfaced to a PC, enabling saliva weight to be recorded continually. The capillary tube between cup and balance was pre-filled with water, eliminating the need to wait for the subject's own saliva to fill the tubing before collection could begin. Priming the collection tube with water did not lead to incorrect saliva weights being recorded during initial collection because the specific gravity of parotid saliva is but trivially different from the specific gravity of the water used to prime the tube: the specific gravity of unstimulated parotid saliva lies in the range 1.0025 to 1.0040 [21], and although some aspects of parotid saliva composition vary with flow rate and duration of stimulation [22], the specific gravity of stimulated parotid saliva remains very close to that of water even during gustatory or masticatory stimulation [23]. The resting temperature of the tongue or palm was obtained using an electronic thermometer which was either held on the anterior tongue with the subject's mouth closed, or placed under the hypothenar of the hand. After approximately 45 s, the temperature reading was taken and used as the neutral temperature (NT) of the subject's tongue or palm. The resting temperature was obtained immediately before the trial that included thermal stimulation at the neutral temperature. Cues to subjects and response scales were presented on a 15″ LCD panel located approximately 60cm in front of the subject. Responses demanded of subjects were made using a cordless joystick which moved a slider on the response scale shown, with selection of a response made by the joystick trigger. 2.3. Design and procedure Thermal stimuli at 10, 22, NT (i.e., resting temperature of tongue or palm) and 44 °C were applied to either the anterior tongue or the hypothenar of the hand (Fig. 1B). Each thermal stimulus was presented for 6-min. The presentation of thermal stimuli was either in increasing (10, 22, NT and 44 °C) or decreasing (44, NT, 22, 10°C) order. Before presentation of a stimulus, the unstimulated salivation rate was measured for 6 min. During this period, there was no stimulus present in the mouth, and the mouth was closed. Thus a trial consisted of two periods, a period where no thermal stimulus was delivered, followed by a period when thermal stimulation was presented to the subject. Between periods of a trial, there was a delay of 1– 2 min. For both unstimulated and stimulated periods, the subject was allowed to swallow as and when they wished. Although the subject's jaws were not fixed in place, the subject was asked to avoid making gratuitous jaw/mouth movements. Testing took place over two days. On one day, stimuli were delivered to the hand, on the other day stimuli were delivered to the mouth. The ordering of which body site was tested on which day was pseudo randomized such that approximately equal numbers of subjects were tested on both possible orderings. Each subject returned to the lab at approximately the same time on each of the two days of testing, to minimize variation in salivation according to time of day [24]. In the hour preceding the experiment, subjects drank 500–700ml of water, to ensure adequate hydration, and the subjects did not eat during this period.
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During both unstimulated and stimulated periods of a trial, subjects rated the perceived wetness or dryness of their mouth or hand. During stimulated periods, subjects were informed that the ratings should reflect the feel of their tongue or palm under the thermal stimulator itself. During unstimulated periods, the wetness ratings reflected wetness of the mouth or hand. Ratings were also made of the perceived temperature of the hand or mouth. During stimulated periods, these ratings represented the perceived temperature of the thermal stimulus, whereas during unstimulated periods of the trials the temperature ratings represented the resting temperature of the hand or mouth. All ratings were made using bisemantic visual analogue scales (Fig. 2). The temperature scale is a linear version of the temperature wheel of Sandick et al. [25], whereas the wetness/ dryness scale is similar in form to the temperature scale. Both scales consisted of 100 discrete steps, as presented by the computerized data collection program. This number of steps was sufficient for the scale to feel continuous to the subjects. 2.4. Statistical analysis Salivary flow was analyzed separately for unstimulated and stimulated periods using mixed-model analysis of variance (ANOVA). Six estimates of salivation rate were extracted during each 6 min period, namely the rates obtained in consecutive 1 min intervals. Both analyses included repeated-measures factors of body site, temperature and time (i.e., corresponding to the six consecutive salivation estimates). Note that for the unstimulated salivation analysis, the body site and temperature factors referred to the site and temperature that were to be subsequently tested. The analysis of stimulated salivation was carried out on relative salivation rates, as has been the case in much previous research [5,13,18]. Each subject's rates were expressed as a proportion of their mean unstimulated rate. As
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Fig. 2. Response scales used to collect (A) subjective impressions of temperature, and (B) perceived wetness/dryness.
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such, the value ‘1’ indicated a stimulated salivation rate no different than the previous unstimulated rate, values greater than one denoted increased salivation, and values less than one indicated decreased salivation. Prior to analysis, all wetness and temperature ratings were converted into the range − 1 to 1. The driest and coldest labels of each scale (‘extremely dry’ and ‘very cold’) were given the value − 1, the wettest and hottest labels (‘extremely wet’ and ‘very hot’) the value 1, and each rating made along the scales assigned a value accordingly. After conversion, the value zero indicated the midpoint of the scale, signifying either a neither wet nor dry feeling, or a neither warm nor cool percept. The ratings were analyzed in similar fashion to the salivary data. Wetness and temperature ratings were analyzed separately, with separate mixed-model ANOVAs carried out for the unstimulated and stimulated data. In all cases the repeated-measures factors were the same as in the saliva analysis. Finally, relationships between salivation rate and ratings of mouth wetness were sought using both within- and betweensubjects correlations. Within-subjects correlations were calculated between the wetness ratings and the salivation rates obtained in the 45s preceding the rating. Each correlation included the wetness ratings and associated salivation rates for the mouth data only, for all combinations of stimulated or unstimulated conditions × time × temperature. Log salivation rates were used, after inspection of the data suggested better fits to linearity with this transform. The hand data was not included because the wetness ratings in this case, being the wetness of the hand, would have no known relationship with the rate of salivation. Between-subjects correlations were calculated for the mouth data, one correlation for the unstimulated data, grouped across the different temperatures, and then one for each of the four stimulated periods at the different temperatures. For the stimulated data, each subject provided four data pairs, consisting of the mean wetness rating and average (log) salivation rate over the 6min stimulus period for each temperature. Additionally, a mixed-model ANOVA was carried out on the stimulated data, with wetness ratings as the dependent variable, and (log) salivation rate, temperature, and their interaction as factors in the model to determine whether overall, salivation rate influenced wetness ratings, and whether any such influence was different for the different temperatures. Any pairwise tests reported were corrected for multiple comparisons using the Tukey–Kramer method. 3. Results 3.1. Parotid salivation Prior to stimulation, the mean unilateral parotid flow did not vary according to either the body site or the temperature of the thermal stimulus subsequently presented. The overall mean unstimulated flow rate was 0.051 g min− 1. In contrast, the stimulated (relative) salivation rates were influenced by the presence of the thermal stimulus, specifically, salivation rates varied according to body site and temperature in an interactive manner (P b 0.02). Fig. 3 illustrates the least-square mean
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Fig. 3. Stimulated relative salivation rates obtained during presentation of a static thermal stimulus to either the anterior tongue or hypothenar of the hand. Values greater than one indicate increased salivation.
relative salivation rates obtained during stimulation for the different body sites and temperatures. Pairwise comparisons of the least-square means indicated that the same relative salivation rate was elicited when the thermal stimulus was present on the hand, regardless of the temperature of the stimulus. However, in the mouth, the 10 °C stimulus led to significantly greater salivation than 22 °C or NT (P b 0.05). Although greater salivation was suggested for the 10°C stimulus than the 44 °C stimulus, the difference fell short of statistical significance (P = 0.078). Of all the relative salivation rates, only that for the 10°C stimulus presented to the mouth was greater than the baseline rate (i.e., a relative salivation rate of N 1). In addition to the effects of stimulus site and temperature, an additional effect was identified, namely that of time (P b 0.001). Inspection of the data revealed that by far the greatest salivation occurred in the first 60s interval of the stimulus trials. The five subsequent minute-long intervals showed salivation rates very similar to those gathered during resting measurements. 3.2. Ratings 3.2.1. Temperature ratings At rest, the mouth and hand were both rated as slightly warm (mean = 0.06, where zero indicates the midpoint of the scale) and did not differ in their perceived warmth. During thermal stimulation, ratings varied according to the temperature of the stimulus (P b 0.0001; Fig. 4), and were similar for both hand and mouth. 3.2.2. Wetness ratings When no thermal stimulus was applied to mouth or hand, the mouth was rated as wetter than the hand (P b 0.0001). The mean wetness ratings for hand and mouth were 0.01 and 0.12,
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binations of stimulus site and temperature. It is clear from the figure that only in the case of the 10°C stimulus delivered to the mouth, did ratings of wetness increase greatly over time. Wetness ratings for the hand were not well differentiated for the different temperature stimuli; there was no evidence that the non-liquid, but cold stimulus led to a perception of wetness on the palm skin under the stimulator. It is also clear that the significant interaction between time and stimulus site was a consequence of certain mouth wetness ratings increasing over time while the hand wetness ratings remained relatively constant.
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Temperature (°C) Fig. 4. Temperature ratings (averaged over hand and mouth body sites) of four different thermal stimuli. Error bars show +1 SE.
respectively. The former value indicates a neither wet nor dry feel, whereas the latter value indicates a feeling approaching ‘slightly wet’; the ‘slightly wet’ label occurs at a value of 0.2 on the wetness response scale (see Fig. 2). Additionally, there was a significant (P b 0.02) interaction between the time at which the rating was obtained and the stimulus site. However, inspection of the ratings indicated that this interaction was not meaningful: Ratings on both hand and mouth were always within 0.04units (2% of the whole scale) of their respective means, and at every time point, the mouth was rated as wetter than the hand. When the tubing was present in the mouth or on the hand, the wetness ratings varied by time (P b 0.001), the interaction between time and stimulus site (P b 0.0001) and the interaction of time, stimulus site and temperature (P b 0.02). Fig. 5 illustrates the ratings made at each of six sample times, for the com-
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Finally, the association between parotid salivation rate and ratings of mouth wetness was investigated. Correlations were calculated for each subject's mouth data, between their wetness ratings and the salivation rates obtained in the 45s preceding the rating. Of the 12 correlations, one for each subject, four were significant (P b 0.05) such that increasing wetness ratings were associated with greater salivation rates. However, these correlations ranged in magnitude from 0.27 to 0.44, suggesting relatively weak relationships. Additionally, three subjects showed an inverse relationship between their wetness ratings and salivation rates, and two of these correlations were statistically significant (r = − 0.30 and − 0.34). These results indicate that overall, for some subjects, the relationship between salivation and wetness ratings was statistically significant, but the relationship was never strong, nor visible in all subject's data.
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Time (min) Fig. 5. Ratings of the wetness of mouth or hand during the course of 6-min trials where a thermal stimulus at 10, 22, NT or 44°C was present on the hand or on the anterior tongue.
Salivation rate (g/min) Fig. 6. Wetness ratings plotted against salivation rate for four temperatures of stimulus, applied to anterior tongue. A least-squares regression line through all data points is shown. A mixed-model ANOVA indicated that the effect of salivation rate on wetness ratings was significant (P b 0.05), but there was no influence of temperature or the interaction of temperature and salivation rate on the ratings.
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Subsequently, the between-subjects relationship between salivation rate and wetness ratings was investigated. For the stimulated data, a mixed-model ANOVA indicated that wetness ratings were influenced by (log) salivation rate (P b 0.006), but not by temperature or the interaction of salivation rate and temperature, indicating that the relationship between wetness ratings and salivation rates was similar for all four stimulated temperatures. A scatterplot of wetness versus salivation rates is shown in Fig. 6. Of the correlations for each of the individual temperatures, all were indicative of higher wetness ratings being associated with greater salivation. These positive correlation values ranged from 0.45 (for the 10 °C stimulus) to 0.62 (NT stimulus). One of the correlations was statistically significant (P b 0.05), namely that for the NT stimulus. For the unstimulated periods, the correlation between salivation rates and wetness ratings was not significant (r = 0.37, P N 0.05). 4. Discussion A non-liquid (10°C) stimulus, positioned statically against the anterior tongue, increases the rate of parotid saliva production, although the magnitude of this increase, namely just more than a doubling on average, appears to be somewhat less than the increase seen with liquids at approximately the same temperature; other researchers have found approximately two- to fivefold increases in parotid salivation for cold temperatures of liquids [5]. Other non-liquid, static stimuli in the range 22–44°C, presented to the mouth, do not increase – and may even decrease – parotid salivation (Fig. 3). Thermal stimuli placed on the hypothenar of the hand do not alter salivation. These results illustrate that although movement of a liquid stimulus around the mouth is not necessary to invoke increased parotid salivation, consideration of the literature suggests that the combination of mechanical stimulation and thermal stimulation leads to greater salivation than thermal stimulation alone. It is likely that proprioceptive input and stimulation of periodontal mechanoreceptors during swishing of a liquid around the mouth [15–17] drives this difference; the ability of a non-liquid thermal stimulus to evoke salivation is clearly rather weak, especially when considering the strength of the most extreme stimuli we used: Both the coldest and warmest temperatures were such that they were the lowest (or highest) that could be reliably tolerated by all subjects, and our stimuli were applied for longer than is typically used for liquids. Yet even at these extremes, the change in evoked salivation was modest. Although the increase in salivation upon stimulation of the tongue with the 10°C challenge is assumed to be a response to the thermal nature of the stimulus per se, one of the reviewers raised the interesting possibility that the salivation may have been mediated by thermally induced taste sensations. Cruz and Green [26] found that cooling the tongue to 10 °C led to, on average, a weak sensation of sourness, with most subjects in the test population showing this thermally induced taste sensation. Given that sour stimuli are potent inducers of parotid salivation, it is possible that any thermally induced sourness could act similarly. We did not collect perceived taste sensations from subjects, and as such we cannot ascertain the magnitude of any
thermal-taste-induced salivation, or indeed even if such salivation actually occurs. The possibility is worth investigating in future studies. A further possible factor mediating the modest salivation changes we found is in the local nature of our thermal stimulus. The stimulus predominately heated or cooled the tongue, with minimal temperature change being delivered directly to many other oral surfaces, such as the labial mucosa. This differs from liquids swilled around the mouth which provide thermal stimulation to most or indeed all of the oral cavity. This wider application of thermal stimulation when using liquid stimuli could well drive greater salivation than for more focal thermal challenges. The results we found broadly agree with those of Pangborn et al. [5] and Brunstrom et al. [13], and differ in some respects from that found by Dawes et al. [14]. In particular, Dawes et al. found that liquid stimuli in the range 0 to 37°C pumped continually through the mouth did not elicit different rates of parotid salivation, whereas we found that sustained application of a 10°C stimulus to the tongue did lead to increased flow. This is perhaps unexpected given that the stimuli of Dawes et al. were at a relatively constant sustained temperature, similar to those we used, by virtue of the continual flow method used by the Dawes et al., as opposed to the sip and spit method often used with liquid stimuli. Note that Dawes et al. did find that stimulation with ice was a relatively potent producer of saliva, and this stimulus appears more similar in some respect to our (cold) thermal stimulus, although ice would presumably provide some periodontal mechanoreceptor stimulation that our thermal device minimized, and the ice would directly stimulate mucosal surfaces in addition to the tongue. Overall, ratings of mouth wetness between-subjects were positively related to salivation rates albeit not especially strongly, with at most 36% of the variance in mouth wetness ratings being accounted for. That is, subjects who salivated relatively more when the thermal tubing was in the mouth did indeed tend to rate their mouth as wetter than subjects who salivated less freely. This relationship also held for some of the within-subjects correlation analyses. Thus the wetness ratings appeared, to at least some extent, to be based on the veridical perception of wetness, although it is possible that a component of the wetness ratings for the 10 °C stimulus was illusory, induced by the pressure of a cold object against the tongue [27]. No illusory wetness on the palm was induced by a cold stimulus pressing on the hand. That the relationships between wetness and salivation rates were not stronger is perhaps unsurprising, for at least two reasons. First, unilateral parotid saliva contributes only a small portion (c. 15%) of the mouth's total salivary volume: unstimulated whole saliva typically flows at a rate of c. 0.3–0.6ml/min [20,28,29], whereas the unilateral unstimulated parotid flow rate lies in the range 0.01–0.09ml/min [3,5,30,31] as found in the current experiment. Submandibular flow rates have found to be similar to those found for parotid saliva [32], with flow from sublingual and minor glands making up the remaining components of whole saliva. Given these different salivary sources, the relationship between wetness and parotid salivation would be expected to be weak if saliva volume
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is the main determinant of perceived mouth wetness, and if the other salivary sources (e.g., sublingual, submandibular) are relatively thermally unresponsive, or if their flow rates are otherwise poorly correlated with the parotid flow rate. There is some evidence this might be so given that some authors have found thermal differences in whole saliva flow rates, but failed to find such differences in parotid flow [14]. It is also true that diverting saliva from one of the parotid glands to the collection tube prevents that saliva from being delivered to the oral mucosa. This would also reduce the potential impact of parotid saliva upon mouth wetness and may have contributed to the relatively weak relationships we found between saliva rates and perceived mouth wetness. Second, the amount of saliva in the mouth at the point of swallowing is probably a critical quantity, because this would be the point at which maximum mouth wetness would be expected. Given that we did not know when the subject swallowed, we were restricted to using ratings that would not necessarily sample these times of maximum mouth wetness. Indeed, it is possible that mouth wetness would remain relatively constant even with increased salivation, by virtue of the subject choosing to swallow more often than when their salivation was less profound. Although all but the 10 °C stimulus suggested a reduction in salivation, the ratings of mouth wetness remained relatively constant, or increased over trials (re. Fig. 5). The basis for this effect could be if the thermal tubing hindered the complete swallowing of accumulated saliva in the mouth. If suppression of salivation occurs in response to its constant presence, this could be a reason for the suggested suppression in salivation seen for the 22–44 °C stimuli. To the best of our knowledge effects of accumulated saliva on salivation has not been studied. If a suppression mechanism is indeed plausible, then the experimental paradigm may have, in fact, underestimated the saliva-stimulating effect of sustained presentation of a static, cold thermal stimulus. A further possible explanation for the presence of reduced salivation is in the relative lack of periodontal mechanoreceptive stimulation in the stimulated versus unstimulated periods. During unstimulated periods the subject's upper and lower teeth could contact each other, whereas during stimulated periods the presence of the stimulus tubing prevented any such direct contact. In conclusion, we find that cooling the tongue with a dry, static stimulus can induce increased parotid salivation, albeit to a lesser extent than is seen when cold liquids are swished around the mouth. This result is consistent with the common suggestion that thermally evoked parotid secretion is primarily a mechanism to protect the oral mucosa against heat or cold induced damage. The parotid salivation rate was but a weak predictor of perceived mouth wetness. Although this may suggest that increased salivation in response to cold fluids is unlikely to be the basis of the high perceived pleasantness of cold water taken into a dry mouth [13], the suggestion warrants caution given that we only considered one component of saliva (i.e., parotid). As expected, thermal stimulation of the hand did not alter salivation, and may be added to the list of other such non-sialagogic stimuli as odors and mental imagery [1,18].
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