AN OLFACTORY-PAROTID SALIVARY REFLEX IN HUMANS?

Transcription

1 Experimental Physiology (1991), 76, Printed in Great Britain AN OLFACTORY-PAROTID SALIVARY REFLEX IN HUMANS? V. M. LEE AND R. W. A. LINDEN Biomedical Sciences Division, King's College London, Strand, London WC2R 2LS (MANUSCRIPT RECEIVED 12 NOVEMBER 1990, ACCEPTED 19 DECEMBER 1990) SUMMARY It is commonly stated that smell elicits a parotid salivary reflex in humans. However, this assumption seems to be supported only by experiments in which either a lemon or an irritating odour has been used as the primary stimulus. In this study six pleasant odours, chocolate, vanilla, peppermint, beef, tomato and lemon, were administered to ten subjects. Air was passed through a solution of the stimulant and the resultant odour was administered to the subject via a face mask. Unilateral parotid saliva was collected via a Lashley cup and salivary flow was measured using an instantaneous flowmeter sensitive to flows as low as ml min-'. Citric acid, lemon juice, chocolate and distilled water were also delivered to the subjects at increasing concentrations. At low concentrations none of these stimuli significantly increased the parotid salivary flow above resting levels. However, an increase in salivary flow was seen when lemon juice or odourless citric acid was sniffed or delivered to the subject at high concentrations, causing irritation in the nasal cavity and/or the back of the throat. Our results suggest that there is no true olfactory-parotid salivary reflex in humans, and that acidic stimuli can cause irritation with a concomitant increase in the salivary flow. INTRODUCTION The presence of food in the mouth provides a powerful stimulus for salivation (Watanabe & Dawes, 1988). The existence of both masticatory- (Lashley, 1916; Weber, 1960; Kerr, 1961; Hector & Linden, 1987; Jensen Kjeilen, Brodin, Aars & Berg, 1987) and gustatory- (Lashley, 1916; Kerr, 1961; Chauncey, Feller & Shannon, 1963; Watanabe & Dawes, 1988) parotid salivary reflexes have been well documented. Since the classical work of Pavlov (1927, 1929) on the conditioned reflex, it has been assumed that the sight, smell and thought of food causes salivation in man. However, the evidence available for the existence of an olfactory-parotid salivary reflex is confusing and inconclusive: Kerr (1961) and Shannon (1974) both reported that olfactory stimulation caused an increase in parotid salivary flow. Pangborn, Witherly & Jones (1979) also reported a significant increase. However, Pangborn & Berggren (1973) had already demonstrated that non-irritating odours had no effect on parotid salivary flow. Furthermore, neither Lashley (1916), Winsor (1928) nor Elsberg, Spotnitz & Strongin (1940) could find any consistent increase in parotid salivary flow in response to non-irritating odours. Despite the fact that these studies provide no conclusive evidence many standard textbooks (e.g. Hendrix, 1980; Osborn, Armstrong & Spiers, 1982; Holland, 1984; Ganong, 1989) state quite clearly that an olfactory-salivary reflex does exist in humans. It should be noted the term olfaction is often used to describe all forms of nasal chemoreception. However, chemical stimuli can be detected by more than one system in the nasal cavity; in humans the principle system other than the olfactory is the trigeminal (Tucker, 1971). In our study smell is defined as nasal chemoreception which includes the

2 348 V. M. LEE AND R. W. A. LINDEN combination or interaction of both the olfactory and trigeminal systems while olfaction is defined as stimulation of the olfactory receptors alone. The aim of the present investigation was to examine more closely the effect of smell on the resting parotid salivary flow in humans. Some of the methods used in this study have been demonstrated to the Physiological Society (Lee & Linden, 1990 a). Preliminary observations on the effect of lemon odour have also been reported (Lee & Linden, 1990b). METHODS Saliva collection Unilateral parotid saliva was collected using a modified Lashley cup (Hector & Linden, 1987). The presence of a flow of saliva into the Lashley cup and cannula was tested using a strong gustatory stimulus (about 05 ml of diluted lemon juice). This was repeated at the end of each experiment to ensure that the cup and cannula remained patent throughout. Instantaneous flowmeter Salivary flow was measured using a modified form of the instantaneous flowmeter described by Burgen (1964). The saliva flowed through the cannula and into a sealed flask causing an increase in pressure. This was measured by an electromanometer (Mercury, Ml 1) in comparison with a sealed reference flask (to minimize the effects of any environmental pressure changes.) To prevent a continual build-up of pressure in the flask a leak was incorporated into the system by way of a 30 gauge needle. The output of the meter was recorded on a pen-recorder (Lectromed) and on magnetic tape (Racal, Store 4 recorder) for future analysis. The flowmeter was calibrated for each experiment; a head of distilled water was used to provide a constant flow which was recorded. The water was collected over a measured period of time and weighed. Thus flow could be correlated to flowmeter reading. The instantaneous flowmeter was sensitive to flows as low as ml min-'. The data were transferred from the magnetic tape to a desktop computer (IBM AT compatible) using an analog-to-digital converter (Computerscope). The total volumes of saliva secreted were calculated by measuring the areas under the curve using an analysis program (Grafix BICERI). In experiments where the flows were very small it was more convenient to calculate the volumes of saliva from the pen-recorder traces using an image analysis program (Digit) in conjunction with a Bit Pad 2 Digitizer Tablet to measure the areas under the curve. There was no significant difference between the results using the two methods. Stimuli The test and control stimuli were produced by filling two nebulizers with a freshly made solution of a test stimulus and distilled water respectively. Air was delivered from a compressed air cylinder through a reduction valve and flowmeter at a constant rate. The air was passed via inert odourless plastic tubing into both the control and test solutions. Both the nebulizers were connected to a threeway tap which allowed instant switching from the test to the control stimuli. This was in turn connected to a plastic face mask via the shortest possible length of common tubing to minimize any cross-contamination. The passage of air through the nebulizer atomized the solution thus resulting in an odour stimulus. Experimental protocol Eighteen healthy volunteers (eleven male and seven female) between the ages of 20 and 41 years participated in these experiments with Local Ethical Committee approval. Recordings were carried out at least 1 h after the subject's last meal. During recordings the subjects were asked to nose breathe as mouth breathing elicits the 'dry mouth reflex' (Jenkins, 1978). Subjects were also encouraged not to swallow or talk as this has been shown to stimulate salivary secretion (Lashley, 1916; Winsor, 1928; Kerr, 1961). The subject sat with the mask close to but not pressing on the face, thus eliminating any possible effect caused by pressure on the parotid duct. Each recording session began with a control period of several minutes. Once a constant resting flow had been recorded for at least 1 min the appropriate test stimulus was administered. This was then followed by a further control period.

3 OLFACTORY-PAROTID SALIVARY REFLEX? In the first experiment, freshly squeezed and filtered lemon juice was administered to ten subjects for a period of at least 1 min. The flow of air through the nebulizers was adjusted for each subject in order to provide a strong lemon odour. In the second experiment, distilled water, lemon juice, odourless citric acid and chocolate solution were administered in a random order to eight subjects. The concentration of the odour being received through the mask was increased at about 15 s intervals by increasing the rate of air passing through the nebulizer. (The flow of air through the nebulizers was adjusted from min-' in steps of 05 to a maximum of min-1.) It should be noted that increasing the rate of administration through the nebulizer does not increase the rate of delivery through the nasal passages, but it increases the concentration of the stimulus that is being inhaled. In the final experiment, chocolate solution, diluted vanilla essence, diluted peppermint essence, tomato juice, and beef stock (Bovril) were each administered to ten subjects by passing air through the stimulus at a constant flow. The test stimulus was then administered twice, each time for a 1 min period followed by a 1 min control period. 349 RESULTS No effect was seen in nine out of ten subjects who received the lemon odour (for examples see Fig. 1), even in a subject who had fasted for 18 h. One subject exhibited an increased flow; however, when the stimulus concentration was lowered the response did not occur although the stimulus was still detectable. Four subjects were asked to smell a freshly cut lemon, as this was the method used to administer the lemon odour in studies where a positive response has been reported (Shannon, 1974; Pangborn et al. 1979). In only one subject was there an increase in flow and it was noticed that this subject was taking deep sniffs of the lemon slice. The subject was therefore asked not to sniff the lemon but to simply breathe in the odour (although deeply enough to be able to smell it), after which no increase in flow was observed (Fig. 2). In both cases where a positive response was seen the subject reported that the odour caused irritation to the nasal passage and/or to the back of the throat. In the second experiment distilled water (odourless and non-irritating), lemon juice (odorous and irritating), citric acid (odourless and irritating) and chocolate solution (odorous and non-irritating) were administered to subjects at increasing concentrations. Six out of the eight subjects exhibited increases in flow in response to the lemon juice and citric acid solution. No effect was seen in response to either distilled water or chocolate solution. Typical responses from a single subject are shown in Fig. 3. The irritating stimulants elicited a salivary response at different concentrations in each subject (the maximum responses occurring between 1 5 and min-1). This may have occurred because the thresholds for odour detection (either olfactory or irritant component) and recognition vary between subjects. Therefore the thresholds for eliciting salivation may also vary. For this reason a direct comparison between subjects at the same rate of administration was impossible. The effects of the stimuli on the parotid salivary flow were assessed by comparing the resting salivary flow with the highest salivary flow that was obtained with the given stimulus. Thus the maximum percentage change in salivary flow for each stimulus could be calculated (Table 1). One subject (subject 5) appeared to show no response to any of the four stimulants at these concentrations. The final subject (subject 8) showed the highest response to the control stimulus with the lowest response to citric acid. However, in this subject the resting values were extremely variable but the maximum flow for all four stimuli showed an effect which was more consistent with the results from the other subjects. During this experiment it was noticed that the subjects appeared to adapt to the high concentrations of odour 13 EPH 76

4 350 o E OL V. M. LEE AND R. W. A. LINDEN f-l._e: o. o 0 Fig. 1. Unilateral parotid salivary flow in two subjects showing the typical response to lemon odour. Air and lemon odour were administered during the periods shown by the open and filled bars respectively. The arrows show when a gustatory stimulus (lemon juice) was administered ct :31-.= C o. co~ c:1- Pz a w _ 9 - g _ u %J I *Il * I Time (s) 0. U hi Fig. 2. The effect, in a single subject, of sniffing and breathing the odour from a cut lemon on parotid salivary flow (cf. parotid flow elicited by tasting lemon juice > ml (30 s)-1). *, control (breathing air); U, sniffing lemon; E, breathing lemon. administration, i.e. if the lemon odour was given before the citric acid, or vice versa, the response to the latter was generally less than the response to the former. In the third experiment various appetizing odours were administered. Resting flow differed between subjects as much as 100-fold. Therefore each subjects' salivary flow was converted into a percentage of the mean of all three control periods. Student's t test for paired observations was carried out on the data and values of P < 0 05 were considered significant. The salivary flow for each of the test periods was compared with the average

5 OLFACTORY-PAROTID SALIVARY REFLEX? 351 1j Distilled water 11 Citrate 0 30 s : l05 E 0 1 Lemon juice Chocolate Fig. 3. Parotid salivary flow in one subject (subject 6) showing the typical response to nebulized distilled water, citrate solution, lemon juice and chocolate solution. Each step shows a min-' increase in the rate of air passing through the nebulizer. The arrow shows when a gustatory stimulus (lemon juice) was administered. Table 1. The effects of varying stimuli on parotid salivary flow Subject Control Citrate Lemon Chocolate (a) (b) (a) (b) (a) (b) (a) (b) 1 0o o (16) (588) (138) (26) (78) (215) (115) (5) (-31) (4960) (1419) (66) (-53) (589) (510) (40) (90) (4) (9) (-32) o (85) (3104) (1425) (8) (43) (741) (6700) (-26) (3006) (182) (582) (651) The effects of odourless, non-irritating (1), odourless, irritating (2), irritating with odour (3), and non-irritating with odour (4) stimuli on parotid salivary flow. Showing resting parotid salivary flow (a, ml min-') and highest salivary flow obtained during administration of the odour (b, ml min-1). The figures in parenthesis show b as a percentage change of a. of the two control periods on either side of it and the three control periods were all compared with each other. There were no significant differences between any of the control periods. None of the five odours caused any significant increase in salivary flow (vanilla, P > 030, chocolate, 13-2

6 352 V. M. LEE AND R. W. A. LINDEN Vanilla E Chocolate _ 2L l b50- _ 0~~~~~~~~~~ U~~~~~~~~~~ 0. 0 _E g 200- Peppermint,, au 100~ ~~~ ~ ~ ~ ~ ~ (0 1 t 2~~~~~- 150X tw ~~~~~~~ Fig. 4. The effect of five appetizing odours on resting parotid salivary flow in ten subjects. The flow for each 1 min period is shown as a percentage of the mean of the three control periods (values represent means + S.E.M. of ten subjects). [1, control stimulus; E, test stimulus. P > 0 40, peppermint, P > 0-30, beef, P > 0-10, tomato, P > 0-35) (Fig. 4). In one subject the beef odour caused salivation; however, it was subsequently discovered that this subject was a vegetarian and the stimulus had caused nausea. In all cases the subjects commented that the odours were quite strong. Therefore there is no doubt that the subjects could smell the stimuli. DISCUSSION Attempts were made in these experiments to bring some clarity to a subject surrounded by much confusion: the so called olfactory-salivary reflex. The results from the first and third experiments indicate that there is no increase above resting parotid salivary flow in humans in response to a variety of odours. All the stimuli had easily distinguishable odours and were considered by most subjects to be quite

7 OLFACTORY-PAROTID SALIVARY REFLEX? appetizing. The only exception was the beef odour which was unwittingly administered to a vegetarian, who found the odour quite nauseating. Nausea is a powerful salivary stimulant (Jenkins, 1978). There is a possibility that an increase in salivary flow is not observed in response to odours in the laboratory because the artificial situation is causing inhibitory affective states in the subjects: Sahakian, Lean, Robbins & James (1981) showed that there was a significant increase in the whole salivary flow in response to sight and smell of a freshly cooked meal presented in restaurant surroundings. However, Kerr (1961) could find no such increase in the whole salivary flow of three hungry subjects in response to the sight and smell of bacon and eggs being cooked in front of them. It is possible that under these types of non-laboratory conditions small increases are caused by unrestricted swallowing and anticipatory mouth movements which cause expulsion of saliva from the dead space of the gland due to muscle contraction (Kerr, 1961). Furthermore, the suggestion that an increased salivary flow will occur only under a certain set of conditions which normally accompany the stimulus implies that 'natural' conditioned salivary reflexes are present. However, Kerr (1961) could find no such increases in parotid, submandibular or even whole salivary flow. In contrast, Jenkins & Dawes (1966) and Holland & Matthews (1970) concluded that small 'natural' conditioned salivary reflexes can be produced in man although the latter found them to be small and inconsistent. However, Holland & Matthews (1970) demonstrated that a substantial conditioned reflex could be established, but only after careful conditioning. This agreed with Pavlov (1927, 1929) who had stressed that a reflex could only be established if a consistent response was obtained with each conditioning stimulus. This suggests that under normal circumstances an odour stimulus would not elicit a consistent conditioned response. Our results show that odours will only elicit an increased salivary flow if the odour is both capable of and strong enough to cause irritation, the latter being achieved either by inhaling deeply or by administering at a high concentration. A similar result was seen by Lashley (1916) who found that out of fifteen odours tested, only two (amyl alcohol and peppermint) showed any signs of stimulating salivary flow and that both of these caused irritation to the mucosa of the pharynx. Lashley (1916) concluded that the salivation was probably due to the irritation rather than the olfactory component of the odour. Pangborn & Berggren (1973) also found that only irritating or unpleasant compounds caused a significant increase in salivary flow. Although these observations are in accord with our results they are in contrast to the results of Shannon (1974) and Pangborn et al. (1979) who both reported a significant increase in parotid salivary flow in response to a lemon odour. However, in their experiments the subjects were instructed to take deep sniffs of a lemon slice and we have shown that sniffing lemon causes irritation. The results from the second experiment indicate that the increased salivary flow in response to higher concentrations of lemon juice was caused by the acid rather than the olfactory stimulus since a similar response could be obtained using odourless citric acid. In humans, chemical stimuli are detected in the nasal cavity principally by the olfactory and trigeminal systems (Tucker, 1971). Olfactory and trigeminal stimulation have traditionally been separated on the basis that trigeminal nerves are only stimulated by noxious chemical stimuli which cause stinging, pungency or pain (Doty, Brugger, Jurs, Orndorff, Snyder & Dale Lowry, 1978). Elsberg et al. (1940) concluded that only odours with a distinct trigeminal effect such as citral, menthol and turpentine caused a marked increase in parotid salivary flow. This suggests that the salivation elicited by administration of acidic odours at high concentration is due to stimulation of trigeminal nerves. 353

8 354 V. M. LEE AND R. W. A. LINDEN Doty et al. (1978) found that subjects lacking olfactory function were able to detect odours and Cain (1974) found that trigeminally deficient subjects exhibited impaired odour detection. Elsberg, Spotnitz & Strongin (1942) found that citral odour elicited a normal parotid salivary response in subjects with impaired olfactory nerve function while abnormalities were seen in subjects with impaired trigeminal or facial nerve function. These studies suggest that trigeminal stimulation occurs with most odours, and at most concentrations. Thus, if the salivary reflex seen in this study was due to pure trigeminal stimulation one would expect salivation to occur in response to a wider range of odours and at lower concentrations. Alternatively, it is possible that high concentrations of the odour stimuli reach the taste buds in the pharynx, this eliciting salivation. Acid causes a greater reflex response than any other gustatory stimulus and this may explain why only the acidic stimuli caused salivation in the present study. Although we could find no evidence of an olfactory-parotid salivary reflex, several subjects were convinced that their mouths were watering. This phenomenon of 'mouth watering' was ascribed by Kerr (1961) to a sudden awareness of saliva already present in the mouth, rather than an increase in the volume. In this study we were only measuring the salivary flow from the parotid gland, but it is possible that any of the other salivary glands may exhibit an increased salivary flow in response to olfactory stimulation. However, Jenkins (1978) and Kerr (1961) showed that a gustatory stimulus and amyl acetate vapour respectively produced higher responses from the parotid gland than the submandibular gland. In conclusion, these data suggest that a true olfactory-parotid salivary reflex does not exist in humans. We would like to thank the subjects for their time and co-operation. V. M. L. is partially supported by the States of Guernsey, Channel Islands. REFERENCES BURGEN, A. S. V. (1964). Techniques for stimulating the auriculo-temporal nerve and recording the flow of saliva. In Salivary Glands and Their Secretions, ed. SREEBNY, L. M. & MEYER, J., pp Pergamon Press, Oxford. CAIN, W. S. (1974). Contribution of the trigeminal nerve to perceived magnitude. Annals of the New York Academy of Sciences 237, CHAUNCEY, H. H., FELLER, R. P. & SHANNON, I. L. (1963). Effect of acid solutions on human gustatory chemoreceptors as determined by parotid gland secretion rate. Proceedings of the Society for Experimental Biology and Medicine 112, DOTY, R. L., BRUGGER, W. E., JURS, P. C., ORNDORFF, M. A., SNYDER, P. J. & DALE LOWRY, L. (1978). Intranasal trigeminal stimulation from odorous volatiles: Psychometric responses from anosmic and normal humans. Physiology and Behavior 20, ELSBERG, C. A., SPOTNITZ, H. & STRONGIN, E. I. (1940). The effect of stimulation by odorous substances upon the amount of secretion of the parotid glands. Journal ofexperimental Psychology 27, ELSBERG, C. A., SPOTNITZ, H. & STRONGIN, E. I. (1942). The olfactory parotid reflex. Archives of Neurology and Psychiatry 47, GANONG, W. F. (1989). Review of Medical Physiology, 14th edn, p Prentice-Hall International Inc., Englewood Cliffs, NJ, USA. HECTOR, M. P. & LINDEN, R. W. A. (1987). The possible role of periodontal mechanoreceptors in the control of parotid secretion in man. Quarterly Journal of Experimental Physiology 72, HENDRIX, T. R. (1980). The secretory function of the alimentary canal. In Medical Physiology, 14th edn, ed. MOUNTCASTLE, V. B., p C. V. Mosby, St Louis, MO, USA.

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