J. Physiol. (1982), 330, pp. 163-174 163With 5 text-figuresPrinted in Great Britain

PAROTID RESPONSES TO STIMULATION OF THE PARASYMPATHETICINNERVATION IN BURSTS IN WEANED LAMBS

BY P.-O. ANDERSSON*, S. R. BLOOMt AND A. V. EDWARDSFrom the Department of Physiology and Biophysics, University of Lund,S6lvegatan 19, S-223 62 Lund, Sweden* the Department of Medicine,

Royal Postgraduate Medical School, Hammersmith Hospital, London W 12tand the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG

(Received 4 December 1981)

SUMMARY

1. The effects of stimulation of the peripheral end of the parotid nerve in 1 secbursts at 10 sec intervals has been investigated in weaned Suffolk lambs 3-4 monthsafter birth.

2. Stimulation in bursts at either 10 or 20 Hz for 10 min produced a significantlygreater flow of parotid saliva, output of Na+ and K+ and fall in parotid vascularresistance (p.v.r) than continuous stimulation at the corresponding constant fre-quency (1 or 2 Hz) for the same period.

3. Stimulation at 40 Hz in bursts for the same period produced a significantlygreater output of K+ and fall in p.v.r. than continuous stimulation at 4 Hz, whereasthe salivary flow rates and Na+ outputs were closely similar.

4. Comparison ofthe effects ofstimulation ofthe parotid nerve in bursts over a widerange offrequencies showed that each ofthese parotid responses was maximal at about80 Hz.

5. Parotid salivary responses to continuous stimulation of the parotid nerve at20 Hz for 10 min were effectively abolished by prior administration of atropine(> 1-0 mg/kg), whereas the percentage fall in p.v.r. was merely reduced from80-2 + 3-6 to 33 1 + 8-3 % under the same conditions. There was no significant changein the mean arterior-venous difference in the plasma concentration of vasoactiveintestinal peptide, substance P or somatostatin, in either the presence or absence ofatropine in these experiments.

6. The results are discussed in relation to the proposition that the enhancement ofparotid responses which is observed during intermittent stimulation of the parasym-pathetic innervation at relatively high frequencies under experimental conditionsalso occurs normally in conscious sheep.

INTRODUCTION

Recent studies have shown that a number of autonomic responses are mosteffectively increased by stimulation of the efferent innervation at relatively highfrequencies (40-100 Hz) when the impulses are delivered in 1 sec bursts at 10 sec

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P.-O. ANDERSON, S. R. BLOOM AND A. V. EDWARDS

intervals. These include the release of vasoactive intestinal peptide (VIP) from nerve

terminals in the submaxillary gland of the cat, the associated fall in submaxillaryvascular resistance and the cholinergically mediated flow of submaxillary saliva(Andersson, Bloom, Edwards & Jirhult, 1981, 1982), the non-cholinergic, non-

adrenergic fall in colonic vascular resistance, which occurs in response to stimulationof the pelvic nerve in the same species (Andersson & JUrhult, 1981) and the releaseof adrenaline from the adrenal medulla during stimulation of the splanchnic nerve

in conscious calves (Edwards, 1981). In contrast, the colonic musculature ofatropinized cats is unaffected by stimulation in bursts at high frequency but readilycontracts in response to continuous stimulation of the pelvic nerve at relatively lowfrequencies (Andersson &Jirhult, 1981).Each of the previous studies of stimulation of parasympathetic nerves in bursts has

involved the use of tissues from which atropine-resistant responses can readily beelicited. Interpretation of the results has therefore been complicated by the likelyinvolvement of more than one transmitter, as virtually nothing is known about theinteractions that may occur between acetylcholine and such peptidergic transmittersas VIP (Lundberg, AnggArd, Fahrenkrug, H6kfelt & Mutt, 1980). Accordingly theparotid gland of the sheep has been investigated in the present study, as both the flowof parotid saliva and the increase in parotid blood flow are reputedly annulled byatropine (Kay, 1958).

Certain of these results have been published previously in a preliminary form(Andersson & Edwards, 1982).

METHODS

AnimalThe experiments were performed on pedigree Suffolk lambs 4-5 months after birth (14-35 kg body

weight). At least a week before any experiment the animals were brought into the laboratory animalhouse, where they were kept in individual pens and maintained on a diet of hay ad lib and 303 AngliaSheep Nuts (BOCM Silcock; 200 g/day). Food but not water was withheld for at least 14 hr beforeeach experiment.

Surgical and experimental proceduresThe animals were anaesthetized with chloralose (80 mg/kg i.v.) following cannulation of the

jugular vein under local anaesthesia. One or other parotid duct was then exposed and cannulatedwith narrow borepolythene tubing. The ipsilateral parotid nerve was dissected free from the duct,ligated, and the peripheral end attached to a fluid electrode. Whenever measurements of parotidblood flow were required, the ipsilateral maxillary vein was ligated just above a convenient parotidtributary and then cannulated below it with a short length of polythene tubing, having first tiedoff any other tributaries entering this segment of the maxillary vein and administered heparin atan initial dose of 1000 i.u./kg i.v.

Parotid blood and salivary flow were recorded continuously by means of photoelectric dropcounters and also measured gravimetrically at intervals. Venous effluent blood in excess of thatrequired for assays was returned to the animal through a cannula inserted into the right femoralvein, by means of a Harvard peristaltic pump, the speed of which was adjusted from time to timein order to match input to output. A second Harvard peristaltic pump was employed to infuseartificial saliva i.v.; Krebs Ringer-bicarbonate solution (Long, King & Sperry, 1968) continuallygassed with 95

%

02/5% C02, at a rate which matched the estimated loss of saliva from both parotidglands. A further polythene catheter was inserted into the right femoral artery and situated so thatthe tip lay in the abdominal aorta. This was used subsequently to monitor heart rate and aorticblood pressure by means of a Devices L221 pressure transducer connected to a Devices M19

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Recorder, and for collection of arterial samples. When required atropine (atropine sulphate B.D.H.)was given by i.v. injection (090% (w/v) NaCl, 0 1 g/100 ml.) at an initial dose of 1P0 mg/kg.

In each experiment a standard 20-30V square-wave stimulus (pulse width 0 5 msec) wasemployed. In one series of experiments the effects of stimulation at 10, 20 and 40 Hz in 1 see burstsat 10 see intervals for 10 min were compared with those following corresponding constant rates ofstimulation (1, 2 and 4 Hz) required to deliver the same total number of impulses over the same10 min period. In a second series the effects of stimulation at various frequencies within the range2-200 Hz, delivered in 1 see bursts at 10 sec intervals for 2-3 min, were investigated. Finally theresponses to stimulation of the parotid nerve continuously at 20 Hz for 10 min were examined inthe presence and absence of atropine.

Particular care was taken to avoid effects which might be attributable to 'spread of stimulus'.Heart rate and aortic blood pressure were continually monitored during each experiment and amean aortic blood pressure in excess of 80 mm Hg was ordained to be a prerequisite forcontinuation. No consistent change in heart rate, blood pressure, or any activity of the facialmusculature was associated with parotid nerve stimulation in any of the experiments that arereported in this study.

EstimationsSamples of arterial and parotid venous blood were collected into chilled, heparinized tubes

containing aprotinin (Trasylol, Bayer: 1000 KIU/ml. blood) for peptide assays. These tubes werecentrifuged at + 4 'C without delay and the plasma then sequestered at -20 'C. Substance P (SP)was measured by radioimmunoassay using antisera raised to synthetic SP, coupled by glutar-aldehyde to bovine serum albumin in rabbits, the most avid of which (SP3) was usable at a finaltitre of 1: 8000; synthetic tyrosine 8-SP was used for iodination. In this assay changes of 5 p-mole/l.plasma between single samples could be detected with 950% confidence. No significant crossreactivity with any other regulatory peptide, including VIP and somatostatin (SRIF) wasdetectable. Somatostatin was measured using an antiserum raised in rabbits to the synthetic ovine14-amino-acid peptide coupled to bovine serum albumin by carbodiimide condensation. Theantiserum used, at 1 in 240,000 dilution with TyrI 11251 label, showed no cross-reaction with otherregulatory peptides or fragments of somatostatin but reacted fully with somatostatin 28. The assaycould detect changes in plasma of 3 p-mole/l. with 95% confidence.VIP was also measured by radioimmunoassay (Mitchell & Bloom, 1978). The arterial and parotid

venous haematocrit was monitored at intervals. Parotid vascular resistance was calculated bydividing the perfusion pressure (taken as mean aortic blood pressure) by the parotid blood flow.Parotid salivary Na+ and K+ concentrations were measured by means of a Corning 435 FlamePhotometer.

Statistical significance was determined by Student's t or by the paired t test where appropriate(Snedocor & Cochran, 1967).

RESULTS

Comparison ofparotid responses to continuous stimulation of the parotid nervefor 10 minwith stimulation in 1 sec bursts at 10 sec intervals for the same periodContinuous stimulation of the parotid nerve at 1 Hz for 10 min in six weaned lambs

caused a small but significant increase in the flow of parotid saliva and in the Na+ andK+ output from the gland. Precisely the same number of impulses delivered in 1 see10 Hz bursts at 10 see intervals significantly enhanced each of these three responses(P < 0 01; Fig. 1 A). Comparison of the effects of continuous stimulation at 2 Hz for10 min with the corresponding frequency in 1 see bursts at 10 see intervals (20 Hz)revealed the same enhancement of each response during intermittent stimulation, asis shown in Fig. 1B (parotid salivary flow: P < 0-02; parotid salivary Na+ output:P < 0-02; parotid salivary K+ output: P <00 1).The output of K+ in the parotid saliva was also significantly increased by inter-

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P.-O. ANDERSON, S. R. BLOOM AND A. V. EDWARDS

mittent stimulation at an even higher frequency (40 Hz, for 1 see at 10 sec intervals),above that which was recorded during stimulation ofthe parasympathetic innervationat the corresponding continuous frequency (4 Hz). In contrast, the changes in boththe flow of parotid saliva and the output of Na+ were closely similar in the same twogroups (Fig. 1C).

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Fig. 1. Comparison of the changes in parotid salivary flow, Na+ and K+ output in responseto stimulation of the parotid nerve either in 1 see bursts at 10 see intervals for 10 min (0)or at the corresponding continuous frequency for the same period (0) in six weaned lambs.Horizontal bars: duration of stimulation. Vertical bars: S.E. of each mean value whereverthis exceeds the size of the symbol. A: 10 Hz in bursts and 1 Hz continuously. B: 20 Hzin bursts and 2 Hz continuously. C: 40 Hz in bursts and 4 Hz continuously.

It proved impossible to collect the whole of the parotid venous effluent in theseexperiments as the isolation of a convenient parotid venous tributary necessitateddissection of and therefore some damage to the gland. This was reflected by a steadyincrease in the K+ output in the parotid saliva presumably because the venous

drainage from part of the gland was occluded and that part became progressivelyhypoxic. Accordingly, the parotid salivary responses of lambs used for vascularstudies have been excluded from consideration. The vascular responses are expressedas percentage changes and relate to responses from an indeterminate part ofthe gland.They were, however, highly reproducible over prolonged periods and can thereforebe supposed to provide an accurate index of vascular changes that would normallyoccur in the whole gland in response to stimulation of the parasympatheticinnervation.

The same protocols that were employed to investigate the parotid salivary responsesshowed that parotid vascular responses were consistently significantly increased bystimulation in bursts (Fig. 2). Thus the fall in parotid vascular resistance at 21 min was20-5 + 4-6 % during stimulation in bursts at 10 Hz and 8-5 + 2-2% during stimulationat 1 Hz continuously, 40-4+ 7-1 % with bursts at 20 Hz and 18-7 + 4-8 % duringstimulation at 2 Hz continuously, 47.9+ 6-9% with bursts at 40 Hz and 34-3 + 7 9%

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PAROTID SECRETORY RESPONSES

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Fig. 2. Comparison ofthe changes in parotid vascular resistance in response to stimulationof the parotid nerve at 1 Hz (A), 2 Hz (B) or 4 Hz (C), continuously for 10 min withstimulation at 10 Hz (A), 20 Hz (B) and 40 Hz (C) for 1 sec at 10 sec intervals for the sameperiod in weaned lambs. Horizontal bar: duration ofstimulation. Vertical bars: S.E. ofeachmean value. n = 8 (A), 5 (B), 6 (C).

during stimulation at 4 Hz continuously (P < 0-02, 0.1 and 0.01 respectively usingthe paired t test).

Parotid responses to stimulation of the parotid nerve for 1 sec at 10 sec intervals, atdifferent frequencies

Stimulation of the peripheral end of the parotid nerve in 1 sec bursts at 10 secintervals for 2-3 min, and over a wide range of frequencies, showed that the outputof both Na+ and K+, together with the flow of parotid saliva, increased steadily withincreasing stimulus frequency between 2 and 80 Hz. None of these responses wassignificantly reduced by further increasing the stimulus frequency between 80 and200 Hz (Fig. 3). A closely similar relation was observed between the change in parotidvascular resistance and stimulus frequency under the same conditions.

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P.-O. ANDERSON, S. R. BLOOM AND A. V. EDWARDS

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Fig. 3. Relation between parotid vascular resistance, salivary flow, Na+ and Ka+ outputand stimulus frequency during stimulation of the parotid nerve in 1 sec bursts, at 10 secintervals, for 2-3 mmn in four weaned lambs. Vertical bars: S.E. of each mean value.

Parotid responses to stimulation of the parotid nerve at 20 Hz continuously for 10 mmnin the presence and absence of atropineThe finding that the parotid vasodilator response to stimulation of the parasym-

pathetic innervation resembles the submaxillary vasodilator response of the atro-pinized cat, in that it is significantly potentiated by intermittent stimulation at highfrequency, prompted us to re-examine the contention that it is atropine-sensitive(Comline &; Kay, 1955; Kay, 1958). This was done by comparing the secretory andvasodilator effects of continuous stimulation of the parotid nerve at a relatively highfrequency (20 Hz) for 10 mmn in the presence and absence of a high dose ( > 1 0 mg/kg)

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PAROTID SECRETOR Y RESPONSES 169

of atropine and measuring the arterio-venous differences in the concentrations ofcertain peptides that are appropriate candidate transmitters to account for non-cholinergic, non-adrenergic responses in salivary glands.

Atropine effectively abolished the parotid secretary responses to stimulation of theperipheral end of the parotid nerve under these conditions, confirming the fact that

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Fig. 4. Comparison of the changes in parotid salivary flow and vascular resistance in eightweaned lambs in response to stimulation of the parotid nerve at 20 Hz continuously for10 min in the presence (0) and absence (0) of atropine (> 1-0 mg/kg). Horizontal bar:duration of stimulation Vertical bars: S.E. of each mean value.

they are entirely dependent upon activation of muscarinic receptors (Fig. 4). In thesame experiments prior adminstration of atropine merely reduced the fall in parotidvascular resistance, showing that some mechanism other than activation ofmuscarinicreceptors is implicated in this particular response to stimulation of the parotidparasympathetic innervation (Fig. 4). There were no significant changes in thedifferences between the mean concentrations of VIP, substance P or somatostatinin the parotid arterial and venous plasma, in response to stimulation of the parotidnerve, in either the presence or absence of atropine (Fig. 5).

P.-O. ANDERSON, S. R. BLOOM AND A. V. EDWARDS

DISCUSSION

It has been claimed that the parotid vasodilator response to stimulation of theparasympathetic innervation to the gland in anaesthetized sheep is 'annulled byatropine' (Kay, 1958). In that study stimulation comprised delivery of ca. 70 pairsof impulses/sec, using either a Palmer induction coil or a neon-tube stimulator, andseems to have been applied for less than 1 min on each occasion. In the present

50

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Fig. 5. Mean parotid arterio-venous differences in the plasma concentrations of vaso-active intestinal peptide (VIP), substance P (SP) and somatostatin (SRIF) in response tostimulation of the parotid nerve at 20 Hz continuously for to min in weaned lambs (n = 8)in the presence (@) and absence (0) of atropine (> 1l0 mg/kg). Horizontal bar: durationof stimulation. Vertical bars: s.E. ofeach mean value wherever these exceed the size of thesymbol.

experiments, continuous stimulation of the parotid nerve at 20 Hz for a much longerperiod (10 min) shows that the parotid vasodilator response, as reflected by the fallin parotid vascular resistance, is not annulled, although it is substantially reduced,by prior administration of atropine (> 1-0 mg/kg). We do not suggest that thisfinding has any particular physiological significance, but just that it shows that theresponse is not completely 'atropine-sensitive', as mechanisms other than activationofmuscarinic receptors clearly contribute to it under certain experimental conditions.The absence of any significant change in the concentration of VIP in the

parotid venous effluent plasma, and consequently in the arterio-venous plasmadifference, indicates that the fall in vascular resistance which occurs in the presenceof atropine cannot be attributed to release of this peptide, as it can in thesubmaxillary gland of the cat (Bloom & Edwards, 1980; Lundberg et al. 1980). Thetime relations of the atropine-resistant vascular responses in the two glands are alsoentirely different during stimulation of the parasympathetic innervation at the same

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frequency and for the same period (20 Hz for 10 min). In the parotid gland of thesheep the response develops gradually over 1-2 min and often persists for more than60 sec after stimulation of the parotid nerve has been discontinued. In thesubmaxillary gland of the cat the vasodilator response is evident within a few secondsand declines with equal rapidity when the stimulus is withdrawn (Bloom & Edwards,1980). Nor is it significantly reduced below that which occurs in the absence ofatropine as it is in the parotid gland of the sheep. The sluggish nature of this parotidvascular response suggested the possibility that it might be due to the gradualaccumulation, and relatively persistent effect, of some enzyme such as kallikrein,which was once thought to account for that atropine-resistant vasodilator responsein the submaxillary gland of the cat (Hilton & Lewis, 1955a, b, 1956). This suggestionis supported by the preliminary finding that the parotid vascular response tostimulation of the parasympathetic innervation, which persists in the presence ofatropine, is progressively reduced during continuous intra-arterial infusions ofaprotinin which would inhibit the formation of kinins such as bradykinin within thegland (A. V. Edwards, unpublished observations). However, it is difficult to reconcilethis proposition with the finding that the parotid gland of the sheep, unlike othersalivary glands in various species, normally contains little or no kallikrein (Werle,1937). We conclude that the evidence at present available is insufficient to determinethe precise mechanism underlying this response.The object of the present study was to investigate the effects of intermittent

stimulation at relatively high frequencies, ofparasympathetic neurones, the responsesto which could be attributed to activation ofmuscarinic receptors, in order to discoverwhether or not this pattern of stimulation enhances those responses as it doesresponses mediated by nicotinic or peptidergic receptors in certain other tissues(Edwards, 1981; Andersson et al. 1982). This objective was realised in the case ofthe parotid salivary responses, and the ouput of both Na+ and Ka+, together withthe flow of saliva, were all significantly increased by stimulation of the parotid nervein 1 sec bursts at 10 sec intervals, at both 10 and 20 Hz, above the responses obtainedduring stimulation at the corresponding continuous frequencies (1 and 2 Hzrespectively). Furthermore each response increased steadily with increasing stimulusfrequency up to 80 Hz, during stimulation in bursts, as did the vasodilator responseunder the same conditions. Presumably autonomic responses reach their maxima atmuch lower frequencies during continuous stimulation (Rosenblueth, 1932; Celander,1954) because the effector cells rapidly become saturated with transmitter; when thefibres are stimulated for a comparatively small fraction of the total time available,as for 1 sec in every 10, the effector cells are able to follow the frequency of activityin the innervation more faithfully.The fibres in the parotid nerve of the sheep are known to be post-ganglionic

(Kay, 1958) and so the enhancement of parotid responses to intermittent stimulationat high frequency cannot be attributed to facilitation of ganglionic transmission. Thefurther question arises as to whether these fibres fire intermittently and at such highfrequencies in the normal conscious animal. The formidable technical difficultiesassociated with the direct recording of activity in single post-ganglionic neurones inthe absence of anaesthesia have so far been overcome only in the case of superficialsympathetic fibres in human subjects. However, single units in the skin have been

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P.-O. ANDERSON, S. R. BLOOM AND A. V. EDWARDS

shown to fire in bursts at instantaneous maximal frequencies of up to 35 Hz (Hallin& Torebj6rk, 1974) and small bundles of human sympathetic post-ganglionic fibresto both skin and muscle characteristically fire in bursts (Hagbarth & Valbo, 1968;Delius, Hagbarth, Hongell & Wallin, 1972a, b, c; Hagbarth, Hallin, Hongell,Torebj6rk & Wallin, 1972; Sundlof & Wallin, 1978; Bini, Hagbarth, Hynninen &Wallin, 1980).

In the case of the parasympathetic system the only records of the activity inconscious animals are those of vagal units obtained by the ingenious, but indirect,technique of Miolan & Roman (1973, 1974) in conscious dogs. These workers reportedthat most of these fibres appear to discharge tonically at low frequencies (<5 Hz),although the activity was found to wax and wane and frequencies as high as 40 Hzwere recorded during gastric relaxation. In anaesthetized animals the dischargefrequency may well be reduced. Even so, vagal units that discharge in bursts atfrequencies of up to 30 and 45 Hz have been described in the rat and the sheeprespectively (Davison & Grundy, 1978; Iggo & Leek, 1967). The activity of thepost-ganglionic parotid neurones in the anaesthetized sheep has also been shown tobe irregular, and spikes regularly appear as doublets or triplets; the interval betweendoublets varies between 8-6 and 14-4 msec (Carr, 1977), equivalent to a maximalinstantaneous frequency of 68-116 Hz. Over longer periods the discharge frequencyof these units increased to over 20 Hz which corresponds precisely with the frequencyrange over which we have found the responses of this gland to be enhanced duringintermittent stimulation. The reflex discharge of E-type units in the chorda tympaniofrabbits anaesthetized with chloralose and urethane, during stimulation of the upperlip, is highly irregular and reaches peak frequencies of 120 Hz over very short periods(Six impulses in 50 msec; Kawamura, Matsua & Yamamoto, (1982). Finally, studiesof the activity of single neurones in Auerbach's plexus have shown that one typecharacteristically discharges in bursts (Wood, 1970, 1973). In cat jejunum theseneurones fired spontaneously in groups of two to thirty-four spikes at a frequencyof 30 Hz with intervals of 0-5-40 msec between the bursts, thus mimicking thepattern we chose to impose in the parotid nerve quite precisely. Wood distinguishedtwo types of such neurones in Auerbach's plexus: one which appeared to beintrinsically driven and another which appeared to be synaptically driven (Wood,1975).The present findings show that at least some muscarinic responses resemble

responses that have been described previously, but which are mediated by activationof other types of receptor, in that they are potentiated by intermittent stimulationat relatively high frequencies. The evidence at present available indicates that thispotentiation occurs at frequencies well within the physiological range.

This work was supported by grants from the British and Swedish (2210) Medical Research Councilsand the Wellcome Trust. It is a particular pleasure to acknowledge the skilled technical assistanceprovided by Miss B. N. O'Connor, Mr P. M. M. Bircham and Mr G. P. MacGregor.

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