The digastric reflex evoked by tooth-pulp stimulation in the cat and its modulation by stimuli applied to the limbs

Brain Research, 336 (1985) 33-43 Elsevier

33

BRE 10775

The Digastric Reflex Evoked by Tooth-Pulp Stimulation in the Cat and its Modulation by Stimuli Applied to the Limbs SAMUEL W. CADDEN Department of Physiology (Oral Biology), The Medical School, University Walk, Bristol BS81 TD (U. K.) (Accepted August 28th, 1984) Key words: digastric reflex - - tooth-pulp - - trigeminal system - - inhibition

The digastric reflex evoked by electrical stimulation of tooth pulp in anaesthetized cats was studied together with the effects on this reflex of stimulating other parts of the body. The threshold for the digastric reflex generally lay in the range of stimulus intensities which would excite a large proportion of the pulpal afferent fibres which suggested that a large amount of central summation was required to evoke the reflex. During the courseof 25/27 experiments, the threshold for the reflex increased. It was also found that repeated application of suprathreshold stimuli produced first an increase and then a decrease in the reflex response. The application of noxious but not of non-noxious mechanical conditioning stimuli to the limbs produced strong, long-lasting depressions of the digastric reflex. Electrical conditioning stimuli applied to the limbs also depressed the reflex; this depression had a latency of onset of 20-50 ms and lasted for up to 500 ms. When conditioning stimuli were applied to the saphenous nerve, the depression of the reflex occurred only when the stimuli were of an intensity sufficient to excite fibres conducting at less than 40 m.s-1; it may be assumed that some of these fibres would have been high threshold mechanoreceptors or nociceptors. These results show that noxious stimulation of anatomically remote structures can depress the activity of a population of trigeminal brainstem neurones. The opiate antagonist, naloxone, had no detectable effect on either the digastric reflex or the depression of the reflex produced by stimulating other parts of the body. The serotonin antagonists, methysergide and cinanserin, strongly depressed the digastric reflex but it was not clear whether these drugs also affected the depression of the reflex by the conditioning stimuli.

INTRODUCTION In a previous study in this l a b o r a t o r y , it was found that, in o r d e r to e v o k e some responses in trigeminal brainstem n e u r o n e s in the cat by electrical stimulation of tooth-pulp, it was necessary to apply electrical stimuli of intensities which would excite the vast majority of the m y e l i n a t e d pulpal afferent fibres 7. It was clear from the latencies of these responses that they were being m e d i a t e d via the m y e l i n a t e d afferent fibres and it therefore s e e m e d likely that a large amount of central s u m m a t i o n was r e q u i r e d to excite the brainstem neurones. Subsequent experiments were carried out to investigate the possibility that this need for s u m m a t i o n was the result of some depression of activity in the trigeminal b r a i n s t e m nuclei. In these experiments, the digastric, or jaw-opening, reflex e v o k e d by t o o t h - p u l p stimulation was used as a

means of monitoring the excitability of a population of trigeminal b r a i n s t e m neurones u n d e r different experimental conditions. A t an early stage, it was found that this reflex could be depressed by sensory inputs arising elsewhere on the body, including those inevitably p r o d u c e d by the surgical p r e p a r a t i o n of the animals. The experiments r e p o r t e d in this p a p e r were p e r f o r m e d to further investigate this type of modulation of the digastric reflex. D a t a relating to some general properties of the reflex will also be presented. Preliminary reports of the results of some of these experiments have been given elsewhereS,6. MATERIALS AND METHODS The experiments were p e r f o r m e d on 28 young adult cats ( 8 - 1 2 months old) ranging in weight from 2.2 to 4.5 kg. A n a e s t h e s i a was induced by an intra-

* Correspondence: S. W. Cadden. Present address: Department of Dental Surgery, The Dental School, Park Place, Dundee, U.K. 0006-8993/85/$03.30 (~ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

34 muscular (24 mg'kg -1) or intravenous (9 mg'kg -1) injection of a mixture of alphaxalone and alphadolone acetate (Saffan, Glaxo Laboratories) and was maintained by a constant infusion (512 mg'kg-l'h -1) of the mixture through a cannula placed in the cephalic or great saphenous vein. The depth of anaesthesia was that which would allow only a very weak flexion withdrawal reflex in response to toe pinch. Intravenous administration of Saffan consistently produced a rise in end-tidal CO2 and it was possible to estimate the stability and depth of anaesthesia by monitoring end-tidal CO2. Saffan was chosen as an appropriate anaesthetic since previous experiments in this laboratory had shown that, in cats anaesthetized with Saffan, it was possible to evoke a digastric reflex by applying to canine tooth-pulp relatively small electrical stimuli corresponding to the thresholds of the lowest threshold pulpal afferent fibres 34,35. Furthermore, in a preliminary series of experiments, it was found that the threshold for the reflex was generally lower in animals anaesthetized with Saffan than in animals anaesthetized with a variety of other agents (urethane, nitrous oxide/halothane, sodium pentobarbitone/a-chloralose). After induction of anaesthesia, cannulae were inserted into the cephalic or the great saphenous vein, the femoral artery and the trachea. In some experiments in which drugs in addition to the anaesthetic were given, these were administered using a second venous cannula in either the cephalic or great saphenous vein, in order to avoid a change in the anaesthetic infusion rate through the other venous cannula. Arterial blood pressure was recorded via the cannula in the femoral artery and end-tidal CO 2 was monitored continuously. Body temperature was maintained at 37 + 0.5 °C by an electric blanket thermostatically controlled from a rectal thermistor. In three experiments the animals were paralyzed with pancuronium bromide (100#g'kg -1 i.v.) and artificially ventilated with room air: in these experiments, the electrocardiogram was also monitored. In most experiments, the head was stabilized by means of a bar which was attached to the skull over the frontal sinus with self-tapping screws and self-curing acrylic resin. The lower jaw was fixed to the upper with the teeth apart by wire staples inserted through holes drilled in the crowns of the molar teeth and reinforced with acrylic resin. In a few experiments, no at-

tempt was made to stabilize the head and jaws, nor were cannulae inserted into the femoral artery or the trachea, for fear that these procedures and the associated surgical trauma may have been influencing the results of the experiments (see Introduction). However, the results obtained from the two types of preparation were essentially the same.

Stimulatingprocedures Two silver/silver chloride electrodes were applied into dentine of one or both upper canine teeth to allow the application of electrical stimuli to tooth-pulp. The design and preparation of these electrodes have previously been described 8. Square-wave constant current stimuli of 0.1 or 1.0 ms duration and of up to 1 mA intensity were applied using an optically isolated stimulator. It had previously been shown that stimuli of up to 1 mA, 1 ms could be applied with this electrode configuration without danger of exciting any extradental nerve fibres 8. In two experiments, the reflex was also studied using mechanical stimulation of the teeth; accurately controlled stimuli were applied using an electromechanical transducer (LTV, Ling Altec, Type V47) and were monitored using a strain gauge interposed between the transducer and the applicator tip of the instrument. The tip of the instrument was attached to the labial surface of the tooth crown with dental 'sticky wax' and etch-bonded dental composite resin. Stimuli of up to 200 g force were applied in a palatal direction. Evidence has been presented that the jaw opening reflex evoked in this fashion is mediated by receptors in the periodontal ligament 2°. In the present study, it is likely that these stimuli were in the non-noxious range since a reflex response of similar magnitude could be obtained by gently tapping the tooth with a hand-held instrument. In addition to these test stimuli, conditioning stimuli were applied to other parts of the body. These consisted of either a sustained toe pinch, electrical stimulation of the skin or electrical stimulation of the exposed saphenous nerve. Square-wave constant current electrical stimuli of 1 ms duration and up to 10 mA intensity were applied to the skin (usually of a hindpaw) through a pair of uninsulated steel needles (0.6 mm diameter, inter-electrode distance 20-40 mm). In order to stimulate the saphenous nerve, a 10-mm length of the nerve was exposed

35 through a skin incision, dissected free with its vascular connective tissue sheath intact and isolated underneath with a strip of polythene sheet. A pair of platinum wire electrodes (0.15 mm diameter, inter-electrode distance 3 mm) were then inserted under the nerve. The exposed nerve was covered with liquid paraffin. Square-wave constant voltage stimuli of 0.1 ms duration and up to 5 V intensity were applied to the saphenous nerve. It should be noted that none of these conditioning stimuli ever produced activity in the digastric muscles, although it was found, in all the experiments, that the conditioning stimuli did cause reflex activity (latency, approx. 50 ms) in the adjacent facial muscles of the lower lip.

Recording techniques Recordings were made from both muscles and peripheral nerves using a low noise amplifier21 with a bandpass of 10 Hz-1 kHz or 10 kHz. The electromyogram (EMG) of the digastric muscle ipsilateral to the stimulated tooth was recorded with a pair of copper wire electrodes (0.25 mm diameter) insulated except for their terminal 5 mm which were inserted transcutaneously into the muscle. In some experiments recordings were also made from the contralateral digastric muscle. In the 3 experiments in which the animals were paralyzed, the reflex was recorded using platinum wire electrodes (0.15 mm diameter) applied to the central cut end of the mylohyoid nerve. This nerve was exposed between the mandibular foramen and the lower border of the mandible by reflecting the skin overlying the ramus and posterior part of the body of the mandible, excising the masseter muscle and removing bone from the mandible. A pool was prepared with the skin flaps and was filled with liquid paraffin. In the experiments in which conditioning stimuli were applied to the saphenous nerve, the evoked compound action potential was recorded from the intact nerve, 35-60 mm central to the stimulating site. Platinum wire recording electrodes were applied in the same way as those for stimulation. The liquid paraffin around the nerve was held at 37 + 0.2 °C using a radiant heat source controlled from a thermistor close to the nerve. Using this preparation, stimulusresponse relationships were determined for the main Aft and A6 components of the compound action potential. Ten successive responses to a given stimulus

were averaged, full-wave-rectified and the integrals of the Aft and At$ components of the compound action potential determined by summation between set points on the waveform (see Fig. 3) using a PDP 11 computer with A R l l interface (Digital Equipment Corporation).

Determination of digastric reflex threshold In preliminary experiments it was found that repeated application of suprathreshold stimuli generally produced changes in the digastric reflex response (see Results). In order to eliminate the effect of these sometimes very large changes in excitability, the reflex threshold was determined by applying stimuli once every 4 s and increasing the stimulus intensity until a response was observed. This stimulus intensity was taken to be threshold. Thresholds were determined routinely using single 0.1 ms stimuli and also, in some experiments, using single 1 ms stimuli or double-shock 0.1 ms stimuli (4 ms separation).

Conditioning-test procedures To quantify the effects of the electrical conditioning stimuli on the digastric reflex, the following procedure was adopted. The reflex was evoked by applying, once every 4 s, test stimuli which were generally 1.5-3 times (and never more than 7.5 times) the threshold for the reflex. It may be noted that, at these stimulus intensities, the responses were submaximal and were little affected, if at all, by the repetitive stimulation rate of once every 4 s (see above and Resuits). As a control, 5 or 10 successive responses were averaged, full-wave-rectified, and the integrals determined as for the compound action potentials (see above). This procedure was immediately repeated using both test and conditioning stimuli and then again using the test stimulus alone (control). This sequence was performed using various conditioningtest (C-T) intervals between 5 and 1000 ms. The integrals of the conditioned responses were expressed as percentages of the means of the two control values obtained immediately before and after.

Administration of drugs In eight experiments drugs were administered intravenously in an attempt to influence the effects of the conditioning procedures described above. The drugs employed were the opiate antagonist, nalox-

36 one, and the serotonin antagonists, methysergide and cinanserin. These drugs were chosen since, in previous studies: (i) the effects of similar conditioning stimuli on dorsal horn neuronal activity had been reduced both by naloxone 28 and by serotonin antagonists10; and (ii) the effects of other conditioning procedures on the digastric reflex had been reduced by naloxone 39 (see Discussion). RESULTS

General properties of the digastric reflex evoked by tooth-pulp stimulation A digastric reflex evoked by electrical stimulation of tooth-pulp was recorded either from the muscle (Figs. 1-3) or from the mylohyoid nerve (Fig. 5) in all the animals. The latency of the muscle response ranged from 6.5 to 9.5 ms. When recording from both digastric muscles, the ipsilateral response generally had a lower threshold, and with suprathreshold stimuli was of greater amplitude, than the contralateral response. The bilateral nature of the reflex was confirmed in the recordings from the mylohyoid nerve in paralyzed animals. Furthermore, the reflex could also be produced by simultaneous application to both upper canine teeth of stimuli which were subthreshold when applied alone. A wide range of thresholds for the reflex was obtained and it was found that the threshold increased markedly during the course of 25 out of 27 experiments. With single 0.1 ms stimuli, the threshold for the ipsilateral response at the start of the recording sessions ranged from 12 to 500/~A (mean 79.9 ~A; S.D., 104.8/~A; n = 26), whereas towards the end of 9 of these experiments (and in the absence of any pharmacological tests) the reflex could not be obtained even with a 1 mA 0.1-ms stimulus. It must be emphasized that these rises in threshold were not accompanied by any deterioration in the condition of the animals as far as could be determined by the monitoring of arterial blood pressure, end-tidal CO2 and body-temperature. In 22 of the experiments, the thresholds for single and double-shock 0.1-ms stimuli were compared and in every case, it was found that the thresholds for double-shock stimuli were lower. For example, at the start of the recording sessions in these experiments, the mean threshold for double-shock stimuli was

24.3/zA (S.D. 16.6 p A ) while that for single stimuli was 84.9 p A (S.D. 112.0 pA). Repeated application of suprathreshold stimuli always caused changes in the magnitude of the reflex. These generally consisted of an initial increase followed by a decrease in the response. For example, when stimuli of 2 - 3 times threshold were applied once per second, the integral of the evoked response would generally increase several-fold during the first minute or two of stimulation and then gradually decline over the next 5-10 min until the response was virtually abolished. Under these circumstances, a response similar in magnitude to that obtained initially, could generally be evoked after a further period o f 10-20 min during which no stimuli were applied. It was because of these changes brought about by repetitive stimulation, which were more marked at higher frequencies of stimulation, that stimuli were always applied as conservatively as possible and generally not more frequently than once every 4 s; it was also for this reason that, when quantifying the effects of a conditioning procedure, the responses were compared with controls performed both immediately before and after. The reflex could also be depressed by increasing the depth of anaesthesia.

Effects of conditioning stimuli In preliminary experiments, the effects on the digastric reflex of pinching a hindpaw were studied. In the example shown in Fig. 1, the reflex evoked by a test stimulus of approximately 2 x threshold, was virtually abolished during a strong toe pinch and was still diminished for several minutes after the pinch had been released. In such experiments, the depression of the reflex was apparent only when the toe pinch was of an intensity which was clearly painful when applied in man. In subsequent experiments, this effect was investigated using electrical conditioning stimuli applied to the hindpaw and a typical set of records from such an experiment is shown in Fig. 2. The top record in Fig. 2A shows the average of 10 responses from the digastric muscle when test stimuli of 30/~A 0.1-ms (approx. 1.5 × threshold) were applied to the ipsilateral upper canine tooth and the subsequent records show the effects on this response of a single conditioning stimulus of 6 mA 1-ms applied to the contra-

37

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10ms Fig. 1. The effect of a sustained toe pinch on the digastfic reflex evoked by tooth-pulp stimulation. El¢ctromyographic recordings obtained from the digastn¢ muscle following the application of 25/~A, 0.1 ms stimuli to the JpsJlateral upper canine tooth. Each record shows 5 successiveresponsesto stimuli applied onceevery4 s.

lateral hindpaw at various intervals before the test stimulus. The reflex was diminished when the conditioning stimulus preceded the test stimulus by 50, 100 or 200 ms. These results are also shown graphically in Fig. 2B in which it can also be seen that the depression of the reflex generally decreased as the conditioning stimulus intensity was decreased. In addition it was found that, for a given conditioning stimulus, the depression of the reflex decreased as the test stimulus intensity was increased. In all 19 preparations in which conditioning stimuli were applied to a hindlimb or a forelimb in this fashion, the reflex was similarly depressed. In one of these experiments, conditioning stimuli were applied separately to the base of the tail and to the abdomen with essentially the same results. Trains of conditioning stimuli always produced a more marked depression of the reflex than did single stimuli and, in 2 preparations, the depression was detectable only when trains of stimuli were applied. The depression was usually obtained only with C-T intervals of 50-200 ms (Fig. 2) although in some experiments it was also found with intervals of 20 and/or 500 ms. In

9 experiments, it was also found that conditioning stimuli applied to the hindlimb could produce increases in the reflex response when C-T intervals of <50 ms or >200 ms were employed. This effect was greatest when the C-T interval was 20 ms and in 6 preparations, occurred with stimulus intensities which were insufficient to depress the reflex. However, in all but 3 of these 9 experiments, this effect was poorly reproducible. In order to determine which groups of nerve fibres had to be excited by the conditioning stimuli to produce the depression of the reflex, experiments were performed in which compound action potentials evoked by conditioning stimuli of different intensities applied to the saphenous nerve, were recorded. It was convenient to do this by first recording the compound action potentials to a range of stimulus intensities and then assessing the effects of these stimuli on the reflex. To ensure that a particular stimulus intensity would have the best chance of producing an effect, the conditioning stimuli were applied as trains (250 s-1) lasting from 200 ms until 50 ms before the application of the test stimulus to the tooth since, using single stimuli, the greatest depression of the reflex occurred with C-T intervals of 50-200 ms. Records from a typical experiment are shown in Fig. 3. Fig. 3A shows compound action potential recordings made from the saphenous nerve following the application of stimuli of various intensities to the nerve. The records in Fig. 3B were obtained from the digastric muscle following the application of test stimuli (approx. 1.5 x threshold) to the ipsilateral upper canine tooth. The top record in Fig. 3B shows the control response obtained when no conditioning stimuli were applied and the subsequent records show the effects on this response of applying to the saphenous nerve, conditioning stimuli of the same intensities as were used to produce the compound action potentials shown in Fig. 3A. It can be seen that the reflex was virtually unaltered by conditioning stimuli which excited only the lowest threshold Aft fibres. However, conditioning stimuli which excited more of the Aft fibres as well as some more slowly conducting fibres (conduction velocities 20-40 m's-l; Fig. 3A record second from top), did produce a depression of the reflex. When the conditioning stimuli were of an intensity sufficient to produce the main A6 component of the compound action potential (Fig. 3A, lower 2 re-

38

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Fig. 2. The effect of electrical conditioning stimuli applied to a hindpaw on the digastric reflex evoked by tooth-pulp stimulation. A: recordings of digastric EMG following the application of 30 g A 0.1-ms test stimuli to the ipsilateral upper canine tooth. Each record shows the average of 10 successive responses to stimuli applied once every 4 s. The top record is the control response when no conditioning stimuli were applied• The subsequent records were obtained when 6 mA 1-ms conditioning stimuli were applied to the contralateral hindpaw at various intervals (C-T intervals) before the test stimuli. B: graphic representation of results shown in A and of results obtained using lower intensities of conditioning stimuli. The digastric EMG recordings were averaged, full-wave-rectified and integrated, and the integrals obtained for responses following the application of conditioning stimuli are expressed as percentages of those obtained for responses to the test stimulus alone (see text).

cords) the reflex was more strongly depressed. Similar results were obtained in 6 other preparations and data from one of these are shown in Fig. 4. Fig. 4A shows stimulus-response relationships for the main Aft and A6 components of the saphenous nerve compound action potential. Not included on this graph are data for the intermediate group of fibres with conduction velocities between 20 and 40 m's -1 (see Fig. 3A). Fig. 4B shows the effects of conditioning stimuli of various intensities on the digastric reflex response to electrical stimulation (approx. 1.5 x threshold) of the ipsilateral upper canine tooth. Comparison of these graphs shows, again, that the digastric reflex was depressed by stimuli which excited a substantial proportion of the Aft fibres, although such stimuli would also have excited some of the intermediate group of fibres. Increasing the conditioning stimulus intensity to excite both the Aft and A6 fi-

bres produced a greater depression of the reflex. The effects of the various conditioning procedures on the digastric reflex evoked by mechanical stimulation of an upper canine tooth were also investigated in two experiments and essentially the same results as those for the reflex evoked by tooth-pulp stimulation, were obtained. To confirm that the effects of the conditioning procedures on the digastric reflexes evoked by electrical and mechanical stimulation of teeth were not dependent upon movement or upon feedback from muscle or joint receptors, 3 further experiments were performed in which the reflexes were recorded from the mylohyoid nerve in paralyzed animals. The results of these experiments were essentially the same as those obtained using E M G recordings and results from one such experiment are shown in Fig. 5.

39

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the threshold for the digastric reflex evoked by toothpulp stimulation. These depressions of the reflex became apparent with doses of approx. 0.14 m g ' k g -1 methysergide and 1.7 m g ' k g -1 cinanserin, each in-

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phenous nerve following the application of stimuli of various intensities, as shown to the fight of the records. Each record is the average of 10 successive responses to stimuli applied once a second. The arrows below the bottom record indicate the components of the wave-form which were integrated when determining the stimulus/response relationships for the A~ and A6 fibres (see text and Fig. 4). B: effect of conditioning stimuli applied to the saphenous nerve on the digastric reflex evoked by tooth-pulp stimulation. Each record shows the average of 5 successive responses in the digastric muscle to 150 gA, 1 ms test stimuli applied once every 4 s to the ipsilateral upper canine tooth. The top record is the control response when no conditioning stimuli were applied. The subsequent records were obtained when trains (250 s-l) of conditioning stimuli of the intensities used to produce the compound action potentials in A, were applied to the saphenous nerve. The conditioning stimuli were applied from 200 ms until 50 ms before the test stimulus.

Effectsofdrugs Naloxone The effect of the opiate antagonist, naloxone, was investigated in 2 experiments. It was found that doses of up to 2 mg" kg-1 administered as single intravenous injections, did not significantly affect either the threshold of the digastric reflex evoked by tooth-pulp stimulation or the depression of the reflex by the conditioning stimuli.

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averaged compound action potentials were full-wave-rectified and integrated, and the integrals expressed as percentages of the maximum obtained for each component. Abscissa as in B (see below). B: the effect on the digastric reflex evoked by test stimuli (T) applied to the ipsilateral upper canine tooth, of trains of conditioning stimuli of various intensities, applied to the contralateral saphenous nerve (250 s-1 from 200 ms until 50 ms before the test stimulus). The digastric EMG recordings were averaged, full-wave-rectified and integrated, and the integrals obtained for responses following the application of conditioning stimuli expressed as percentages of those obtained for responses to the test stimuli alone (see text).

40 A.

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Fig. 5. Recordings made from the mylohyoid nerve following the application of electrical (90/~A, 1 ms) test stimuli to the ipsilateral upper canine tooth-pulp (A) or mechanical test stimuli to the same tooth (B). The record at the bottom right showsthe time course and magnitude of the mechanical stimuli. Each record shows 5 successive responses to a test stimulus applied once every 4 s. The records in the middle row were obtained immediately after the corresponding records in the top row, and show the effect of trains of conditioning stimuli applied to the contralateral hindpaw (250 s-~ from 200 ms until 50 ms before the test stimulus). The records in the bottom row were obtained immediately after the correspondingrecords in the middle row. diovascular systems as judged by the cessation of spontaneous breathing (necessitating artificial ventilation) and marked decreases in blood pressure. The changes in the reflex responses produced by these drugs made it difficult to determine whether they also affected the pathways involved in the depression of the reflex by the conditioning procedures. After the administration of either drug, the reflex could still be depressed by the conditioning stimuli, but for conditioning stimuli of a given intensity, the effects appeared to be weaker. DISCUSSION

General properties of the digastric reflex The general properties of the digastric or jawopening reflex which were found in these experiments were consistent with those previously reported in that the reflex could be evoked both by electrical stimulation of tooth-pulp and by mechanical stimulation of teeth (for reviews see refs. 15 and 31) and was predominantly ipsilateral as originally described by Sherrington 40. A contralateral response could be recorded but this always had a higher threshold than the ipsilateral response as previously reported for the

reflex evoked by electrical stimulation of the face and oral mucous membrane in the dog24. It has also been reported that the reflex evoked by electrical stimulation of the inferior alveolar nerve in the cat can be depressed by repetitive stimulation19; in the present experiments, it was found that repeated stimulation generally caused first an increase in the reflex response and then a gradual decline until the response was less than its initial level. The latency of the digastric reflex evoked by toothpulp stimulation indicated that the afferent limb of the reflex involved myelinated fibres and the thresholds for the reflex generally lay in the range of stimulus intensities which would have excited a large proportion of these myelinated afferent fibres8. These findings suggest that the digastric reflex required the central summation which can result from synchronised activity in a large population of primary afferent fibres (cf. ref. 7) and this hypothesis was supported by the finding that the reflex could be produced by double-shock stimuli of very much smaller intensities than those of threshold single stimuli. Such a need for summation may also have explained why in some experiments performed in conjunction with the present series, it was found that digastric reflex activity could be produced by thermal stimulation of cat canine teeth only when the stimuli were sufficient to produce a large amount of synchronized activity in the intradental nerves and that such reflex responses were both weak and labile. The finding that the reflex threshold rose in the course of almost every experiment is consistent with previous findings in this laboratory and has been attributed to an accumulative effect of the anaesthesia 34. However, in view of the present findings, it seems possible that repeated stimulation of the teeth and other inputs to the central nervous system, such as those arising from the surgical preparation or the conditioning stimuli, may also have produced a longterm decrease in the excitability of some cells and thus a rise in the reflex threshold. This hypothesis is consistent with the fact that the lowest thresholds for the digastric reflex evoked by tooth-pulp stimulation which have been obtained in this laboratory, were obtained in experiments in which cats with chronically implanted tooth electrodes were anaesthetised with Saffan and then, without any surgical preparation on the day of the experiment, the reflex thresh-

41 old was determined immediately34,as. It is also possible that experimental procedures may have been responsible, at least in part, for the high thresholds of unitary responses in the brainstem 7 referred to in the Introduction since in those experiments, recordings were made only after several hours of surgical preparation and repeated applications of intense electrical 'search' stimuli to tooth-pulp.

Effects of conditioning stimuli The majority of the data collected in the present experiments were concerned with the effects on the digastric reflex of applying conditioning stimuli to other parts of the body. It was found that the application of either noxious mechanical or electrical conditioning stimuli could produce a depression of the reflex evoked by electrical stimulation of tooth-pulp. This finding is consistent with some early studies of the digastric reflex evoked by electrical stimulation of the tongue in which it was found that this reflex was depressed by repetitive stimulation of the sciatic nerve3,25. In view of the finding in some of the present experiments that the conditioning stimuli could produce both a depression and a facilitation of the reflex, it is also of interest that Cardot and his colleagues 9 in 1923 reported that the reflex evoked by stimulation of the tongue in dogs, was generally facilitated by stimulating the skin of the hindlimb or the sciatic nerve but that, under certain circumstances, these stimuli inhibited the reflex. The depression of the reflex in the present experiments is also similar to the previously reported effects of anatomically remote electrical stimuli, on the digastric reflex evoked by electrical stimulation of the inferior alveolar nerve in the cat 26 and of various orofacial structures including tooth-pulp in the rat 42. In this last study 42, Tal and his colleagues reported that electrical stimulation of the limbs was followed first by a brief period of facilitation and then by a longer period of inhibition of the digastric reflex which again is similar to the findings in some of the present experiments. However, as acknowledged by these authors 42 and others22, 23, electrical stimulation of tooth-pulp in the rat generally results in excitation of extradental (periodontal) as well as intradental (pulpal) nerve fibres and reflex responses to such stimuli must therefore be interpreted with caution. For this reason, Tal and his colleagues 42 also made a brief reference to experiments which

they had performed on the digastric reflex evoked by tooth-pulp stimulation in the cat and in which they had obtained essentially similar inhibitory, but apparently not facilitatory effects. Finally, the present findings are compatible with the results reported by several groups who have used the digastric reflex as a model for studying the effects of acupuncture-like procedures in animals 16-18, although in the context of these studies, it should be noted that there is some dispute as to the effectiveness of acupuncture-like stimulation of the limbs, in relieving dental pain in man (see ref. 1). It has been reported that the digastric reflex evoked by electrical stimulation of tooth-pulp in the cat can be depressed by stimulation at selected sites in the brainstem (e.g. periaqueductal gray matter (PAG), nucleus raphe magnus (NRM); see e.g. refs. 32, 33, 38, 39). The time course of the effects of PAG or NRM stimulation reported by Sessle and his colleagues38,39 was similar to that reported for the phenomenon studied in the present experiments and these authors also reported that the period of depression of the reflex was sometimes preceded by a period of facilitation which is again consistent with the present findings. In view of these similarities, one might propose that the effects of the conditioning stimuli in the present experiments, were mediated by neural pathways involving the PAG and/or NRM. However, this hypothesis is not supported by the finding that the effects of PAG or NRM stimulation on the digastric reflex could sometimes be blocked by the opiate antagonist naloxone 39 since in the present experiments, naloxone had no effect,on the depression of the reflex. The present findings may also be related to those from some studies on neurones in the trigeminal sensory nuclei. It has been reported that stimuli applied to remote areas of the body can inhibit some toothpulp evoked responses in trigeminal nuclei oralis and caudalis in the cat12,37,39,43 and both these nuclei have been implicated in the digastric reflex pathway 2,15,36,41. This phenomenon may be the same as that referred to as diffuse noxious inhibitory controls (DNIC) whereby the activity of certain cells in the spinal dorsal horn29,30 and trigeminal nucleus caudalisl2,13 of the rat can be inhibited by noxious stimulation of remote parts of the body. There are several similarities between the present findings and those

42 related to DNIC. First, in both cases, the conditioning and test stimuli are applied to quite separate areas of the body. Secondly, D N I C can influence neuronal activity evoked by either noxious or nonnoxious cutaneous stimuli, while in the present study, the depression of the digastric reflex was similar regardless of whether the reflex was evoked by toothpulp stimulation at intensities which would generally be regarded as being noxious or by relatively gentle mechanical stimulation of a tooth. Finally, D N I C are produced only by noxious conditioning stimuli 12,13,27,29 and in the present experiments, the digastric reflex was depressed by mechanical conditioning stimuli only when these were of noxious intensities, and by electrical conditioning stimuli only when these recruited fibres with conduction velocities between 20 and 40 m's -1, some of which would normally be excited exclusively by intense mechanical stimuli 4. In contrast to these similarities, the present findings differ from those for DNIC in that the depression of the digastric reflex was not affected by naloxone although this drug has been reported to greatly reduce the effects of DNIC 28. Serotonergic systems have also been implicated in DNIC10A 4 but, as explained above (see Results), it was not clear whether serotonin antagonists influenced the effects of the conditioning stimuli in the present experiments since these drugs yery strongly depressed the digastric reflex itself. This latter finding is open to various explanations including the possibility that the drugs were depressing some tonically active excitatory influence on the reflex pathway; evidence for such a tonically ac-

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tive pathway, originating from higher centres, has been obtained in other experiments in this laboratory in which very large increases in the digastric reflex threshold have been found following mid-collicular decerebration (e.g. ref. 11). Despite the above similarities between the present findings on the modulation of the digastric reflex and existing data for neuronal activity in the trigeminal sensory nuclei, it is possible that the effects of the conditioning stimuli in the present experiments may have resulted from synaptic events not in the sensory nuclei but in the trigeminal motor nucleus. However, although inhibitory post-synaptic potentials have been reported in jaw elevator (masseter, temporalis) motorneurones following stimulation of nerves in the limbs26 there is no evidence that this also occurs in digastric motorneurones (see ref. 36). ACKNOWLEDGEMENTS The work was supported by the Medical Research Council. I wish to thank Professor B. Matthews for his advice and encouragement in the course of these studies. My thanks are also due to I. P. Rogers and R. H. Harris for technical assistance, to C. Makepeace and D. Johanson for photography and to M. A. Knapp and S. Heywood for typing this manuscript. The naloxone, methysergide and cinanserin used in these experiments were gifts from Endo Laboratories, Sandoz Products Ltd., and Squibb Europe Ltd., respectively.

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