Presynaptic inhibition of jaw-opening reflex by high threshold afferents from the masseter muscle of the cat

BRAIN RESEARCH

193

P R E S Y N A P T I C I N H I B I T I O N OF J A W - O P E N I N G R E F L E X BY H I G H T H R E S H O L D A F F E R E N T S F R O M T H E MASSETER M U S C L E OF T H E CAT

Y. N A K A M U R A AYD C. Y. WU*

Department of Neurophysiology, Institute of Brain Research, School of Medicine, University of Tokyo, Tokyo (Japan) (Accepted April 28th, 1970)

INTRODUCTION

Afferent fibers from muscle spindles of the masseter muscle have excitatory synaptic linkages with masseteric motoneurons 16,1s,21,2s. The threshold of these afferent fibers is lower than that of axons of masseteric motorteuronslS, 21. These afferents are assumed to belong to group Ia fibers in analogy with the spinal monosynaptic reflex pathway. In the spinal cord, high threshold muscle afl'erents are known to produce not only reflex actions on motoneurons but also depolarization of the central terminals of primary afferent fibers (PAD) s 11. In the trigeminal system, whereas various cutaneous branches have been reported to give primary afferent depolarization of other cutaneous branches 5,19 no information is available on whether or not proprioceptive afferents produce the PAD. Intricate coordination is found between movements of the tongue and the jaw; stimulation of the tongue or the lingual nerve evokes a jaw-opening reflex2,15,17. In the present study we have tried to elucidate the role ofproprioceptive afferents from the masseter muscle in the regulation of the inflow of afferent impulses from the tongue to the brain. Evidence will be presented that group II and III afferents of the masseter muscle depolarize the central terminals of the lingual mechanoreceptor fibers and exert a presynaptic inhibitory action on the jaw-opening reflex evoked by these lingual afferents (linguo-digastric reflex). METHODS

General

The experiments were done on 14 anesthetized and 23 unanesthetized cats. Tracheotomy and cannulation into the left femoral vein were performed under initial * Visiting investigator from the Department of Neurophysiology, Taipei Medical College, Taipei, Taiwan, Republic of China. Brain Research, 23 (1970) 193-211

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administration of pentobarbital sodium (30 mg/kg i.p.) in the first group or under ether inhalation in the second. In anesthetized cats pentobarbital sodium (5-10 mg/kg) was added i.v. when necessary. The cerebellum was removed from all cats of this group. Precollicular transection was performed in 5 cats. In unanesthetized cats, thiopental sodium (7-10 mg/kg) was injected intravenously after cessation of ether inhalation, and anesthesia was maintained by repeated injections of thiopental sodium (3-5 mg/kg) every 10-15 rain throughout the period of surgical operation. Artificial ventilation was started at the time of initial administration of thiopental. After local application of procaine, the spinal cord was transected at the level between C2 and Cz. Precollicular transection was performed in all cats in this group, and the cerebellum was also removed except in 4 cats. At the beginning of recording, which was 2 h after the last injection of thiopental, gallamine triethiodide (Flaxedil) was injected and pneumothorax was made bilaterally. Animals were immobilized by repeated injection of gallamine triethiodide throughout the experiments. The rectal temperature was kept at 36-38°C with a heating pad and an infrared lamp.

Preparation of nerves The left lingual nerve and the nerve supplying the anterior belly of the left digastric muscle (digastric nerve) were exposed by ventral approach and isolated from the surrounding tissue. The digastric and mylohyoid muscles were partially removed to make a mineral oil pool for these nerves. The head of the cat was fixed on a stereotaxic apparatus. The masseteric nerve was then prepared bilaterally as described in a previous paper 21. In 5 cats the floor of the frontal sinus was removed and the left supraorbital nerve was isolated. All these nerves were ligated at the peripheral end and sectioned.

Exposure of the semilunar ganglion To make a partial selective lesion or to place a recording electrode just medial to the motor root of the left trigeminal nerve in the semilunar gangtion under direct vision, the ganglion was exposed on the left side by removing the overlying brain structures by suction. During aspiration of the brain tissue rostral to the precollicular transection, the carotid arteries were occluded temporarily. Later they were released after the application of a gelatin sponge to the exposed portion of the brain.

Stimulation and recording The cat's head was tilted with the left side down. Two pools were made using the skin flaps of the mandibular and temporal regions: one for the lingual and digastric nerves, the other one for the masseteric nerve. The peripheral ends of these nerves

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Fig. 1. Schematic representation of experimental setup, a, Bipolar silver wire electrode used either for conditioning stimulation of masseteric nerve or for recording responses to mesencephalic stimulation, b, Bipolar silver wire electrode for recording linguo-digastric reflex, c, Bipolar silver wire electrode used either for recording antidromic responses to stimulation of spinal nucleus or for stimulation of lingual nerve, d, Monopolar steel needle electrode for recording evoked volleys in semilunar ganglion, e, Concentric bipolar stimulating electrode in trigeminal mesencephalic nucleus. f, Concentric bipolar stimulating electrode in nucleus oralis for evoking antidromic responses in lingual nerve; core used for monopolar recording of responses to lingual nerve stimulation. Abbreviations: MASS, masseteric nerve; DIG, nerve to anterior belly of digastric muscle (digastric nerve); LING, lingual nerve; SEMI, semilunar ganglion; MES, trigeminal mesencephalic nucleus; MOT, trigeminal motor nucleus; SP.O, trigeminal spinal nucleus (nucleus oralis); SP.C, trigeminal spinal nucleus (nucleus caudalis). Efferent fibers from motor nucleus are shown by broken lines.

as well as the supraorbital nerve were placed on bipolar silver wire electrodes (polar distance, 3-4 ram); the whole isolated portion was then immersed in mineral oil, and the recording was made monophasically (Fig. 1). Concentric needle electrodes (inner core, 0.2 ram; sheath, 0.5 ram; polar distance, 0.5 ram) were inserted stereotaxically into the left trigeminal mesencephalic nucleus and the left spinal nucleus. The electrode tips were placed at the optimal points for evoking direct antidromic responses in the masseteric and lingual nerves, respectively. The same type of electrode was inserted into the left semilunar ganglion near the medial border o f the m o t o r r o o t to m o n i t o r the incoming volleys f r o m the masseteric nerve or the volleys elicited by stimulation of the mesencephalic or spinal nucleus o f the trigeminal nerve. The potential was recorded monopolarly between the core and the neck muscle. Similarly, the potential in the left spinal nucleus, evoked by stimulation o f the left lingual or masseteric nerve, was recorded m o n o p o l a r l y using the core of the electrode as the active pole. Electrical pulses generated by electronic stimulators (Nihon Koden, MSE 3 and MSE 40) were used for stimulation t h r o u g h isolation transformers. Pulses of 0.05-0.2 msec in duration were applied to the spinal and mesencephalic nucleus, and pulses o f 0.02-0.03 msec in duration were Brain Research, 23 (1970) 193-211

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applied to the masseteric nerve. When pulse trains were applied to the masseteric nerve, their interval was set at 2-4 msec. Responses were displayed on a cathode ray oscilloscope (Nihon-Koden VC7) or on the cathode ray oscilloscope of an averaging computer (Nihon-Koden ATAC510-10) by superimposing 10-20 responses. The effects of conditioning stimulation to the masseteric nerve as well as to the trigeminal mesencephalic nucleus were studied on the preterminal excitability of the lingual primary afferent fibers using Wall's technique 3°. The effects were estimated by 2 procedures: (1) 10 records were made of both the control and the conditioned responses, and the F test was used to find ally significant difference between them (significance level, 5 ~ ) ; or (2) averaged amplitudes of conditioned antidromic spikes on ATAC were divided by those of control responses expressing the control value as 100 O/~o.The same procedures were applied to testing effects on lingually evoked potentials in the trigeminal spinal nucleus and on tile linguo-digastric reflex.

Application of drugs Administration of pentobarbital sodium (10 mg/kg), strychnine (0.1-0.3 mg/kg) and picrotoxin (0.5-2 mg/kg) were made intravenously.

Lesion technique In 6 cats a partial transection was made, including the trigeminal spinal nucleus and tract at the level of the obex on the side of stimulation of the spJnat nucleus using a small knife made from a razor blade. The same tool was used for selective section of the motor root of the trigeminal nerve in the exposed semilunar ganglion near the exit of the mandibular nerve (4 cats).

Determination of electrode tip locations and extents of lesions Post mortem examination included measurements of distances between stimulating and recording electrodes. The extent of the lesions in the brain stem or in the trigeminal ganglion was determined histologically. RESULTS

Antidromically evoked activities in the lingual nerve by stimulation of ipsilateral trigeminal spinal nucleus Stimulation of the trigeminal spinal nucleus (nucleus oralis) evoked :in the ipsilateral lingual nerve an antidromic spike followed by a rhythmic potential consisting of 3-4 peaks (Fig. 2A). The latency of its onset and peak was 0.9 _~ 0.1 msec (mean ~ S.D.,n = 11)and 1.2 -t- 0. t msec (n = 11), respectively. The first peak of the rhythmic potential appeared with a latency of 4.1 ~-~ 1.2 msec (n = 9) followed by

Brain Research, 23 (1970) 193-211

197

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Fig. 2. Antidromic responses in the lingual nerve evoked by stimulation of ipsilateral trigeminal spinal nucleus (nucleus oralis). A, Directly evoked antidromic spike (DEAS) and synaptically evoked antidromic response (trigeminal dorsal root reflex, TDRR). Stimulation: left spinal nucleus (0.5/sec, 0.15 msec, 3.5 V). Record: left lingual nerve, 10 sweeps superimposed. Time base: 1 msec for upper two, 5 msec for lower two records. Calibration: 100/iV for all records. B, DEAS and T D R R in lingual nerve in relation to intensity of stimulation of spinal nucleus. Abscissa: intensity of stimulation (0.5/sec, 0.1 msec) in volts. Ordinate : amplitude of DEAS (circles) and the first peak of T D R R (triangles), as shown in inset A and B from an unanesthetized decerebrate cat.

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Fig. 3. A, Simultaneously recorded responses in semilunar ganglion (upper record) and lingual nerve (lower record) to stimulation of ipsilateral spinal nucleus (0.5/sec, 0.1 msec, 16 V). 13, Simultaneously recorded responses in semilunar ganglion (upper record) and masseteric nerve (lower record) to stimulation of ipsilateral mesencephalic nucleus (0.5/sec, 0.1 msec, 3.1 V). C, Responses in semilunar ganglion to stimulation of ipsilateral masseteric nerve (0.5/sec, 0.02 msec, 1.4,2.5 and 4.9 V from top to bottom). Time base and calibration: 2 msec and 100 #V for all records. A and 13 obtained from the same cat, C from another; both anesthetized with pentobarbital. All records consist of 10 superimposed sweeps. Monopolar recording in the semilunar ganglion; negativity shown as upward deflection.

Brain Research, 23 (1970) 193-211

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2 or 3 peaks at 2-4 msec intervals. The duration of this antidromic potential was 15-20 msec. The conduction velocity of the fibers responsible for the antidromic spike was found to be 50-66 m/sec from the ratio of the conduction distance versus the latency difference between the onset of the lingual antidromic spike and the positive peak of the antidromic potential in the semilunar ganglion (Fig. 3A). The rhythmic potential was very susceptible to changes in the frequency of stimulation, whereas the spike was consistently induced. The former was usually completely suppressed with a stimulation of 4-5/sec, but the latter could follow high frequency stimulation up to 100/sec without reduction of its amplitude. The rhythmic potential appeared at 1.2-1.6 times the threshold ( x T) and reached the maximum at 1.8-2.0 /: T for evoking the direct response. The amplitude of the antidromic spike, on the other hand, continued to increase almost directly proportionally with the stimulus intensity up to 4-5 T, reaching the maximal amplitude at 5--8 ~ T (Fig. 2B). Thus, a directly evoked antidromic spike (DEAS) and a trigeminal dorsal root reflex (TDRR), similar in all respects to those described by King and Meagher ~9,

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Fig. 4. Lingual PAD in relation to intensity of masseteric nerve stimulation. A. Lingual PAD induced by masseteric nerve stimulation. Left: control response. Right: conditioned response. Test stimulus: left spinal nucleus (0.5/sec, 0.1 msec. 13 V). Conditioning stimulus: left masseteric nerve (0.02 msec. 4.8 × T, 10 pulses in 2 msec interval). Conditioning-test interval: 40 msec from the first conditioning shock. Record: left lingual nerve, average of 10 responses. B, Responses in semilunar ganglion to masseteric nerve stimulation. Stimulation: left masseteric nerve (0.5/see, 0.02 msec); applied intensities expressed by numerals representing multiples of nerve threshold { × T) in each record. Record: left semilunar ganglion, monopolar, upward deflection negative. C, Effect of masseteric nerve stimulation on lingual PAD and on responses in semilunar ganglion. Circles: amplitudes of conditioned DEAS in per cent (control DEAS: 100%). Triangles, peak-to-peak amplitude of responses in semilunar ganglion to masseteric nerve stimulation. Abscissa: intensities of masseteric nerve stimulation represented as multiples of nerve threshold. Ordinates: left applies to circles, right to triangles. A, B and C obtained from an unanesthetized deserebrate cat. A is sample record of C.

Brain Research, 23 (1970) 193-211

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were recorded in the lingual nerve by stimulation of the ipsilateral trigeminal spinal nucleus (nucleus oralis). EFfects o f masseteric nerve stimulation on the antidromic responses in the lingualnerve

The effects of masseteric nerve stimulation on the excitability of the central terminals of the lingual primary afferent fibers were studied using Wall's technique 30. The test stimulus was ajusted to set the amplitude of the control lingual DEAS at about one-third of its maximum. Conditioning stimulation of the masseteric nerve induced an increase of the DEAS in the lingual nerve bilaterally, usually accompanied by suppression of the T D R R (Fig. 4A). Thus, the stimulation of the masseteric nerve induced an increase in excitability of the central terminals of lingual afferents presumably owing to primary afferent depolarization (PAD). The maximal increase of the DEAS was obtained at the conditioning-test interval of 30-50 msec. Short trains of pulses to the masseteric nerve were more effective in inducing the PAD than were single shocks. The PAD of the lingual nerve was never detected by masseteric nerve stimulation if the intensity was less than 1.5 × T even with a conditioning train of as many as 10 pulses. The relation between the conditioning volley to the masseteric nerve and the lingual PAD was studied by monitoring the evoked potential in the masseteric nerve at the ipsilateral semilunar ganglion near the motor root (Fig. 3C, 4B). Stimulation of the masseteric nerve evoked a triphasic response in the ipsilateral semilunar ganglion. The latency of the first positive peak was 0.3 ± 0.04 msec (n 14). The amplitude from the positive to the negative peak increased with stimulus intensity up to 2.0-3.2 > T, reaching a quasi-plateau (triangles in Fig. 4C). The conduction velocity of fibers yielding the earliest potential was found to be 95.8 ± 12.5 m/sec (n - 7). With a stimulation of above 2.0 × T, an inflection was detected in the descending phase of the negative component of the triphasic potential (Fig. 3C). On the assumption that the earliest peak and this inflection were produced by 2 different component potentials in different sized nerve fibers, the conduction velocity of the fibers contributing to the inflection was estimated as 44.0 ± 4.1 m/sec (n = 5). Fig. 4C shows an example of the relationship between the conditioning nerve volley and the PAD effect. A slight increase of DEAS amplitude was detected at 1.5 × T, and the amplitude reached 1 2 0 ~ of the control at 2.4 × T where the amplitude of the nerve potential reached a quasi-plateau. At higher intensities the DEAS was still markedly increased, reaching more than 150~ at 6.0 × T. This increase continued up to 10 x T, which was the highest intensity applied to the nerve during this study. The time course of the PAD was studied in 8 unanesthetized decerebrate cats (Fig. 5). The masseteric nerve was stimulated with a train of 3-5 pulses of 3-5 × T intensity. The increase in amplitude of the DEAS began at the conditioning-test interval of 13-14 msec and reached a peak in 30-50 msec; after this peak the effect gradually decreased, reaching the control level at 150-200 msec; then a slight decrease in amplitude of the DEAS followed for about 100 msec (Fig. 5Af, B) in 4 of Brain Research, 23 (1970) 193-211

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Fig. 5. Time course of masseterically induced lingual PAD and effects of pentobarbital andstrychnine. A, Upper row: control responses in left lingual nerve to stimulation of left spinal nucleus (0.5/sec, 0.1 msec, 12 V). Lower row: responses conditioned by masseteric nerve stimulation (0,02 msec, 5 × T, 3 pulses at 2 msec interval). All are averaged records of 10 responses. Numerals in each column represent conditioning-test intervals, a-f correspond to circles in B labeled as a-f, respectively: B: Time course of lingual PAD. Abscissa: conditioning-test intervals in msec~ Ordinate: amplitude of conditioned DEAS in per cent (control, 100K). Circles: before application of drugs. Squares: after i.v. injection of pentobarbital, 10 mg/kg. Triangles: after i.v. injection of strychnine, 0.1 mg/kg, applied 25 rain after i.v. injection of pentobarbital at I0 mg/kg. C, D: Sample records at conditioning test interval of 40 msec after injection of pentobarbital and strychnine, labeled as g andh ;in B i respectively. Time base and calibration in D applies to all records in A, C and D. A-D obtained from an unanesthetized decerebrate cat.

the 8 cats investigated. A t c o n d i t i o n i n g - t e s t intervals o f l o n g e r t h a n 300 msec, no effects o f c o n d i t i o n i n g s t i m u l a t i o n were found. T h e effects o f p e n t o b a r b i t a l s o d i u m on the lingual P A D were studied on 5 u n a n e s t h e t i z e d cats. T h e time course o f the P A D did n o t change essentially after i.v. injection o f p e n t o b a r b i t a l s o d i u m (10 m g / k g ) in either the onset o r p e a k latency, b u t the l a t e r p a r t o f the P A D was p a r t i c u l a r l y e n h a n c e d (Fig. 5B squares). In 2 cats strychnine nitrate (0.1-0.3 m g / k g ) was injected i n t r a v e n o u s l y following i.v. injection o f p e n t o b a r b i t a l s o d i u m . This d r u g m a r k e d l y e n h a n c e d the lingual P A D (Fig. 5B triangles). O n the c o n t r a r y , p i c r o t o x i n p r o f o u n d l y reduced or a b o l i s h e d the P A D effects o f masseteric nerve stimulation. A f t e r i.v. injection o f p i c r o t o x i n (0.5-2 mg/kg) to 4 cats, the a m p l i t u d e of the c o n t r o l lingual D E A S was g r a d u a l l y i n c r e a s e d for 20--30 min a n d t h e T D R R was m a r k e d l y depressed. T h e a m p l i t u d e o f the D E A S was increased when a s t r o n g e r test s t i m u l a t i o n was applied, b u t t h e c o n d i t i o n ing effects o f masseteric nerve s t i m u l a t i o n u p o n the D E A S was depressed or even abolished. Brain Research, 23 (1970) 193-211

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Stimulation of one cutaneous branch of the trigeminal nerve is known to induce PAD in another trigeminal cutaneous branch with a time course similar to that of the lingual PAD induced by stimulation of the masseteric nervea,5,2L In order to exclude the possibility of spread of stimulating current to any of these trigeminal cutaneous branches, the conduction of the masseteric nerve was blocked at a portion proximal to the stimulating electrode either by local application of procaine to the nerve (4 cats) or by mechanical use of fine forceps (7 cats), the PAD effect of the masseteric nerve stimulation of 5 and 10 x T being thereby compared before and after the conduction block. The blocking effect was checked by recording the potential in the semilunar ganglion evoked by masseteric nerve stimulation. After the block, the PAD effect of the masseteric nerve stimulation disappeared completely. When the conduction had recovered from procainization, the PAD effects of masseteric nerve stimulation reappeared. Partial transection of the brain stem, at the level of the obex or 1-2 mm rostral to it including the trigeminal spinal nucleus and tract, caused the enhancement of the amplitude of the DEAS (30-50 %) as well as the abolition of the T D R R . On the other hand, the PAD effects of masseteric nerve stimulation on the lingual nerve were greatly reduced or abolished. The abolition of PAD could not be ascribed to occlusion because the size of the DEAS was still less than half its maximum after transection.

Effects of stimulation of the trigeminal mesencephalic nucleus upon the antidromic potential in the lingual nerve Afferent fibers from both primary and secondary endings of muscle spindles of the masseter muscle are reported to have their cell bodies in the trigeminal mesencephalic nucleus 2s. If group Ia or II from the masseter muscle takes part in the lingual PAD, stimulation of the mesencephalic nucleus should evoke a PAD in the lingual nerve similar to that evoked by masseteric nerve stimulation. In fact, stimulation of the mesencephalic nucleus (4 anesthetized cats) evoked the PAD in the ipsilatelal lingual nerve with almost the same time course as that evoked by stimulation of the masseteric nerve (Fig. 6). The trigeminal mesencephalic nucleus was stimulated, and the evoked antidromic response was recorded from the peripheral portion of the ipsilateral masseteric nerve. The difference in the latencies was measured between the onset of this potential and the positive p e a k ' o f the triphasic response in the semilunar ganglion to mesencephalic stimulation in 4 cats (Fig. 3B). This difference in latencies coincided with the latency of the positive peak of the response in the semilunar ganglion to masseteric nerve stimulation (Fig. 3C) with a discrepancy of less than 1 0 ~ in each cat. Thus, the conduction velocity of the fibers responsible for this antidromic potential was comparable to that of the fastest group of fibers in the masseteric nerve, i.e., group I fibers. The minimal PAD effect of mesencephalic stimulation was detected when the stimulus intensity was 1.5-2.0 times the threshold for evoking the antidromic response in the masseteric nerve. The effect was increased in parallel with stimulus intensity in the supramaximal range for production of this antidromic response.

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Fig. 6. Time course of lingual PAD induced by stimulation of trigeminal mesencephahc nucleus. A, sample records of lingual PAD produced by stimulation of mesencephalic nucleus: Tes t stimulus : left spinal nucleus (0.5/sec, 0.1 msec, 20 V). Conditioning stimulus: left trigeminat mesencephalic nucleus (0.05 msec, 4.8 V, 4 pulses at 2 msec intervals). Record: left lingual nerve. Control response shown at farthest left, conditioned responses shown with each conditioning-test interval in msec. Time base and calibration applies to all records. B, Time course of lingual PAD, Abscissa: conditioning-test intervals in msec. Abscissa: amplitudes of conditioned DEAS in per cent (control 100 %). Cat anesthetized with pentobarbital.

This finding agrees with the proposition that group II fibers from the masseter muscle participate in the lingual PAD. Stimulation of the mesencephalic nucleus evoked a small direct response in the spinal nucleus, as could be anticipated from morphological studies '~8,29.

Effects of selective section of the motor root of the trigeminal nerve upon the lingual PAD induced by masseteric nerve stimulation The m o t o r root of the trigeminal nerve in the cat leaves the brain stem in the ventro-medial part of the fifth nerve, travels in this position to the semilunar ganglion and crosses underneath the ganglion to join the mandibular nerve f r o m the semilunar ganglion; the m o t o r root at first occupies the medial part of the mandibular nerve: the mesencephalic fibers travel in the motor root mixed with the m o t o r fibers 29. This anatomical arrangement gave us the possibility of selective sectioning of the m o t o r root including the mesencephalic fibers at the exit of the mandibular nerve. The selective section of the motor root was successful in 2 decerebrate cats, on which the effects of masseteric nerve stimulation were compared before and after the section. Before section of the trigeminal motor root. recordings were made simultaneously f r o m both the masseteric and lingual nerves during stimulation of the mesencephalic (Fig. 7A) and spinal nuclei (Fig. 7C) alternately every second. With the aid Brain Research, 23 (1970) 193-21 l

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Fig. 7. Effects of selective section of trigeminal motor root upon lingual PAD induced by stimulation of masseteric nerve and of mesencephalic nucleus. A-D, Simultaneously recorded responses from left masseteric nerve (upper record) and left lingual nerve (lower record) to stimulation of left trigeminal mesencephalic (A, B) and spinal nucleus oralis (C, D) before transection of motor root in left semilunar ganglion (A, C) and after transection (B, D). A, B, 10 superimposed records of responses to stimulation of mesencephalic nucleus (0.5/sec, 0.1 msec, 1.4 V). C, D, 10 superimposed records of responses to stimulation of spinal nucleus (0.5/sec, 0.1 msec, 14 V). Faster sweep speed in left columns of A-D. E, F, Graphic representation of lingual PAD induced by conditioning stimulus of left masseteric nerve (E) and of left mesencephalic nucleus (F) before (circles) and after (squares) transection of motor root. Abscissa: intensity of conditioning stimulus in multiples of nerve threshold. Ordinate: amplitudes of conditioned DEAS in lingual nerve in per cent (control: 100K). Test stimulus: left spinal nucleus oralis (0.5/sec, 0.1 msec, 15 V). Conditioning stimulus: left masseteric nerve (0.02 msec, 8 pulses at 2 rnsec intervals), or mesencephalic nucleus (0.1 msec, 8 pulses at 2 msec intervals). Conditioning-test interval: 35 msec. G, Sample record of test response. A - G from a decerebrate cat anesthetized with pentobarbital.

of a small knife made from a razor blade, a lesion was made in a frontal plane in the semilunar ganglion stepwise from medial to lateral at the proximity of the exit of the mandibular nerve. In each step the amplitude of antidromically evoked potentials in the masseteric as well as the lingual nerve was observed. The lesion was complete when a marked reduction occurred in the amplitude of the antidromic potential in the masseteric nerve evoked by mesencephalic stimulation (Fig. 7B), while that of the DEAS of the lingual nerve evoked by stimulation of the spinal nucleus remained unchanged (Fig. 7D). At such a stage a small antidromic potential in the masseteric nerve evoked by stimulation of the spinal nucleus (Fig. 7C upper record) was also almost completely or totally abolished (Fig. 7D upper record). Brain Research, 23 (1970) 193-211

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Fig. 8. Effects of masseteric nerve stimulation upon responses in spinal nucleus to stimulation of lingual nerve. A, Time course of lingual PAD induced by masseteric nerve stimulation. Test stimulus: left trigeminat spinal nucleus (0.5/sec, 0.1 msec. 4.5 V); position used for recording of series in C. Conditioning stimulus: left masseteric nerve (0.02 msec, 3 × T. 3 pulses at 2 msec intervals). Abscissa: conditioning-test intervals in msec, common with B. Ordinate: amplitudes of conditioned lingual DEAS in per cent (control. 100 ~). B, Time course of changes in amplitudes of lingually evoked negative complex in spinal nucleus induced by masseteric nerve stimulation. Test stimulus: left lingual nerve (0.5/sec, 0.01 msec, 4.5 V); position used for recording of A. Conditioningstimulus same as in A. Abscissa: conditioning-testintervals in msec. Ordinate: amplitudes of negative complex in per cent (control: 100K), triangles and circles representing first and second peaks of negative complex. C, Sample records of effects shown in B. Upper and lower rows show controland conditioned responses, respectively, in spinal nucleus. All obtained by averaging 10 responses. In each column conditioning-test intervals shown by numerals, a-e correspond to points in B labeled as a--e. A-C obtained from an unanesthetized decerebrate cat. Time base and calibration: 10 msec and 200/~V. After this selective section, the effects of mesencephalic s t i m u l a t i o n remained u n c h a n g e d (Fig. 7F). But s t i m u l a t i o n of the masseteric nerve did n o t induce any lingual P A D so long as the intensity of s t i m u l a t i o n was kept less t h a n 5 x T (Fig. 7E). W i t h stronger s t i m u l a t i o n P A D was i n d u c e d in the lingual nerve, a l t h o u g h the effect was definitely reduced c o m p a r e d with that o b t a i n e d before section with the same stimulus parameters (Fig. 7E squares).

Effects of masseteric nerve stimulation upon lingually evoked potentials in the trigeminal spinal nucleus S t i m u l a t i o n of the lingual nerve evoked, in the ipsilateral trigeminal spinal nucleus, a short-latency positive-negative potential followed by a negative complex with u s u a l l y 2 or 3 peaks (Fig. 8B insets). T h e latency of the positive peak o f the

Brain Research, 23 (1970) 193-211

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LINGUAL PAD BY MASSETERIC AFFERENTS %

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Fig. 9. Effects of masseteric nerve stimulation on the linguo-digastric reflex. A, Time course of lingual PAD (triangles) and of linguo-digastric reflex (circles) induced by masseteric nerve stirnulation. Conditioning stimulus: left masseteric nerve (0.02 msec, 3 >< T, 3 pulses in 2 msec interval). Test stimulus: left spinal nucleus (0.5/sec, 0.1 msec, 10 V) for antidromic responses in left lingual nerve; left lingual nerve (0.5/sec, 0.01 msec, 1.5 V) for evoking linguo-digastric reflex. Abscissa: conditioning test intervals in msec. Ordinate: amplitudes of conditioned lingual DEAS and area of linguo-digastric reflex discharges (control, 100%). Area of reflex discharge is defined as that surrounded by averaged line of reflex discharge and by direct lines connecting one positive peak to next, as shown by inset in A. B, Sample records of linguo-digastric reflex discharges. Each is the average of 10 responses. Upper and lower rows illustrate control and conditioned reflex discharges. Conditioning test intervals shown by numerals in each column, a-e correspond to circles in A labeled a e, respectively. C, Linguo-digastric reflex discharges after i.v. injection of picrotoxin (1 mg/kg). Upper and lower records: control and conditioned responses, respectively. Test and conditioning stimuli are the same as used for A and B. Conditioning-test interval: 40 msec. Five msec and 500 yV apply to each time base and calibration in B and C. A-C obtained from an unanesthetized decerebrate cat.

positive-negative potential was 0.7 0.9 msec, and the latency of onset of the first negative potential was 1.3-1.6 msec. The amplitude of the positive-negative potential was stable and followed 100/sec stimulation with only 20-25 °//o reduction. On the other hand, the negative wave was variable in amplitude and shape, and an increase of stimulus frequency up to 100/sec induced a reduction of 85 ~o of the amplitude compared with that evoked by 0.5/sec stimulation. Thus, the short latency potential and the succeeding negative complex represented the activities of the presynaptic and postsynaptic components in the spinal nucleus, respectively, as reported by Erickson et al. re. Masseteric nerve stimulation depressed the lingually evoked negative complex with essentially the same time course as the lingual PAD. An example is shown in Fig. 8B, in which the masseteric nerve was stimulated with 3 shocks at 2 msec intervals, and the time course of lingual PAD was examined (Fig. 8A). Single shocks were then applied to the lingual nerve through the same electrode as used for recording the DEAS, and the evoked potential was recorded with the same electrode in the same position as used for evoking DEAS in the lingual nerve. The same conditioning stimuli were applied to the masseteric nerve as were used for inducing the lingual Brain Research, 23 (1970) 193-211

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PAD, and the effects were observed upon the lingually evoked negative complex in the spinal nucleus (Fig. 8B, C). The amplitude was increased at a short interval of 8-10 msec, but this tendency was reversed at 15 msec. Thereafter, the amplitude was depressed with the peak at 30 msec which was the same as the peak latency for lingual PAD in this cat. After this peak, the amplitude gradually recovered. After reaching the control level, the amplitude was then slightly increased above the control level. The peak of this facilitation corresponded with that of depression of the DEAS. Thus, the time course of the change in amplitude of the negative complex in the spinal nucleus was like a mirror image of that of the lingual PAD, except that tile former showed a phase of facilitation preceding the depression. Effects of masseteric nerve stimulation upon the linguo-digastric reflex Single shocks to the lingual nerve induced a reflex discharge in the ipsilateral digastric nerve with the onset latency of 4. I :c 0.6 msec In -- 5). These reflex discharges appeared after lingual nerve stimulation of weak intensity as low as 1.2 T. An increase in the stimulus intensity caused a shortening of the onset latency as well as increase in amplitude and number of peaks of the reflex discharges. The response reached a maximum with 4-5 A T stimulation. Masseteric nerve stimulation depressed this reflex discharge with essentially the same time course as that of the lingual PAD. Fig. 9A illustrates an example in which the effect of conditioning stimuli of the masseteri cnerve was tested upon the lingual DEAS (Fig. 9A triangles). Next, the effects of the same conditioning stimuli were tested upon the reflex discharges evoked from the lingual nerve (Fig. 9A circles). A phase of facilitation was found at around 8-10 msec intervals, which was followed by a prolonged phase of depression. The time course of this depression corresponded with that of the lingual PAD as to onset. peak and duration. The facilitation preceding the depression was not accompanied by any lingual PAD (Fig. 9A), but the onset latency of the reflex discharge was shortened (Fig. 9Ba). In fact. masseteric nerve stimulation itself could evoke the reflex discharge in the digastric nerve with a latency of around 7-8 msec. if a short train of pulses were used with an intensity of more than 2 T. The reflex discharge was depressed by i.v. injection of picrotoxin (Fig. 9C" note the difference in amplification between B and C), but the effect of masseteric nerve stimulation was also markedly reduced or completely abolished (Fig. 9C) coincidentally with reduction or abolition of the lingual PAD effect of masseteric nerve stimulation. DISCUSSION

P A D in the lingual nerve The effects of masseteric nerve stimulation upon the excitability of the central terminals of lingual primary fibers were tested using Wall's method, and it was found that augmentation of the amplitude of the directly evoked antidromic spikes was Brain Research, 23 (1970) 193-211

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produced by masseteric nerve stimulation. The time course was similar to that found in other trigeminal nerve terminals evoked by stimulation of trigeminal cutaneous nerves, muscular and cutaneous nerves of fore- and hindlimb, and stimulation of the brain stem as well as the cerebral cortext,5,~, la, although in the present study the duration of the lingual PAD was slightly shorter. The effect of transection at the obex level upon the DEAS and PAD in the lingual nerve was also the same as in other cutaneous branches 27. These observations suggest a common mechanism for PAD both in the lingual nerve and in other trigeminal nerves so far reported. |n the spinal cord it was repotted that the negative dorsal root potential was followed by a phase of low positivity 7, which corresponded to a phase of decreased excitability in the presynaptic terminal of primary afferent fibers 2°. In the trigeminal system, however, no such diphasic changes of excitability were reported in either cutaneous or proprioceptive nervesl,a,6,1a,2a, ~a 27. In the present study with unanesthetized decerebrate cats, however, it was found that a phase of decreased excitability of the central terminals of lingual fibers followed a phase of PAD in half the cats in which the masseteric nerve was stimulated with an intensity of 5 >; T. The decrease of excitability was less than 10% but consistent. This decrease in excitability was more marked after the application of strychnine. The phase of decrease in excitability of lingual preterminal fibers was also found when a conditioning stimulation was applied to the supraorbital nerve. The most probable reason for the disagreement between the present study and other studies, therefore, is the difference between the anesthetic condition in our experiments and in those reported by other investigators. In our experiments unanesthetized decerebrate cats were used, while other researchers used animals under pentobarbital (more than 35 mg/kg) or ~tchloralose anesthesia. The cortically induced PAD in the masseteric nerve in unanesthetized cats 26 showed a time course of short duration similar to our observation of the lingual PAD, although it is not clear whether the PAD phase was followed by a decreased excitability.

Lingual fibers receiving PAD effect of masseteric nerve stimulation The lingual nerve contains fibers that come from mechanoreceptors in the tongue as well as those that mediate taste, temperature and pain sensation from the tongue. Taste fibers reach the nucleus of the solitary tract via the chorda tympani. Among those fibers that end in the spinal nucleus, the thicker ones originate from the mechanoreceptors of the tongue, and temperature and pain are mediated by thin fibersla, :~1. Hensel and Zotterman 14 estimated that the mechanoreceptor fibers were 8-10 and 12-15 ,urn in diameter. Porter z2 reported 2 groups of mechanoreceptor fibers in the lingual nerve: the group of superficially situated, rapidly adapting mechanoreceptors had faster conduction velocities (mean value, 40 m/sec) than the presumably deeply situated, slowly adaptive endings. The conduction velocity of the fibers contributing DEAS in the lingual nerve and thus receiving the PAD effect was estimated to be 50-66 m/sec in this study. It is, therefore, concluded that masseteric nerve stimulation induced P A D in the mechanoreceptor fibers in the lingual nerve.

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Afferent fibers in the masseteric nerve producing lingual P A D Masseteric nerve stimulation evoked, in the ipsilateral semilunar ganglion, a triphasic potential. The conduction velocity of the fibers responsible for this potential was estimated at about 96 m/see on average. The amplitude reached a quasi-plateau at a stimulation intensity of 2.0-3.2 ~," T. This triphasic response is assumed to be produced by activation of group I fibers of the masseteric nerve. The lowest intensity necessary for inducing a lingual P A D was 1.5 /.: T. This suggests that the lowest threshold afferents from the masseter muscle, i.e. group Ia, did not contribute appreciably to lingual P A D production.However, it is possible that group la afferents contributed to the PAD, but that the effect as a whole was not marked owing to the small number of group Ia fibers in the masseteric nerve 4. The lingual P A D remained at less than 30 ~ when the stimulus intensity was raised to 2.0-3.2 ~: T. which was necessary to evoke the maximal group I response. The lingual PAD was augmented proportionally with the increase of stimulus intensity within the supramaximal range for group 1 fibers. This clearly shows the participation of high threshold muscle afferents. Participation of group II fibers was confirmed by the presence of the lingual P A D induced by stimulation of the mesencephalic trigeminal nucleus as welt as by the abolition of the lingual PAD after selective section of the motor root of the trigeminal nerve. The lingual P A D induced by intense stimulation of the masseteric nerve after selective section could be ascribed to a small number of the high threshold group II afferents that were left intact because the section was not complete. But the effect seems rather to be chiefly due to group iII afferents which conduct in the sensory root presumably among afferents other than those from muscle spindles of the masseter muscle and thus were free from the selective section of the motor root. Since the P A D was not found with an intensity of less than 5 /. T after the selective section, no positive evidence for the participation of group Ib fibers from the masseter m uscle was obtained in the present study. It is possible, however, that group lb fibers were sectioned concomitantly with group I I fibers during the selecttve section of the t~igeminal motor root, taking the recent report into consideration that the soma of the afferent fibers from tendon organs of masticatory muscles is located in the mesencephalic nucleus 3. These findings appear to be consistent with a recent report of the PAD effect of muscle nerves of fore- and hindlimbs upon the supraorbital nerve, that P A D in the supraorbital nerve was produced by activation of group II and III fibers of muscular nerves, and that no effects were observed when only group I muscle fibers were activated 1.

Effects on jaw-opening reflex Masseteric nerve stimulation depressed the response of the second order neurons in the trigeminal spinal nucleus (nucleus oralis) as well as the linguo,digastric reflex with essentially the same time course as that of lingual PAD. This depression could be abolished by application of picrotoxin coincidentally with the abolition of the

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209

lingual PAD induced by masseteric nerve stimulation. These effects indicate that masseteric nerve stimulation inhibited the linguo-digastric reflex presynaptically at the first synaptic relay of this reflex pathway. On the other hand, the facilitation of the linguo-digastric reflex preceding the inhibitory phase was not accompanied by any sign of presynaptic hyperpolarization or of other presynaptic change. The facilitation may be ascribed to the postsynaptic excitatory effect of masseteric nerve stimulation. Recently, Sauerland and Mizuno 24 reported that stimulation of the masseteric nexve produced a prolonged suppression of the linguo-hypoglossal reflex. The time course of this masseteric effect is similar to that found in this study, suggesting that this is because of a presynaptic inhibition. SUMMARY

(1) The effects were studied of masseteric nerve stimulation upon the excitability of the central terminals of lingual primary afferent fibers in cats, using Wall's method, and upon lingually evoked potentials in the trigeminal spinal nucleus as well as upon the linguo-digastric reflex. (2) Stimulation of the masseteric nerve with intensities of more than 1.5 times threshold induced an increase in amplitude of antidromic spikes (conduction velocity, 50-66 m/sec) directly evoked by stimulation of preterminal fibers of the lingual nerve in the trigeminal nucleus oralis. This increase in excitability of preterminal fibers started around 15 msec after conditioning stimulation of the masseteric nerve, reached its peak after 30-50 msec, and returned to the control level after 100-150 msec. The effect was increased by raising the stimulus intensity in the supramaximal range for low threshold fibers in the masseteric nerve (conduction velocity, 96 m/sec in average). (3) Partial transection of the brain stem at the obex level, including the trigeminal spinal nucleus and tract, as well as application of picrotoxin reduced or abolished the facilitation of the antidromic spike. (4) Selective section of the trigeminal motor root at the semilunar ganglion abolished the observed effects of masseteric nerve stimulation so long as the intensity was kept below 5 times the nerve threshold, but the effect of stronger stimulation persisted. (5) Responses of second order neurons in the spinal nucleus (nucleus oralis) to lingual nerve stimulation were depressed, and the linguo-digastric reflex was diminished with increase in excitability of lingual preterminal fibers. (6) It is concluded that group II and III afferents in the masseteric nerve induce preterminal depolarization in the mechanoreceptor fibers of the lingual nerve by intervention of the trigeminal nucleus caudalis, and that they exert a presynaptic inhibitory action on the jaw-opening reflex. ACKNOWLEDGEMENTS

The authors wish to express their appreciation to Dr. T. Tokizane for his guidance and support, and to Drs. H. Shimazu and T. Hongo for their critical review of our manuscript. Brain Research, 23 (1970) 193-211

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REFERENCES

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28 SZENT,g,GOTHAI, J., Anatomical considerations of monosynaptic reflex arcs, J. Neurophysiol., l I (1948) 445~,54. 29 THELANDER, H. E., The course and distribution of the radix mesencephalica trigemini in the cat, J. comp. Neurol., 37 (1924) 207-220. 30 WALL, P. n., Excitability changes in afferent fiber terminations and their relation to slow potentials, J. Physiol. (Lond.), 142 (1958) 1 21. 31 ZOTTERMAN, Y., Specific action potentials in the lingual nerve of cat, Skand. Arch. Physiol., 75 (1936) 105-119.

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