Non-reciprocal facilitation of trigeminal motoneurons innervating jaw-closing and jaw-opening muscles induced by iontophoretic application of serotonin in the guinea pig

Bram Research, 515 (1990) 126-134

126

Elsewer BRES 15410

Non-reciprocal facilitation of trigeminal motoneurons innervating jaw-closing and jaw-opening muscles induced by iontophoretic application of serotonin in the guinea pig Ikufumi Kurasawa*, Kazuo Toda and Yoshio Nakamura Department of Physiology, Faculty of Denttstry, Tokyo Medtcal and Dental Untverstty, Tokyo (Japan) (Accepted 3 October 1989)

Key words. Tngeminal motoneuron, Iontophoresls; Serotonln; Methyserg~de, Glutamate, Guinea p~g

Effects of lontophoretlcally apphed serotonm (5-HT) and its antagonist, methyserglde (MS), on masseter (a jaw-closer, MA.MNs) and antenor digastnc motoneurons (a jaw-opener, AD.MNs) were studied m paralyzed guinea pigs, chloralose-anesthetized or decerebrate. Umtary activity was recorded with mulUbarrel capdlary electrodes from MA.MNs and AD.MNs tdentlfied by anUdromlc spikes evoked by stimulation of the masseter and anterior digastnc nerves, respectively. Under chloralose anesthesia, both MA-MNs and AD.MNs were almost quiescent, and application of 5-HT alone induced no changes m discharge of either of them. However, lontophoreUcally apphed 5-HT increased the frequency of discharge induced by iontophoretlc apphcation of glutamate m 26 of 34 MA-MNs (76%) and 17 of 30 AD.MNs (59%) tested. MS depressed the glutamate-induced actwity m 17 MA.MNs and 3 AD-MNs, respectwely, in which 5-HT exerted a facditatory effect on the glutamate-reduced activity. In decerebrate preparations, the firing index of spikes of MA.MNs monosynapUcally evoked by stimulation of the tngeminal mesencephahc nucleus was increased by 5-HT and decreased by MS 5-HT also enhanced the discharge of MA.MNs reduced by a ramp-and-hold stretch of the masseter muscle. We conclude that 5-HT alone does not excite either MA.MNs or AD.MNs, but potentmtes the effect of excitatory inputs to them' 5-HT exerts a modulatory facdltatory action on trigemmal motoneurons INTRODUCTION Intravenous administration of precursors of serotonin (5-HT) is known to facilitate the spmal monosynaptic reflex 3'4'12'49, to increase a- and ~,-motoneuron discharges 2"2°'38, and to facilitate the tonic vibration reflex of the spinal cord 1°. H o w e v e r , studies of iontophoretic application of 5-HT to spinal m o t o n e u r o n s revealed rather inconsistent results of either excitation 7"s8 or inhibition 21"22'45. In addition, no reports have so far been available of w h e t h e r the effects of 5-HT are reciprocal or not on the m o t o n e u r o n s innervating the antagonistic muscles. Serotonergtc innervation has been d e m o n s t r a t e d in the b r a m s t e m m o t o r nuclei as well as the spinal ventral horn ~8"23'42. 5-HT iontophoretically applied to facial m o t o n e u r o n s was shown to facihtate the effects of excitatory inputs to these m o t o n e u r o n s 3s. Likewise lntracellular studies d e m o n s t r a t e d that 5-HT causes a slow depolarization of facial m o t o n e u r o n m e m b r a n e s and an increased input resistance which leads to an increase m excitability of these m o t o n e u r o n s 57. Though this facihta-

tlon seems to be general regardless of the muscles these facial m o t o n e u r o n s innervate, it has not been demonstrated as yet whether or not the effect of 5-HT is discrete among the facial m o t o n e u r o n s innervating various facial muscles. The trigeminal m o t o r nucleus contains the m o t o n e u rons innervating the jaw muscles which are antagonistic to each other: the jaw-opening and jaw-closmg muscles. Immunohlstochemical studies have elucidated that trigeminal m o t o n e u r o n s are s u r r o u n d e d by dense terminals of serotonergic fibers and suggest a rich serotonergic innervation of trigeminal m o t o n e u r o n s 16's3. The trigeminal m o t o r nucleus accordingly seems to be suitable for the study of whether 5-HT exerts reciprocal or nonreciprocal effects on the m o t o n e u r o n s innervatmg the antagonistic muscles. The present study tried to d e t e r m i n e : (1) the m o d e of action of 5-HT on trigeminal m o t o n e u r o n s innervating the mutually antagomstic masseter (a jaw-closing muscle) and anterior digastric muscles (a jaw-opening muscle); and (2) the effects of 5-HT on the monosynaptic response of masseter m o t o n e u r o n s to stimulation of the trigeminal

* Present address. Department of Prosthodontlcs I, Matsumoto Dental College, Shlolm, Nagano Prefecture 399-07, Japan

Correspondence Y. Nakamura, Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental Umverslty, 5-45 Yushlma 1-chome, Bunkyo-ku, Tokyo 113, Japan 0006-8993/90/$03 50 t~) 1990 Elsevier Science Pubhshers B.V (Biomedical Dwlsion)

127 m e s e n c e p h a l i c n u c l e u s as well as stretch o f the m a s s e t e r m u s c l e in t h e g u i n e a pig. T h e results i n d i c a t e that 5 - H T alone

can

excite

neither

digastric m o t o n e u r o n s , the

effects

of

the

masseter

nor

anterior

but n o n - r e c i p r o c a l l y facilitates

excitatory

inputs

to

these

trigeminal

motoneurons. P r e l i m i n a r y r e p o r t s o f this study a p p e a r e d in abstract f o r m 29,30.

MATERIALS AND METHODS

Surgtcal procedures Experiments were performed in 42 adult guinea pigs weighing 400-650 g They were initially anesthetized with pentobarbital sodmm (35 mg/kg, i.p ). After tracheal cannulation, 5 pmrs of bipolar copper wire electrodes (diameter: 100 pm; varnish insulated except for 500/~m at the tip; interpolar distance: 1.0 mm) were implanted m each of the masseter and anterior digastric muscles on the right side, to record EMG and to stimulate antidromically the axons of masseter (MA.MNs) and antenor digastrlc motoneurons (AD.MNs) The animals were mounted on a stereotaxic apparatus after R6ssner's method 'z, and the skull and dura over the cerebral and cerebellar cortices were removed. The sinus transversus was ligated, cut and reflected. The exposed cortical surface was covered with cotton winks soaked m saline. A burr hole was drilled in the midline of the ventral surface of the mandible of 5 animals, and a screw was implanted in the hole. This screw was later connected with a steel wire to a mechanical device to pull the mandible downwards to various positions with constant velocities (i.e., a ramp-and-hold stretch of the jaw closing muscles) They were decerebrated at the mid-thalamlc level (A10.0). In the remaining 37 animals anesthesia was maintained with a-chloralose (10-15 mg/kg/h, i.p.) throughout the experiment. Body temperature was kept at 37 °C with radiant heat from above, and the ECG was continuously monitored during the experiments. The depth of anesthesia was periodically monitored by checking the pupil size and pulse rate during strong pinching of the forepaw. The experiments were terminated by i.p. injection of an overdose of pentobarbital.

Stimulatmg and recording procedures An electropolished tungsten microelectrode (tip resistance: 0.51.0 MD at 1 kHz) was stereotaxlcally inserted into the tngeminal mesencephalic nucleus on the right side at a 20° anterlor-to-postenor angle of approach, and was fixed to the point at which stimulation evoked EMG response in the ipsilateral masseter muscle at the lowest intensity. A stretch reflex was evoked in MA.MNs of the 5 decerebrate animals by a ramp-and-hold depression of the mandible. The mandible was depressed with a constant velooty at 5-10 mm/s of 0.5 s and held in the displaced posinon for 4.0 s. Unitary recordings were made from MA.MNs and AD.MNs under artificial ventilation after paralysis (pancuronlum bromide 1.2 mg/kg, i.m every 5 h) with 5-barrel glass microelectrodes (tip: 5-8 /zm). The central recordmg barrel was filled with 4 M NaCI (resistance' 2-3 MD at 1 kHz). The remaining 4 barrels contained, for iontophoretic apphcation, serotonin creatimne sulfate (Sigma, 0.12, M, pH 4.0), methysergide hydrogen maleate (Sandoz, 0 03 M, pH 6.0), monosodium L-glutamate (Sigma, 2.0 M, pH 8 0) and NaCI (Wako, 2.0 M, pH 7.0), respectively. To retain the drugs in the electrodes, 10 nA braking current was continuously applied to each drug-containing barrel except during periods of drug apphcations The multibarrel electrode was inserted stereotaxically into the right trlgemmal motor nucleus at a 30° posterior-to-anterior angle. The anttdromic field potentmls evoked by stimulation of the massetenc and anterior digastric nerves were used as a guide to

locate the masseter and anterior digastric motoneuron pools in the trigeminal motor nucleus, respectively. Unitary activity of trigeminal motoneurons was displayed on an oscilloscope and recorded on magnetic tape. The output of the oscilloscope was fed through an amplitude discrinunator into a frequency meter The reciprocal of each consecutive spike interval was displayed on an ink-wntang recorder. Permanent photographic and ink-written records were obtained from the data recorded on tape.

Data analysts To study the effects of 5-HT and its specific antagonist, methysergide (MS), the 60 s immediately preceding the onset of drug application was divided into 6 periods of 10 s, and spike numbers during each period of 10 s were counted before and after local application of the drugs. The mean of the discharge rate in the 6 periods immediately preceding the start of iontophoretlc application of these drugs was taken as the control rate. Likewise the mean firing rate was calculated for every 10 s after the onset of drug application. The effect of the drugs was taken to be facilitatory or inhibitory, when the maximum or minimum mean firing rate after drug application was higher than the mean control rate +2 S.D. or lower than the mean control rate -2 S.D. In the study of the effects of these drugs on the discharge of MA-MNs induced by stimulation of the tngeminal mesencephalic nucleus (1 Hz), stimulus intensity was set at the level which evoked a spike potential in 4-6 trials in 10 consecutive trials. The 60 s immediately preceding the onset of drug application was divided into 6 periods of 10 s. The finng index (i.e. the probability of spike firing in 10 consecutive trials) was obtained for each of the 6 periods. The mean of the 6 firing indices was compared with that of the 6 firing indices obtained from trials at 1 Hz for 60 s from 60 to 120 s after the onset of 5-HT application or from 30 to 90 s after the onset of MS application. To study the effects of 5-HT on the discharge of MA-MNs induced by stretch of the masseter muscle, a ramp-and-hold depression (law-opening) was applied to the jaw 6 times at intervals of 15 s immediately before the onset of drug apphcation and from 60 s after the onset of drug application. The numbers of spikes induced during the 0.5 s of a stretch at a constant velocity (dynamic phase) and during 0.5 s of a constant displacement from 0.5 s after the end of the dynamic phase (static phase) were counted. The means of the number of sp~kes during the dynamic and static phases before drug application were compared with those after drug application. To exclude current artifacts, positive current was applied through the 2 M NaCl-filled electrode at an intensity 1.2 times that which was used to eject drugs. The trigeminal motoneurons which responded with any change in discharge to this test were not included in this paper. RESULTS

Effects of 5-HT on spontaneous and glutamate-induced discharge of trigeminal motoneurons M A . M N s and A D . M N s w e r e i d e n t i f i e d by a n t i d r o m i c spike p o t e n t i a l s e v o k e d by s t i m u l a t i o n o f t h e m a s s e t e r i c and a n t e r i o r digastric n e r v e s , r e s p e c t i v e l y , which app e a r e d after a short, c o n s t a n t l a t e n c y o f 0 . 9 - 1 . 2 ms, with an a l l - o r - n o n e fashion at the t h r e s h o l d intensity, and f o l l o w e d r e p e t i t i v e s t i m u l a t i o n at 100 H z . U n d e r the a n e s t h e s i a u s e d in this study, t h e s e m o t o n e u r o n s scarcely showed spontaneous

discharge.

N o c h a n g e s w e r e de-

t e c t e d in r e s p o n s e to i o n t o p h o r e t i c a p p l i c a t i o n o f 5 - H T alone, even though the intensity of applied anodal c u r r e n t was i n c r e a s e d to 200 n A . A c c o r d i n g l y the effects

128 of 5-HT were tested on the stable discharge of trigeminal motoneurons induced by iontophoretic application of glutamate at an intensity of 5-50 nA. The ejection of glutamate started ca. 30 s in advance of the onset of recording of the control activity and was terminated more than 3 min after cessation of the ejection of 5-HT. The pattern of the response of MA.MNs and AD.MNs to tontophoretically applied 5-HT was a facilitation, with an exception of inhibition in 4 AD.MNs (Table I). Fig. 1 illustrates a facilitation of firing rate of 3 MA-MNs induced by application of 5-HT as a function of current mtensity used for ejection. Since the maximal effects were obtained at an intensity of 60-80 nA, the highest current intensity used for 5-HT application in the present study was set at 80 nA, as was the highest current intensity for MS ejection. The current intensities used for 5-HT application to the trigeminal motoneurons shown in Table I ranged from 30 to 80 nA in MA.MNs (mean: 64.0 nA, n = 34) and from 40 to 80 nA in AD.MNs (mean: 65.6 hA, n = 30). There was no difference in the mean current intensities for 5-HT ejection between MA.MNs and AD.MNs (Wilcoxon's rank sum test, P > 0.05). Likewise the current intensities used for MS ejection ranged from 30 to 80 n A m MA.MNs (mean: 65.6 nA, n = 34) and from 40 to 80 nA in AD.MNs (mean: 66.7 nA, n -- 30). No difference was found in the mean current intensities for MS ejection between the former and latter groups of trigeminal motoneurons (P > 0.05, Wilcoxon's rank sum test). When the highest or lowest firing rate during 5-HT 5.0 © A A

4.0

[]

i 3.~t 0

iio I

2O

©

©

[]

[]

[] (

I

40

'

I

60

'

I

80

'

InA I O0

current intensity

Fig. 1. Effects of 5-HT on discharge of MA.MNs reduced by local apphcatlon of glutamate Abscissa, current intensity for election of 5-HT; ordinate, firing rate dunng 5-HT apphcanon represented as multiples of the control rate. Circles, triangles and squares represent 3 different MA.MNs

TABLE I Effects of 5-HT and MS on dtscharge acttvmes of mgeminal motoneurons reduced by tontophoretlcally apphed glutamate Each column and line of field represent the numbers of MA.MNs (upper panel) and AD.MNs (lower panel) showing respectwe response to 5-HT (columns) and MS (hnes), respectwely 5-HT Facthtatton Inhtbaton

No response

Total

MA MNs ( Faohtation 0 J Inhibmon 17 MS ~ No response 9 [. Total 26

0 0 0 0

0 3 5 8

0 20 14 34

AD. MNs Facditation 0 Inhibition 3 MS | No response 14 ~, Total 17

1 0 3 4

0 3 6 9

1 6 23 30

or MS application at the intensity of 80 nA did not exceed the control firing rate +2 S.D., the drugs were judged ineffective (No response in Table I). In MA.MNs the observed effects were invariably facilitation (26 of 34, 76%), and inhibition was mduced in no MA.MNs (Table I). The firing rate started to rise ca. 30-60 s after the onset of 5-HT application, reached a peak ca. 60-90 s after the onset of 5-HT application, and recovered the same level as the control before 5-HT application ca. 30-180 s after the cessation of 5-HT application (Fig. 2Aa). 5-HT also induced facilitation in 17 of 30 (59%) AD.MNs tested (Table I), the time course of which was similar to that in MA.MNs (Fig. 2Ba), and inhibition in 4 AD.MNs (Table I). On the other hand, iontophoretic application of MS inhibited the glutamateinduced discharge in 17 MA.MNs and 3 AD.MNs, in which 5-HT exerted facilitation of discharge (Table I). This inhibition started ca. 10-30 s after the onset of application, reached a peak ca. 20-30 s, and returned to the control level ca. 10-30 s after termination of MS application (Fig. 2Ab,Bb). ThUS the incidence of facilitation with 5-HT was higher in MA.MNs (26/34, 76%) than AD.MNs (17/30, 59%) (P < 0.05, xE-test), as was the incidence of inhibition with MS: 17/34 (50%) m MA.MNs and 3/30 (10%) in AD-MNs (P < 0.05, X2-test). Opposite effects between 5-HT and MS were seen in 17/34 MA.MNs and 3/30 AD.MNs. The incidence of the opposite effect was also different between MA.MNs and AD.MNs (P < 0.05, X2-test). Correlation o f 5 - H T effects with the firing rate o f trigemmal motoneurons durmg the control period Effects of 5-HT on discharges of tngeminal motoneu-

129

A i

I

~-',,~,mim ....

B

a t 7 .......

|

II

a

mim m__mm_maim !!m_!m ma~!= !!!!!Lm _m.!

...............

i

Illll

II

Ill

II

'

i

.....

,

.....

i

tl

b I I l m I I I l I I ) ---1

I III I II

...........................

II IIlII

I I U l I l m I ~ l l I I I l l I m m ~

,.

I I

'

i

i

i

i

II

ilU

)! 6 ....

....

flllllll

I

I, ,~,...i. ............

•llllllll

40Hz

.I A .

i

30spikesv's

=la u~.., tJb.,t...&,.=..... u,.~b. . . . . ]

I fl~ i l I I l i l l i I l l l a l l l i

I I llill

:

i

...........................

...... ,~ ....... J ..... i .

:=~ =,, t.-'~]--,

I

i I

.....

:i

30splkes/s I

~_~..... L~,La, tJ.J-,~=,,

=

60 s 60 s Fig. 2. Effects of 5-HT and MS on discharges of a MA.MN and an AD.MN induced by local apphcatlon ot glutamate. A,B: umtary actavlties of a MA.MN and an AD.MN, respectively, a,b: effects of 5-HT and MS, respectwely. In each panel, traces show spike activity shown by uniform pulses obtained as outputs from a window discriminator (top), instantaneous finng frequency (middle) and mean firing rate during 1 s immediately preceding each spike (bottom) Left and right dotted hnes show the onset and cessation of application of 5-HT (a) and MS (b), respectively.

rons tended to vary with their control firing rates before 5-HT application. To study a possible correlation of the facilitatory effects with the control firing rate in individual motoneurons, effects of application of the same amount of 5-HT were tested on discharges at various rates in 3 M A - M N s and 2 A D . M N s . The control discharges at various rates were induced by iontophoretic application of glutamate at different current intensities. Fig. 3 illustrates the firing rates during the 10 s period including the maximum facilitation or inhibition, represented as the multiples of the control rates during 10 s before 5-HT application (ordinate) against the control firing rates (abscissa). Thus the lower the control firing rate, the stronger the facilitatory effect of 5-HT in each M A . M N as well as A D . M N . In the range of a control rate of 15-20 Hz, there were no neurons that showed the firing rate exceeding twice the control. Some neurons that showed a remarkable facilitatory response at a lower control rate exhibited no responses at a control rate of higher than 15 Hz.

onset of 5-HT application was 0.42 + 0.08 (mean + S.D., n = 6) (Fig. 4 A left, column, filled square), and increased to 0.65 + 0.05 after 5-HT application (Fig. 4A middle column, filled square) (P < 0.05, t-test). This neuron decreased its firing index from 0.58 + 0.08 during the control period (Fig. 4A left column, open square) to 0.35 + 0.10 during MS application (Fig. 4A middle column, open square) (P < 0.05, t-test). The firing index showed

9.0"

8.07.0-

6.0-

~ 5.0~4.03.0.

I..

~.

2.0.

Effects of 5-HT and MS on MA.MN discharge evoked by stimulation of the trigeminal mesencephalic nucleus Stimulation of the trigeminal mesencephalic nucleus evoked a spike potential after a latency of 1.0-2.0 ms in the ipsilateral M A . M N s . We observed the effects of 5-HT and MS iontophoretically applied to M A . M N s on monosynaptically driven spike potentials when the latency was 1.5 ms or less 4°. Fig. 4 shows an example of M A . M N s that responded with an increase in the firing index to 5-HT and a decrease to MS. In this neuron, the firing index during the 60 s period immediately preceding the

i.o. o.o

o 6 i'o r5 z'o Hz Fig. 3 Facilitatory effects on the rate of glutamate-induced discharge of trigemmal motoneurons as a function of the firing rate during the control periods Diamonds, circles and triangles connected with solid hnes represent 3 MA.MNs, and squares and inverted triangles connected with broken lines represent 2 AD.MNs. Filled symbols indicate that the firing rate exceeded the mean control firing rates plus 2 S.D Abscissa, the firing rate during the control period; ordinate, the highest and lowest finng rates during 5-HT apphcation represented as multiples of the control finng rate.

130

A

1.0-

control

A

~v

4--"t . . . .

5-HT MS

~,~..~....................... IU'ffrll r[ . . . .

u~

recovery

I

.....

L.~

~v _'3 40C,j

B ~ ,.~,,.~,,.. ,,. ,. . . . . . J ' :i:: . r l q ~ r l l ) r [ - r l ] 1"3~ I" I

q ' : : ='-:~'""

0.8o -o

0.6-

o

._

o o

0.4-

Go

N

•

o

" ;"t

o

b

0.2-

o

~

o

N

•

e

,

,-,.I

01

'

dynamicf

0.0

'o

so' 'o

B a.

so'

'o

"

ffo,vbcon.o.I/

Sos

oo,v

Fig. 4. Effects of 5-HT and MS on firing index of spikes of a MA.MN monosynaptically evoked by stimulation of the tngemmal mesencephalic nucleus (I/s, 0.1 ms m duration, 150 pA) A: finng radices before (left), during (middle) and after application (right) of either 5-HT (filled circles) or MS (open orcles). The left was recorded during 60 s immediately preceding the onset of drug application, the middle dunng 60 s from 60 to 120 s after the onset, and the right during 60 s from 300 to 360 s after cessation of drug applicatmn. Filled and open squares with bars m the left, middle and right panels represent the mean firing indices w~th 1 S.D. before, dunng and after drug applicatmn, respectively B: spikes of the MA.MN (10 sweeps superimposed), the firing indices of which are shown in A. a: antidromic spikes evoked by stimulatmn of the masseter nerve (l/s, 0.1 ms in duration, 1.0 V). b: spikes recorded before and during 5-HT application; c: those recorded before and during MS application The finng indices in B b and B c correspond to circles in&cated by arrows b and c m the left and middle panels of A

I__i,. , "

2'

3~

=siat,c '

d~/nam)c

static

Fig. 5. Effects of 5-HT on stretch reflex discharge of a MA-MN reduced by a ramp-and-hold depression of the mandible. A and B illustrate &scharges before and during 5-HT application; top: reflex discharge (single sweep); middle: displacement of the mechanical dewce, the law depressmn Is shown upward and 0 mm corresponds to the rest positron; bottom: histogram of the sum of number of spikes induced by 6 trials of the jaw depression (50 ms per bin); dynamic and static' periods during which numbers of spikes were counted. that t h e r e was an increase in r e s p o n s e to 5 - H T in 3 of 7 MA.MNs

t e s t e d ( P < 0.05, t-test) a n d a d e c r e a s e in

r e s p o n s e to M S in 3 of 6 M A . M N s t-test) (Table II). T w o M A . M N s

t e s t e d ( P < 0.05,

s h o w e d an o p p o s i t e

r e s p o n s e to t h e s e drugs, consisting o f an i n c r e a s e to 5 - H T and a d e c r e a s e to MS. E v e n t h e 3 M A . M N s s h o w i n g no significant increase in t h e firing i n d e x after 5 - H T application s h o w e d a t r e n d t o w a r d s an i n c r e a s e in t h e firing i n d e x after 5 - H T application.

Effects o f 5 - H T on phasic and tonic stretch reflex discharge o f M A . M N s T h e effect of 5 - H T was s t u d i e d on t h e d i s c h a r g e of 8 M A . M N s d u r i n g t h e d y n a m i c a n d static phases of stretch

TABLE II

Effects of 5-HT (upper panel) and MS (lower panel) on firing of MA MNs monosynapttcally evoked by sumulaaon of the tngemmal mesencephahc nucleus Columns represent, from left to right, kinds of drugs mntophoretlcally applied to MA.MNs, reduced effects on firing indices, current intensity used for electron of drugs, control firing in&ces ± S D before apphcation and those dunng application of respective drugs.

Drug

Effects

Current (nA )

Before apphcauon

Dunng apphcat~on

5-HT

Facditatmn (n=3)

50 60 40 50 80 80 40

0 58 ± 0 08 036±005 0~±0.08 0.52±017 054±005 0.27±0.07 0.38±0.~

0.76 ± 0.13 046±005 0.65±005 073±023 0.65±021 0.36±0.05 0.39±012

Inhibition (n=3)

45 50

Nochange (n=3)

25 40 60

065±010 031±0.11 0.58±008 076±013 0.30±014 045±007

048±008 0.10±0.10 0.35±010 060±008 045±015 0.53±007

Nochange (n=4)

MS

TABLE III

Effects of f-HT on stretch reflex dlscharge of MA. MNs Columns represent, from left to right, ldentlficaUon number of tested neurons, current intensity used for ejection of 5-HT, mean spike numbers + S D. during the dynamic phase and those during the staUc phase, control and during 5-HT represent mean spike numbers without and during applicaUon of 5-HT, respecnvely. The mean spike number ± S D. was obtained by averaging the number of sp,kes reduced during 6 trials m each neuron. * Indicates no slgmficant &fference between control and durmg 5-HT (P > 0.05, t-test)

Neuron no

Current Dynamtc phase (nA) Control Dunng 5-HT

Stauc phase Control

Durmg 5-HT

1 2 3 4 5 6 7 8

80 50 60 60 20 20 10 30

119±41 70±40 0.6±09 32±1.6 1.2±04 1.7±27 01±04 02±0.4

190±2.1 12.7±40 8.0±33 187±5.4 3.3±18 42±1.2" 2.3±3.5* 3.7±23

18,3±29 285±2.5 13.4±34 182±32 4.2±18 9.2±19 78±17 223±33 40±17 80±3.4 02±04 12±08 13±11 47±23 17±1.5 40±18

131 reflex of the masseter muscle induced by a rampand-hold depression of the jaw in 5 decerebrate animals. Fig. 5 illustrates a typical example. This MA.MN showed 4.0 and 1.1 spikes on the average during the dynamic and static phases of 6 trials, respectively (Fig. 5A). During 5-HT application, the number of the induced spikes during the dynamic and static phases of the same stretch reflex increased to 8.0 and 3.3 on the average, respectively (Fig. 5B). The effects of 5-HT on 8 MA.MNs tested are shown in Table III. Since glutamate was not applied to these MA.MNs, their spontaneous activity was low, no change in discharge was induced even after 5-HT application when the stretch was not applied to the masseter muscle. As seen in Table III, 5-HT increased the number of the induced spikes during the dynamic phase of stretch reflex in all the 8 MA.MNs tested (P < 0.05, t-test) and during the static phase of 6 of 8 MA.MNs tested (P < 0.05, t-test), respectively. Thus 5-HT exerted a strong facilitatory effect on static as well as phasic stretch reflex of MA.MNs. DISCUSSION

Effects of 5-HT on trigeminal motoneurons The present study has demonstrated that the main effect of iontophoretically applied 5-HT on MA.MNs and AD.MNs is a non-reciprocal facilitation of their glutamate-induced discharge, and that 5-HT alone does not excite either MA.MNs or AD.MNs. The results are consistent with some reports of local application of 5-HT to facial as well as spinal motoneurons 35'56'5s, but do not agree with other earlier reports of 5-HT effects on spinal motoneurons 22'45. The difference in intensities of currents for 5-HT ejection might be a factor contributing to the discrepancy: application of a small amount of 5-HT (25 nA ejection current) caused an increased excitability in facial motoneurons 57, while application of a large amount of 5-HT (150 nA) to spinal motoneurons is reported to cause a decreased excitability44. In the present study the current for 5-HT ejection was set to 80 nA or less. It has also been shown that a specific antagonist of 5-HT, methysergide (MS), could inhibit the glutamate-induced discharge of MA.MNs and AD.MNs, which is consistent with the effects of another specific antagonist of 5-HT, metergoline, on spinal motoneurons5s. 5-HT showed a consistent effect of facilitation on glutamate-induced discharge of MA-MNs whenever any effect was observed, while 5-HT facilitated the glutamate-induced discharge of about a half of AD.MNs but inhibited it in a few of them. MS exerted a facilitatory effect on one of the latter group of AD.MNs, though it showed no effects on the remaining 3 AD.MNs Based on

the molecular, biochemical and physiological studies, 5-HT receptors have been classified into several subtypes: 5-HTIA_O, 5-HT 2 and 5-HT 3 receptors 43'44. The 5-HT 1 receptors may mediate inhibitory responses while the 5-HT 2 receptors trigger excitatory responses 35'43'44. Thus the great majority of 5-HT receptors richly distributed on MA-MNs may consist of 5-HT 2 receptors leading to their excitation. On the other hand, a substantial number of 5-HT receptors on AD.MNs may be 5-HT1 receptors in addition to 5-HT 2 receptors. This may cause either excitation or inhibition of these motoneurons as an overall effect. In our study a majority of MA.MNs was facilitated with 5-HT and the incidence of the facilitation with 5-HT application was significantly higher in MA.MNs than AD.MNs. One may argue that the 5-HT applied to AD.MNs was less than that to MA.MNs. This possibility can be excluded, because there was no difference in the range of intensity of current for ejection of 5-HT between both groups of trigeminal motoneurons. Another possibility is that AD.MNs may be tonically so strongly facilitated by endogenous 5-HT that they may not respond to exogenous 5-HT. This is also unlikely, because the incidence of inhibition induced by MS was far less in AD.MNs than in MA.MNs. Probably AD.MNs have a lower sensitivity to 5-HT because of their fewer 5-HT receptive sites and/or the lower incidence in AD.MNs that have 5-HT receptors than in MA.MNs. In his histochemical study, Steinbusch ~3 reported that 5-HTpositive fiber terminals are denser in the rostral portion than m the caudal portion of the trigeminal motor nucleus. Anatomical studies of myotopical organization of trigeminal motor nucleus in various species, including the guinea pig, have shown a consistent pattern in this nucleus: MA.MNs are located in the dorsolateral portion of the nucleus throughout its rostrocaudal extent, while AD.MNs are located in the ventromedial portion at the caudal two-thirds of this nucleus 31'34'36'37'48'56. Thus, a less serotonergic innervation to AD.MNs may be a factor for the lower sensitivity of AD.MNs to 5-HT. The MA.MNs which showed no response to 5-HT may have been recorded from the caudal portion of the masseter motoneuron pool where serotonergic innervation is sparse. The raphe nuclei can be a possible source of the serotonergic innervation of the trigeminal motor nucleus, because fluorescent neurons containing 5-HT were located in almost all the raphe nuclei 8"17. In addition, direct projections to the trigeminal motor nucleus were demonstrated from the nuclei raphe centralis superior and raphe magnus in the cat by an anterograde autoradiographic study of injection of radioactwe leucine into the raphe nuclei, though these projections cannot be inter-

132 preted as a pure demonstration of 5-HT projections9. Stimulation of the raphe nuclei was reported to suppress the jaw-opening reflex evoked by electrical stimulation of the trigeminai sensory nerves including the tooth pulp in the cat 19'41'51. The raphe stimulation was shown to suppress also the responses to innocuous as well as noxious trigeminal afferent stimulation of the neurons in the cat trigeminal spinal nucleus 47'5~'52, where the excitatory interneurons mediating the jaw-opening reflex were reported to be located 55. The projections from the nuclei raphe centralis superior, raphe magnus and raphe pontis to the spinal nucleus were also demonstrated by the autoradiographic study mentioned above 9. If the results of the present study are taken into consideration that 5-HT facilitates the response of AD.MNs to excitatory inputs, the suppression of the jaw-opening reflex by the raphe stimulation could be ascribed to inhibition of the interneurons in the trigeminal spinal nucleus involved in the jaw-opening reflex.

Effects of 5-HT on stretch reflex discharge of MA.MNs In the present study, iontophoretically applied 5-HT facilitated the discharge of MA-MNs during the phasic and tonic phases of passive jaw-depression. Jaw-depression induces a condylar rotation of the temporomandibular joint (TMJ) as well as stretching of the jaw-closing muscles, which in turn generate impulses in the afferent fibers from the muscle spindles in the jaw-closing muscles 14"15'26and from the joint receptors 27. Mechanical stimuli including the condylar rotation of the TMJ activate low-threshold mechanosensitive TMJ primary afferents, leading to inhibition of the jaw-closer motoneurons including the a-MA.MNs 1. Thus, the discharge of MA.MNs induced by passive jaw-depression would primarily be due to excitation of MA.MNs by the muscle spindle afferents originating from the stretched jawclosing muscles. The jaw-closing muscles are richly supplied with muscle spindles 2s, and their activity has been demonstrated during mastication and jaw-jerk 5"13'33. Stimulation of the trigeminal mesencephalic nucleus monosynaptically excites a-MA.MNs 28'39, and di- or trlsynaptically y-MA.MNs 5°. It has also been demonstrated that Group Ia and II fibers from the muscle spindles of the masseter muscle make monosynaptic excitatory connections with MA.MNs 6. The MA.MNs studied in the present work were monosynaptically driven by stimulation of the trigeminal mesencephalic nucleus and responded with spikes to the stretch of the masseter muscle. In addition, it was reported that no responses were induced by passive jaw movement in y-MA.MNs 33.

These reports indicate that ~t was the stretch reflex discharge of a-MA-MNs induced by Group Ia and II muscle spindle afferents from the jaw-closing muscles that was facilitated by locally applied 5-HT.

Roles of 5-HT in control of trtgemmal motoneuron activity As stated above, 5-HT non-reciprocally facilitates the glutamate-induced discharge of MA.MNs and AD.MNs and the stretch-induced discharge of MA.MNs. However, 5-HT alone evoked no discharges in either MA.MNs or AD.MNs. This mode of action of 5-HT was reported with respect to spinal and facial motoneurons 35' 58. It is likely that 5-HT exerts a modulating action on excitatory inputs rather than a direct excitatory action on trigeminal motoneurons. Recently Chandler has suggested that an excitatory amino acid, activating nonNMDA receptors, mediates some component of synaptic transmission from the mesencephalic trigeminal nucleus to jaw-closer motoneurons 11. A specific antagonist of 5-HT (MS) depressed the glutamate-induced discharge in 20 of 34 MA.MNs and in 6 of 30 AD.MNs, suggesting that 5-HT tonically exerts its modulating action on trigeminal motoneurons, readily to respond to excitatory inputs. In this regard, the result is to be noted that the lower the rate of glutamate-induced discharge, the stronger the facilitatory effect of 5-HT on it. 5-HT seems to play the role of 'a gain setter to enhance the effects of excitatory afferent inputs' to motoneurons 35, with such a variable gain as to set the level of activity of the motoneuron in a certain range. Strahlendorf et al. 54 reported a similar correlation between the spontaneous firing rate of cerebellar Purkinje cells and the action of iontophoretically applied 5-HT, and suggested that the overall qualitative effects of 5-HT were to set Purkinje cells at a preferred firing rate. Another finding to note in this study is that 5-HT exerts a more consistent enhancing effect on MA.MNs than on AD-MNs. It was demonstrated that stretch reflex of jaw-closing muscles plays a critical role for maintenance of jaw-position during locomotion of terrestrial mammals 24'32. Serotonerglc excitatory inputs to jawcloser motoneurons may contribute to stabilizing the jaw-position by facilitating the stretch reflex of jawclosing muscles during locomotion.

Acknowledgements. This study was supported by a Grant-m-Aid for Specml Project Research from the Ministry of Education, Science and Culture of Japan. We wish to express our thanks to Prof. E. Flucklger and Dr H Frledli, Pharmazeutlsches Department, Sandoz A G., Basel, Switzerland for kindly having provided us with methysergide We are grateful to Dr A Bingham and Dr M Nol for improving the Enghsh expressions in our manuscript.

133

REFERENCES 1 Abe, K., Takata, M. and Kawamura, Y., A study on inhibition of masseteric a-motor fibre discharges by mechanical stimulation of the temporomandibular joint in the cat, Arch Oral. Btol, 18 (1973) 301-304 2 Ahlman, H., Grlllner, S. and Udo, M , The effect of 5-HTP on the static fusimotor activity and the tonic stretch reflex of an extensor muscle, Brain Research, 27 (1971) 393-396. 3 Anden, N.E and Jukes, M.G.M. and Lundberg, A , Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 4 Anderson, E G. and Shlbuya, T., The effects of 5-hydroxytryptophan and L-tryptophan on spinal synaptic activity, J Pharmacol. Exp. Ther., 153 (1966) 352-360. 5 Appenteng, K., Monmoto, T. and Taylor, A , Fuslmotor activity in masseter nerve of the cat during reflex jaw movements, J Physiol. (Lond.), 305 (1980) 415-431 6 Appenteng, K., O'Donovan, M.J., Somjen, G , Stephens, J.A. and Taylor, A., The projection of jaw elevator muscle spindle afferents to fifth nerve motoneurones in the cat, J Physiol. (Lond.), 279 (1978) 409-423. 7 Barasi, S. and Roberts, M.H.T., The modification of lumbar motoneurone exotabihty by stimulation of a putative 5-hydroxytryptamine pathway, Br J. Pharmacol., 52 (1974) 339-348. 8 Bj6rklund, A , Falck, B. and Stenevl, U., Classification of monoamine neurons in the rat mesencephalon: distribution of a new monoamine nervous system, Brain Research, 32 (1971) 1-17 9 Bobdlier, P., Seguin, S., Petitjean, E, Salvert, D , Touret, M. and Jouvet, M , The raphe nuclei of the cat bralnstem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976) 449-486 10 Carp, J S. and Rymer, W . Z , Enhancement by serotonin of tonic vibration and stretch reflexes in the decerebrate cat, Exp. Brain Res., 62 (1986) 111-126 11 Chandler, S . H , Evidence for excitatory amino acid transmission between mesencephahc nucleus of V afferents and jaw-closer motoneurons in the guinea pig, Brain Research, 477 (1989) 252-264 12 Cllneschmldt, B , Pierce, J.E. and Sjoerdsma, A., Interactions of tncyclic antidepressants and 5-hydroxylndolealkylamine precursors on spinal monosynaptlc reflex transmission, J. Pharmacol Exp Ther., 179 (1971) 312-323 13 Cody, FW.J., Hamson, L.M. and Taylor, A., Analysis of activity of muscle spindles of the jaw-closing muscles during normal movements in the cat, J. PhystoL (Lond), 253 (1975) 565-582 14 Cody, FW.J., Lee, R W.H. and Taylor, A., A funcUonal analysis of the components of the mesencephahc nucleus of the fifth nerve in the cat, J Phystol (Lond.), 226 (1972) 249-261 15 Corbin, K.B and Harnson, E, Function of mesencephallc root of fifth cranial nerve, J Neurophyslol., 3 (1940) 423-435 16 Cropper, E C., Elsenman, J S and Azmitia, E C , 5-HTimmunoreactive fibers in the trigemlnal nuclear complex of the rat, Exp. Brain Res., 55 (1984) 515-522 17 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamlne-contalnlng neurons in the central nervous system I Demonstration of monoamlnes in the cell bodies of brain stem neurons, Acta Phystol Scand., 62, Suppl. 232 (1964) 1-55. 18 Dahlstrom, A and Fuxe, K , Evidence for the existence of monoamlne neurons in the central nervous system. II Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta Physiol. Scand, 64 Suppl. 247 (1965) 1-36 19 Dostrovsky, J.O., Hu, J W , Sessle, B.J. and Sumlno, R., Stimulation sites in perlaqueductal gray, nucleus raphe magnus and adjacent regions effective in suppressing oral-facial reflexes, Brain Research, 252 (1982) 287-297 20 Ellaway, P H and Trott, J R , The mode of action of 5-

hydroxytryptophan in faclfitatlng a stretch reflex in the spinal cat, Exp Brain Res., 22 (1975) 145-162. 21 Engberg, I , Flatman, J.A. and Kadzielawa, K., Lack of specaficity of motoneuron responses to microiontophoretically applied phenolic amines, Acta Physiol. Scand., 96 (1976) 137-139 22 Engberg, I. and Ryall, R.W., The inhibitory action of noradrenaline and other monoamines on spinal neurones, J Phystol. (Lond.), 185 (1966) 298-322. 23 Fuxe, K , Evidence for the existence of monoamine-containing neurons in the central nervous system IV. The distribution of monoamine terminals in the central nervous system, Acta Physiol. Scand., 64 Suppl. 247 (1965) 37-85 24 Goodwln, G . M , Hoffman, D. and Luschel, E.S., The strength of the reflex response to slnusoidal stretch of monkey law closing muscles during voluntary contraction, J Physiol. (Lond.), 279 (1978) 81-111 25 Hosokawa, H., Proprioceptlve innervation of striated muscle in the territory of the cranial nerves, Texas Rep. Biol Med., 19 (1961) 405-464. 26 Jerge, C R , Organization and function of the tngeminal mesencephalic nucleus, J. Neurophysiol., 26 (1963) 379-392. 27 Kawamura, Y. and Abe, K., Role of sensory information from temporomandlbular joint, Bull Tokyo Med. Dent. Umv., 21 (1974) Suppl 78-82. 28 Kidokoro, Y., Kubota, K., Shuto, S. and Sumino, R., Reflex organization of cat masticatory muscles, J. Neurophysiol., 31 (1968) 709-716 29 Kurasawa, I., Toda, K. and Nakamura, Y , Effects of local apphcation of serotomn on the trigeminal motoneuron activity in the guinea pig, J. Phystol. Soc Jpn., 49 (1987) 412. 30 Kurasawa, I., Toda, K. and Nakamura, Y., Serotomn effects on trigeminal motoneurons are dependent on baseline activities, J. Dent Res., 67 (1988) 735. 31 Limwongse, V and DeSantis, M , Cell body locations and axonal pathways of neurons innervating muscles of mastication in the rat, Am J. Anat, 149 (1977) 477-488. 32 Lund, J P, Drew, T and Rosslgnol, S , A study of jaw reflexes of the awake cat dunng mastication and locomotion, Brain Behav. Evol, 25 (1984) 146-156. 33 Lund, J.P, Smith, A . M , Sessle, B.J. and Murakami, T Activity of trigeminal a- and ~-motoneurons and muscle afferents during performance of a biting task, J. Neurophystol., 42 (1979) 710-725. 34 Matsuda, K , Uemura, M , Kume, M,, Matsushima, R. and Mizuno, N., Topographical representation of masticatory muscles in the motor trigeminai nucleus in the rabbit: a HRP study, Neuroscl Lett., 8 (1978) 1-4 35 McCall, R.B. and Aghajanlan, G.K., Serotonerglc facilitation of facial motoneuron excitation, Brain Research, 169 (1970) 11-27. 36 Mizuno, N., Konishl, A. and Sato, M., Locahzation of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 164 (1975) 105-116 37 Mlzuno, N , Matsuda, K., Iwahon, N., Uemura-Sumi, M., Kume, M and Matsushlma, R., Representation of the mastlcatory muscles in the motor tngeminal nuclus of the macaque monkey, Neurosct Lett., 21 (1981) 19-22. 38 Myhnskl, N R. and Anderson, E.G., The effect of serotonin precursors on alpha- and gamma-motoneuron activity, J. Pharmacol Exp. Ther, 204 (1978) 19-26 39 Nakamura, Y , Goldberg, L J. and Clemente, C D., Nature of suppression of the masseteric monosynaptic reflex induced by stimulation of the orbital gyrus of the cat, Brain Research, 6 (1967) 184-198 40 Nozakl, S., Irlki, A. and Nakamura, Y., Trigeminal mesencephalic neurons innervating functionally identified muscle spindles and involved in the monosynaptlc stretch reflex of the lateral pterygoid muscle of the guinea pig, J Comp. Neurol., 236 (1985) 106-120

134 41 Ohveras, J L., Gudbaud, C. and Besson, J.M., The use of tooth pulp stimulation to evaluate the analgesia induced by stimulation of raphe nuclei in the cat In D.J. Anderson and B Matthews (Eds.), Pam m the Trtgernmal Region, Elsevier, Amsterdam, 1977, pp. 319-330. 42 Palkovits, M., Brownstem, M and Saaverda, J . M , Serntonm content of the brainstem nuclei m the rat, Brain Research, 80 (1974) 237-249 43 Peroutka, S J., 5-Hydroxytryptamme receptor subtypes, molecular, biochemical and physiological characterization, Trends Neurosci., 11 (1988) 496--500. 44 Peroutka, S.J., Lebovitz, R.M. and Snyder, S.H., Two distract central serotonm receptors with different physiological functions, Science, 212 (1981) 827-829. 45 Phillis, J.W., Tebecis, A K. and York, D . H , Depression of spinal motoneurones by noradrenahne, 5-hydroxytryptamme and histamine, Eur. J. Pharmacol., 4 (1968) 471-475 46 Rossner, W., Stereotakttscher Htrnatlas vorn Meerschwemchen, Pallas Verlag, Lochham bei Munchen, 1965, 31 pp. 47 Sasa, M., Munekiyo, K and Takaori, S., Dorsal raphe st~mulauon produces inhibitory effect on tngemmal nucleus neurons, Brain Research, 101 (1975) 199-207. 48 Sasamoto, K , Motor nuclear representation of masticatory muscles in the rat, Jpn. J Physiol., 29 (1979) 739-747 49 Sastry, B S.R and Sinclair, J . G , Serotonm involvement m the blockade of buibospmal mhibmon of the monosynaptlc reflex, Brain Research, 115 (1976) 427-436. 50 Sessle, B J , Identification of alpha and gamma tngemmal motoneurons and effects of stimulation of amygdala, cerebellum, and cerebral cortex, Exp Neurol, 54 (1977) 303-322

51 Sessle, B J and Hu, J W, Raphe-induced suppression of the jaw-opening reflex and single neurons in tngeminal subnucleus orahs, and influence of naloxone and subnucleus caudahs, Pam, 10 (1981) 19-36. 52 Sessle, B J., Hu, J.W., Dubner, R and Lucler, G.E., Functional properties of neurons m cat trigemmal subnucleus caudahs (medullary dorsal horn) II. Modulauon of responses to noxious and nonnoxlous stimuli by perlaqueductai gray, nucleus raphe magnus, cerebral cortex, and afferent influences, and effects of naloxone, J Neurophystol, 45 (1981) 193-207 53 Stembusch, H . W M , Distribution of serotonin-tmmunoreactlwty m the central nervous system of the rat - - cell bodies and terminals, Neurosctence, 6 (1981) 557-618 54 Strahlendorf, J C., Lee, M and Strahlendorf, H . K , Effects of serotonm on cerebellar Purkmle cells are dependent on the baseline firing rate, Exp Brain Res., 56 (1984) 50-58. 55 Summo, R., Central neural pathways revolved in the lawopening reflex m the cat. In R. Dubner and Y. Kawamura (Eds), Oral-Faczal Sensory and Motor Mechanisms, AppletonCentury-Crofts, New York, 1971, pp 315-331 56 Uemura-Sumi, M , Takahashl, O., Matsushlma, R., Takata, M., YasuL Y. and Mizuno, N , Locahzatlon of mastlcatory motoneurons in the trlgemmal motor nucleus of the guinea pig, Neurosct. Len, 29 (1982) 219-224 57 VanderMaelen, C P. and Aghajaman, G K., Intracellular studles showing modulation of facial motoneurone excltabdity by serotomn, Nature (Lond.), 287 (1980) 346-347. 58 White, S R. and Neuman, R . S , Facihtatlon of spinal motoneuron exc~tablhty by 5-hydroxytryptamme and noradrenaline, Brain Research, 188 (1980) 119-127