55
Brain Research, 560 (1991) 55-62 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116969N
BRES 16969
Evaluation of vagal afferent modulation of the digastric reflex in cats W. Maixner,
D.F.
Bossut
and E.A.
Whitsel
Dental Research Center and the Departments of Pharmacology and Endodontics, University of North Carolina, Chapel Hill, NC 27599-7455 (U.S.A.)
(Accepted 30 April 1991) Key words: Reflex; Vagus; Vagal afferent; Baroreceptor; Pain; Nociception; Tooth pulp; Analgesia; Cardiovascular
In the present study, we have examined the relative ability of cervical, thoracic, cardiac and diaphragmatic vagal stimulation to modulate the digastic reflex produced by tooth-pulp stimulation in anesthetized cats. The right maxillary tooth pulp was stimulated and the digastric reflex was recorded from the fight digastric muscle. Cervical vagal stimulation produced a biphasic effect on the digastric reflex. The reflex was facilitated at conditioning test intervals less than 20 ms and inhibited at conditioning test intervals between 100 ms and 500 ms. Cardiac and thoracic vagal stimulation did not significantly facilitate the digastric reflex but inhibited the reflex at conditioning test intervals between 50 ms and 500 ms with maximum inhibition observed at 200 ms. In contrast, diaphragmatic vagal stimulation produced a weaker inhibition of the digastric reflex. The relative ability of different vagal segments to inhibit the digastric reflex was: thoracic = cardiac = cervical > diaphragmatic. The inhibitory effects were not related to cardiovascular responses to vagal afferent stimulation. These findings suggest cardiopulmonary vagal afferents represent an important source of vagal afferents which modulate the digastric reflex in the cat. INTRODUCTION The electrical stimulation of afferents which course in the cervical vagus produces both facilitatory and inhibitory effects on reflex responses to noxious stimuli 9A°'28' 33,34. Depending upon stimulation parameters, cervical vagal stimulation produces either a facilitation or an inhibition of the digastric reflex in the cat and reflex (i.e. tail-flick reflex) responses to noxious radiant heat in the rat 9A°'28"33. Several recent electrophysiological studies have also demonstrated that electrical stimulation of either cervical or thoracic vagal afferents produces either facilitatory, inhibitory or both effects on dorsal horn neurons in the rat, cat and m o n k e y 4'16'34"36. The anatomical origins of the vagal afferents which modulate somatic reflexes evoked by noxious stimuli have not been extensively examined. It has been suggested that vagal afferents originating from cardiopulmonary tissues represent a major source of vagal afferents which modulate nociceptive reflexes 28"3°. In support of this view, the physiological stimulation of afferents, induced by vascular volume expansion, inhibits the tailflick reflex in the rat 2°. Similarly, the pharmacological stimulation of vagal afferents, produced by the intravenous administration of non-selective opiate-receptor agonists, results in an immediate and profound inhibition of the tail-flick reflex. The antinociceptive effects pro-
duced by these two experimental procedures are attenuated by cervical vagotomy 29'32. These findings, though suggestive, do not provide conclusive evidence that cardiopulmonary vagal afferent stimulation produces antinociception because their conclusions are based on experimental manipulations of the cervical vagus which contains afferents originating from anatomical regions both rostral and caudal to cardiopulmonary tissues. It has been demonstrated recently that abdominal vagal afferents modulate reflex responses to noxious stimuli in the rat 33. In contrast, electrophysiological studies conducted on cats and monkeys have found that abdominal vagal afferent stimulation produces very weak inhibitory effects on the responses of spinal sensory neurons to noxious stimuli 4'5"16. Thus, the degree to which different sub-populations of vagal afferents influence reflex and neuronal responses to noxious stimuli remains unclear. In addition, the relative ability of vagal afferents originating from different peripheral sites to modulate polysynaptic reflexes other than the tail-flick reflex has not been systematically evaluated. In the present study, we have examined the relative ability of cervical, thoracic, cardiac and diaphragmatic vagal afferent stimulation to modulate the digastric reflex produced by tooth-pulp stimulation in cats. Since vagal stimulation produces a variety of cardiovascular responses 24'25, which may in some way contribute to the
Correspondence: W. Maixner, Rm 03 Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455, U.S.A. Fax: (1)
(919) 966-3683.
56 inhibitory effects on reflexes produced
by noxious stim-
uli, t h e r e l a t i o n s h i p b e t w e e n t h e c a r d i o v a s c u l a r r e s p o n s e s to vagal stimulation and the effects on the digastric reflex h a s a l s o b e e n e x a m i n e d . P r e l i m i n a r y r e p o r t s o f t h e s e findings have appeared
e l s e w h e r e ~9
MATERIALS AND M E T H O D S
General preparation Eighteen adult cats weighing from 1.6 to 3.9 kg (2.56 _+ 0.11 kg), were anesthetized with pentobarbital sodium (40-45 mg/kg, i.p.) and placed in a supine position. Rectal temperature was maintained between 36.5 and 37.5 °C by a thermostatically controlled electric heating blanket. The trachea was cannulated and the cat was ventilated artificially with room air. Stroke volume and respiration rate were adjusted to maintain expired CO 2 (measured with an infrared capnometer) levels between 3,5 and 4.5%. The left femoral vein and artery were cannulated and arterial blood pressure and heart rate were monitored. Deep anesthesia was produced with a-chloralose (60 mg/kg, i.v.) and maintained with an intravenous infusion of a-chloralose (3-5 mg/kg/h) or supplemental bolus doses as required. Adequacy of anesthesia was evaluated by monitoring arterial blood pressure, checking for blink reflex, and testing for hindlimb flexion reflex in response to strong paw pinch.
Tooth preparation On the facial surface of the right maxillary canine, 1-2 mm coronal to the gingival border, a small pit preparation was made in the enamel with a high speed dental drill until the dentino-enamel junction was reached. A hand held carbide bur was used to deepen the preparation while irrigating with isotonic saline until the pulpal vasculature was just visible. A male Amphenol ® pin was placed in contact with the bottom of the preparation and secured to the tooth with UV light-cured composite resin. The pin was insulated from the surrounding tissue using dental baseplate wax.
Tooth pulp (TP) stimulation Constant-current rectangular pulses (1.0 ms) were delivered from the cathodal pole to the TP once every 5 s to produce a reflex contraction of the ipsilateral digastric muscle. The cathodal pole was attached to the TP and the anodal pole was connected to the right ear. The intensity of the current delivered to the TP was monitored on a storage oscilloscope by assessing the voltage drop across a 100 f2 resistor in series with the cathodal pole. Fibers originating in the TP produced the reflex since pulpectomy abolished it at intensities up to 5× threshold.
stimulated. In 8 cats, the right cervical vagus was deshcathcd and isolated from the cervical sympathetics and carotid artery. The aortic depressor nerve was isolated from the cervical vagus when thcy appeared as two separate nerve trunks. In 3 preparations the w~gus was transected and in the remaining 5 preparations the vagus was left intact. Since no significant qualitative or quantitative differences were found between the effects of conditioning intact w~gus vs. the central ends of transected vagus, the data obtained from both preparations were combined for analysis. In a separate group of 10 cats, a right thoracotomy in the fourth intercostal space was performed to expose the right cardiac branch of the vagus and the thoracic bundle of the vagus just cranial to the cardiac branch. To facilitate exposure, the azygous vein was transected and the apical lobe of the right lung was resectcd. In 5 of the l0 cats, a right thoracotomy in the ninth intercostal space was also performed to isolate the vagus just cranial to the diaphragm. The vagus was left intact at these last three conditioning sites. All vagal conditioning sites were immersed in mineral oil warmed to 37 °C.
Vagal conditioning The vagus was stimulated with a bipolar platinum electrode with a pole-to-pole separation of 2-3 mm at the conditioning sites shown in Fig. 1. Anodal current was passed through the pole most distal to the brain to minimize anodal blockade. Current intensities were monitored as described for TP stimulation. All vagal sites were stimulated or conditioned with 333 Hz, 20 ms trains of pulses which were 1.0 ms in duration at 0.0 mA, 5.0 mA or 10.0 m A intensities. At the end of 3 experiments, the effects of conditioning the intact thoracic vagus were compared to effects of conditioning the peripheral end of the transected thoracic vagus. This was done to assess the degree to which vagal efferents contributed to the observed outcomes.
Experimental procedures First, baseline or control reflex magnitude was determined by signal-averaging l0 individual E M G recordings which were elicited at an intertrial interval of 5 s. Then, TP stimulation (i.e., test stimulus) resumed and 10 E M G curves were signal-averaged following the delivery of conditioning stimuli to the vagus at conditioning test intervals (CTIs) ranging from 5 to 1000 ms. The effects of vagal conditioning were evaluated at each intensity and CTI for each vagal conditioning site.
CERVICAL VAGUS
Electromyogram (EMG) recording The digastric reflex was monitored by transcutaneously implanting two stainless steel electrodes into the anterior belly of the right digastric muscle approximately 1 cm from each other. Differential recordings were obtained with a low-noise amplifier with a bandpass of 10 Hz to 1 kHz. All electromyographic activity usually occurred within 20 ms of TP stimulation and was displayed on a storage oscilloscope. With the aid of a computer, digitized (50 kHz) traces were displayed on-line, signal-averaged, and stored for offline data analysis.
THORACIC VAGUS
Digastric reflex threshold determination Analysis of threshold was performed frequently throughout the experiment. Pulse duration was set at 1.0 ms and pulse intensity was increased until 4 of 5 consecutive EMG responses to TP stimulation were obtained. The pulse intensity which produced four traces was defined as the threshold current. All data collected involved TP stimulation at 2 - 3 x threshold.
Vagal preparation Fig. I displays the four vagal segments that were isolated and
Fig. 1. Anatomical sites of vagal conditioning.
57 Data analysis Signal-averaged E M G ' s were rectified and quantified by integration using software developed by Modular Instruments, Inc. E M G integrals were expressed as a percentage of the baseline (%BL) E M G integral for each vagal site, intensity, and CTI. The effects of various conditioning parameters on the derived percentage values were analyzed by an appropriate analysis of variance. A repeated measures design was used to assess within-group main effects and a complete block design was used to assess between-group main effects. Duncan's multiple range test was used when appropriate. Cardiovascular data and the differences between the responses to stimulating the intact vs. the peripheral end of the thoracic vagus were analyzed with a paired t-test. In all cases, significance was assumed at an a ~
E
0.0 -0.5
I
I
I
I
1
I
I
I
|
Gins
200 ms
0.5 ~::
0.0 -O.G
I
I
1
I
I
I
I
1000 ms
0.0 -0.5 0
I 4
I 8
I 12
I 16
I ~
0
I 4
I 8
ms
I 12
I 111
I 20
ms
Fig. 3. The effects of cervical vagal conditioning on the digastric reflex induced by tooth-pulp stimulation. Each electromyographic trace represents the averaged response to 10 successive stimuli applied to the ipsilateral tooth-pulp at 2× threshold intensity applied at a rate of 0.2 Hz. The first trace (upper left corner) is the control response in the absence of cervical vagal conditioning. The subsequent traces were obtained when a train of stimuli (5 mA, 1.0 ms pulses delivered at 333 Hz for 20 ms) was delivered to the central end of the cervical vagus at various intervals (5-1000 ms) prior to the test stimulus. The test stimulus artifact has been removed.
The effects of stimulating the cardiac branch of the
of conditioning stimuli to the diaphragmatic vagus did
v a g u s at 5.0 a n d 10.0 m A a r e s h o w n in Fig. 2C. C o n d i -
n o t f a c i l i t a t e t h e d i g a s t r i c reflex at b r i e f C T I s a n d p r o -
tioning of the cardiac branch of the vagus produced a
d u c e d a w e a k b u t significant i n h i b i t i o n o f t h e d i g a s t r i c
p r o f o u n d i n h i b i t i o n o f t h e reflex b u t d i d n o t f a c i l i t a t e t h e
reflex at t h e l o n g e r C T I s . S i g n i f i c a n t i n h i b i t i o n r e l a t i v e
reflex r e l a t i v e t o s h a m c o n d i t i o n i n g . A s s e e n w i t h t h o -
t o s h a m s t i m u l a t i o n was o b s e r v e d at 100 m s ( F ( 2 , 7 ) =
racic v a g a l c o n d i t i o n i n g , l o n g e r C T I s f r o m 50 to 500 m s
4.76) a n d 500 m s ( F ( 2 , 7 ) = 4.87) C T I s . T h e i n h i b i t o r y
r e s u l t e d in a p r o f o u n d i n h i b i t i o n o f t h e reflex ( f o r 50 ms:
effects w e r e e q u i v a l e n t at b o t h t h e 5.0 a n d 10.0 m A
F ( 3 , 1 8 ) = 6.01; F o r 100 ms: F ( 3 , 1 8 ) = 8.80; f o r 200 ms:
conditioning intensities.
F ( 3 , 1 8 ) = 13.96; f o r 500 ms: F ( 3 , 1 8 ) = 6.86). T h e inh i b i t o r y effects w e r e e q u i v a l e n t at 5 a n d 10.0 m A .
A s s h o w n in Fig. 4, t h e r e l a t i v e ability o f v a r i o u s vagal b r a n c h e s to i n h i b i t t h e d i g a s t r i c reflex at t h e 200 m s
T h e e f f e c t s o f s t i m u l a t i n g t h e c e n t r a l e n d o f t h e dia-
C T I (5 m A ) v a r i e d as a f u n c t i o n o f c o n d i t i o n i n g site
p h r a g m a t i c v a g u s is s h o w n in Fig. 2 D . T h e a p p l i c a t i o n
( F ( 3 , 2 9 ) = 11.14). T h e r e l a t i v e efficacy o f i n h i b i t i o n w a s
TABLE I
The effect of vagal stimulation on blood pressure and heart rate Each value represents the mean ± 1 S.E.M.
Systolic (ram Hg)
Diastolic (mm Hg)
Heart rate (BPM)
baseline
vagal
baseline
vagal
baseline
vagal
Cervical (n = 8)
5 mA 10 mA
122 ± 10 120 + 7
135 -+ 12" 138 ± 9*
88 ± 9 86 ± 7
101 ± 10" 104 +_ g*
255 -_ 7 251 --- 5
257 +- 7 251 +- 7
Thoracic (n = 9)
5 mA 10mA
112 ± 10 112 ± 10
135 ± 7* 144 ± 7*
7l -+ 9 71 ± 9
93 -+ 7* 99 ± 7*
203 ± 15 204 ± 17
206 ÷ 14 209 -+ 15
Cardiac branch (n = 10)
5 mA 10 mA
112 ± 9 108 -+ 10
130 ± 9* 129 ± 8*
76 ± 9 73 ± 10
92 ± 9* 94 ± 8*
212 ± 19 209 ± 19
223 ± 16 224 ± 17
Diaphragmatic (n = 5)
5 mA 10 mA
94 -+ 5 97 ± 9
118 ± 18 126 ± 25
58 ± 6 63 ± 11
80 ± 18 86 -+ 23
206 ± 32 206 -+ 32
212 ± 33 215 ± 33
*, Significantly different from corresponding baseline value; Student's paired t-test (P diaphragmatic. Similar findings were o b t a i n e d when the various vagal branches were conditioned with 10 m A pulses.
A
CTI = 5
CTI = 200
200
Effects of stimulating the peripheral end of the thoracic vagus
100 iE
0
2~ Iota ,,,,
2~
B
o. f--A 0.5
-0.5
The effects of vagal stimulation on b l o o d pressure and heart rate are shown in Table I. Stimulation of either the cervical, thoracic o r cardiac vagus p r o d u c e d a pressor response in all experiments at both 5 and 10 m A intensities and p r o d u c e d variable effects on heart rate. Diaphragmatic vagal stimulation increased b l o o d pressure in 3 of 5 p r e p a r a t i o n s but, as shown in Table I, did not achieve statistical signficance. Cervical vagal stimulation increased heart rate in 5 of 8 preparations, thoracic vagal stimulation increased heart rate in 5 of 9 p r e p a r a tions, cardiac vagal stimulation increased heart rate in 5 of 10 preparations, and diaphragmatic vagal stimulation increased heart rate in 3 of 5 preparations. The observed inhibitory effects on the digastric reflex p r o d u c e d by vagal stimulation were i n d e p e n d e n t of changes in arterial b l o o d pressure and heart rate. A s shown in Fig. 5, the cardiovascular responses to thoracic vagal afferent stimulation were essentially identical at 5 ms and 200 ms CTIs, yet the digastric reflex was essentially abolished at the 200 ms CTI and was still evident at the 5 ms CTI. F o r a given p r e p a r a t i o n , cervical, thoracic or cardiac vagal nerve stimulation p r o d u c e d equivalent cardiovascular responses at each CTI while producing m a r k e d l y different effects on the digastric reflex.
I'
rm
0
4
8
IIl,,S,,,,
The effects of stimulating the intact thoracic vagus or the p e r i p h e r a l end of the transected thoracic vagus were evaluated in 3 preparations. Stimulation of the intact thoracic vagus at a 200 ms C T I inhibited the digastric reflex to 94% of the control value and increased both b l o o d pressure and heart rate in all three preparations. In contrast, stimulation of the peripheral end of the thoracic vagus at a 200 ms CTI did not alter the digastric reflex or b l o o d pressure from pre-stimulation values and increased heart rate from pre-stimulation values in 2 of 3 preparations.
,,,m~,o,~w
, 12 18 20
msec
• 0
DISCUSSION
, 4
, 8
1'2 18
20
msec
Fig. 5. Blood pressure and heart rate responses to thoracic vagal conditioning at condition test intervals of 5 ms (left panel) and 200 ms (right panel) are shown in A. Each tic mark denotes the time when a test stimulus was delivered to the tooth-pulp. The lower electromyographic traces (B) represent the composite or averaged trace obtained in response to the 10 test stimuli. Note that equivalent cardiovascular responses were associated with different effects on the digastric reflex.
Previous research has d e m o n s t r a t e d that stimulation of cervical vagal afferents modulates several nociceptive reflexes including the digastric reflex. In general it has been assumed that vagal afferents originating from card i o p u l m o n a r y structures represent the p r i m a r y population of afferents that m o d u l a t e somatic reflexes. Previous studies have p r o v i d e d only suggestive evidence that c a r d i o p u l m o n a r y vagal afferent stimulation suppresses somatic reflexes e v o k e d by noxious stimuli. F o r exam-
60 pie, Chase and co-workers ~ have proposed that cardiopulmonary vagal afferents represent the primary group of vagal afferents modulating the digastric reflex in the cat. This suggestion is based on the observation that alterations in the magnitude of the digastric reflex occur when vagal afferents with conduction velocities similar to those originating from cardiac tissues are stimulated. Similarly, based on the outcomes of a series of physiological and pharmacological studies, it has been proposed that cardiopulmonary vagai afferent stimulation impairs the nociceptive tail-flick reflex in rats. Both vascular volume expansion TM (i.e. physiological activation of cardiopulmonary baroreceptors) and the atrial administration of opiate-receptor agonists 2~'32 (i.e., pharmacological activation of cardiopulmonary baroreceptors) impair the tail-flick reflex in rats. The antinociceptive effects of these procedures are attenuated by cervical vagotomy. Although suggestive, these studies have not provided direct or definitive evidence that vagal afferents originating from cardiopulmonary structures mediate the observed inhibitory effects on nociceptive somatic reflexes. The results of the present study clearly and conclusively demonstrate that stimulation of cardiopulmonary vagal afferents inhibits the digastric reflex. Furthermore, the outcomes of the present study provide additional evidence that vagal afferents originating from cardiopuimonary structures represent the primary vagal afferent population mediating antinociceptive effects in the cat. The present findings are also consistent with previous reports that cervical vagal conditioning produces a biphasic effect on responses to noxious stimuli. For example, cervical vagal afferent stimulation produces a CTIdependent facilitation and inhibition on the digastric reflex in immobilized encephala isole cats 9'1°. CTIs less than 10 ms produce facilitation while CTIs greater than 10 ms produce inhibition of the reflex. Similarly, electrical stimulation of cervical vagal afferents produces an intensity-dependent biphasic effect on the tail-flick reflex in rats 33. Several recent electrophysiological studies have also demonstrated that electrical stimulation of either cervical or thoracic vagal afferents produces facilitatory, inhibitory, or both effects on spontaneous and evoked activity of spinothalamic, spinoreticular and non-classified dorsal horn neurons to nociceptive stimuli in the rat, cat and monkey 4'16'34"36. However, it should be noted that the observed effect of vagal afferent conditioning on spontaneous and evoked activity of spinal dorsal horn neurons is quite complex and appears to be dependent on a variety of factors such as the species examined, vagal site stimulated and stimulation p a r a m e t e r s 4" ~6"34"36. The mechanisms by which cervical vagal afferent stimulation produces facilitation of the digastric reflex is not evident. It is possible that vagal afferents originating
from the upper airways contribute t o the facilitatory effect. Alternatively, it is possible that the stimulation ot the aortic depressor nerve or sympathetic efferents, which are frequently ensheathed within the cervical vagal trunk 2, produces a facilitation of the reflex. In support of this view, aortic depressor nerve stimulation facilitates the digastric reflex in rabbits ~ and cervical sympathetic nerve stimulation facilitates the digastric reflex in cats 14. In this regard, Randich and co-workers have suggested that the facilitatory effects of cervical vagal stimulation on the nociceptive tail-flick reflex in the rat may result from the unintentional stimulation of the aortic depressor nerve 3L. The observation that diaphragmatic vagal afferent stimulation produces rather modest inhibitory effects on the digastric reflex in cats is consistent with the results from previous studies showing that diaphragmatic vagal stimulation produces relatively weak inhibitory effects on responses to noxious stimuli in both cats and monkeys 4'5'1°'~6. In the present study, the delivery of conditioning stimuli to the cervical, thoracic and cardiac branches of the vagus at intensities that clearly and profoundly inhibited the digastric reflex produced much less inhibition when applied to the diaphragmatic vagus. It should be noted, however, that a more profound inhibition of the reflex by diaphragmatic vagal conditioning may occur using stimulation parameters different from those evaluated in the present study. Thus, it is not possible to conclude that diaphragmatic vagal afferent stimulation is less able to produce antinociceptive effects than more rostral vagal branches until more extensive parametric studies have been conducted. The mechanism by which vagal afferent stimulation alters nociceptive reflexes and neural responses to noxious stimuli has not been extensively investigated. Recent studies have provided evidence that vagal afferent stimulation may produce antinociception by activating polysynaptic pain inhibitory networks within the central nervous system. Many of the central nervous system loci which receive vagal afferent input via the nucleus of the solitary tract modulate the responses of dorsal horn nociceptive neurons and mediate the modulatory effects of cervical vagal stimulation on the tail-flick reflex 27'28'33 35,3,~. The observation that maximum inhibitory effects occur at a relatively long CTI (i.e. 200 ms) is consistent with the view that vagal afferent stimulation impairs responses to noxious stimuli by engaging polysynaptic central nervous system networks. The long duration of the inhibition produced by brief vagal conditioning is consistent with the findings reported by Ammons and co-workers 5. Thoracic vagal afferent conditioning produces inhibition of A-6 and C-fiber evoked responses of thoracic spinothalamic cells receiv-
61 ing viscerosomatic convergence. Inhibition starts at a CTI of approximately 40 ms and lasts for at least 200 ms. The long duration of inhibition produced by vagal conditioning is consistent with a presynaptic inhibitory mechanism. In general presynaptic inhibition, which results from the depolarization of primary afferents, lasts for several hundred ms 11 while postsynaptic inhibition is thought to occur with a duration of 50 ms or less 12. However, we cannot conclude that the inhibitory effects produced by vagal stimulation are mediated by a presynaptic process since long duration (i.e., lasting several hundred ms) postsynaptic inhibition of nociceptive dorsal horn neurons can occur following the stimulation of either the nucleus raphe magnus or periaqueductal gray TM. The effects of vagal conditioning were not secondary to the cardiovascular or peripheral responses produced by vagal stimulation. In the present study, vagal stimulation produced equivalent increases in both blood pressure and heart rate regardless of the CTI, yet produced CTI-dependent effects on the digastric reflex (see Fig. 5). In addition, no clear relationship between the magnitude of the pressor or tachycardiac responses with the modulatory effects on the digastric reflex was observed. The pressor responses to vagal stimulation likely resulted from the stimulation of myelinated cardiac vagal afferents. In cats, high-frequency stimulation (i.e., greater than 10 Hz) of cardiac vagal afferents produces modest pressor responses while low-frequency stimulation (i.e. less than 10 Hz) of non-myelinated cardiac vagal afferents produces depressor responses 24. Since the stimulation intensities used in this study were clearly able to activate both myelinated and non-myelinated vagal afferents it is not possible to conclude that the effects of vagal conditioning are mediated by myelinated cardiac vagal afferents. However, previous research has indicated that myelinated cardiac vagal afferents with conduction velocities around 15 m/s mediate the inhibitory effects on the digastric reflex 9'1°. The modulatory effects of vagal afferent stimulation were not mediated by peripheral vagal efferents because stimulation of the peripheral end of the thoracic vagus did not influence the digastric reflex. Furthermore, no quantitative or qualitative difference in the modulatory effects of stimulating either the intact cervical vagus or the central cut end of the cervical vagus was observed and our findings are consistent with those of previous studies that have evaluated the effects of stimulating the central cut end of the cervical vagal on the digastric reflex in the cat 9A°. It is also noteworthy that high-frequency stimulation of the peripheral end of the thoracic vagus did not produce parasympathetic mediated responses (i.e. bradycardia and hypotension). This was not
an unexpected finding since: (1) preganglionic vagal efferents have a critical refractory period of 5 ms and do not entrain stimulation frequencies above 200 Hz22; (2) ganglionic blockade or transmission failure occurs with high frequency stimulation6; and (3) vagal escape occurs with high-frequency vagal stimulation 23. Collectively, these findings demonstrated that the effects of vagal stimulation on the digastric reflex cannot be attributed to peripheral mechanisms. Our findings also support the view that there are a variety of species differences related to the temporal effects of vagal stimulation on somatic reflexes. In rats, cervical vagal afferent stimulation produces an initial inhibition and late facilitation of neuronal responses of lumbar dorsal horn neurons to noxious heat 34. In the present study, and in agreement with previous findings 9" 10, the facilitatory effects produced by cervical vagal stimulation occur prior to the inhibitory effects. Species differences in the vagal afferent fiber types which modulate reflex and neuronal responses to noxious stimuli also appear to exist. In rats it has been proposed that the facilitatory effects associated with low intensity cervical vagal results from the stimulation of myelinated afferents while the inhibitory effect associated with high intensity cervical vagal stimulation is mediated by the stimulation of non-myelinated C-fibers 33'34. In cats, myelinated vagal afferents with conduction velocities around 15 m/s mediate the inhibitory effects on the digastric reflex9"1°. In further support of species differences, the stimulation of the diaphragmatic vagus impairs the tailflick response in rats 3"33'37, but fails to profoundly impair responses to noxious stimuli in cats and monkeys 4'5'16. Collectively, these tindings suggest that the modulatory effects of vagal afferent stimulation on somatic reflexes may be species-dependent and our understanding of the neuromechanisms which mediate these effects depend upon a number of factors including the species examined. The present findings provide additional evidence that stimulation of cardiopulmonary vagal afferents modulates somatomotor reflexes evoked by noxious stimuli. It has been argued that this system may play an important role in integrating the sensory and vascular responses to environmental stressors 2°'28'3°'39. In this regard, it seems quite plausible that cardiopulmonary vagal afferent pathways contribute to much more than the simple modulation of visceral and somatic reflexes but instead play a much broader role in modulating the integrative function of the central nervous system. For example, vagal afferents and carotid sinus baroreceptors produce profound changes in electrocortical activity, alter consciousness, and have been demonstrated to alter the perception of a variety of sensory stimuli 7'8"13"15"17"21'26'38. Thus, it is
62 likely that a f f e r e n t i n f o r m a t i o n o r i g i n a t i n g f r o m cardiop u l m o n a r y structures c o n t r i b u t e s to the h o m e o s t a t i c regulation of an o r g a n i s m ' s b e h a v i o r during a d a p t i v e and m a l a d a p t i v e states.
REFERENCES 1 Aars, H. and Brodin, P., Reflex changes in sympathetic activity affect the tooth tap-digastric reflex in rabbits, Proc. Finn. Dent. Soc., 85 (1989) 379-382. 2 Agostoni, E., Chinnock, J.E., De Burgh Daly, M. and Murray, J.G., Functional and histological studies of the vagus nerve and its branches to the heart, lungs and abdominal viscera in the cat, J. Physiol., 135 (1957) 182-205. 3 Aicher, S.A., Lewis, S.J. and Randich, A., Antinociception produced by electrical stimulation of vagal afferents: independence of cervical and subdiaphragmatic branches, Brain Research, 542 (1991) 63-70. 4 Ammons, W.S., Blair, R.W. and Foreman, R.D., Vagal afferent inhibition of primate thoracic spinothalamic neurons, J. Neurophysiol., 50 (1983) 926-940. 5 Ammons, W.S., Blair, R.W. and Foreman, R.D., Vagal afferent inhibition of spinothalamic cell responses to sympathetic afferents and bradykinin in the monkey, Circ. Res., 53 (1983) 603-612. 6 Bard, P., Medical Physiology, 10th edn., Mosby, St. Louis, MO, 1956, 70 pp. 7 Bartorelli, C., Bizzi, E., Libretti, A. and Zanchetti, A., Inhibitory control of sinoearotid pressoceptive afferents on hypothalamic autonomic activity and sham rage behavior, Arch. ltal. Biol., 98 (1968) 308-326. 8 Bonvallet, M., Dell, P. and Hiebel, G., Tonus sympathique et activit6 61ectrique corticale, Electroencephalogr. Clin. Neurophysiol., 6 (1954) 119-144. 9 Chase, M.H., Nakamura, Y. and Torii, S., Afferent vagal modulation of brain stem somatic reflex activity, Exp. Neurol., 27 (1970) 534-544. 10 Chase, M.H., Torii, S. and Nakamura, Y., The influence of vagal afferent fiber activity on masticatory reflexes, Exp. Neurol., 27 (1970) 545-553. 11 Eccles, J.C., Kostyuk, P.G. and Schmidt, R.E, Presynaptic inhibition of the central action of flex or reflex afferents, J. Physiol., 161 (1962) 258-281. 12 Eccles, J.C. and Sherrington, C.S., Studies on the flexor reflex. 1I. The reflex response evoked by two afferent volleys, Proc. R. Soc. Brit., 107 (1931) 535-556. 13 Gellhorn, E., Principles of Autonomic-Somatic Interactions, University of Minnesota Press, Minneapolis, MN, 1967. 14 Grassi, C. and Passatore, M., Effect of cervical sympathetic nerve stimulation on nociceptive jaw opening reflex in the cat, Funct. Neurol., 2 (1987) 547-552. 15 Heymans, C. and Neil, E., Baroreceptor reflexes other than circulatory. In C. Heymans and E. Neil (Eds.), Reflexogenic Areas of the Cardiovascular System, Churchill, London, 1958, pp. 95-100. 16 Hobbs, S.F., Oh, U.T., Chandler, M.J. and Foreman, R.D., Cardiac and abdominal vagal afferent inhibition of primate T9-$1 spinothalamic cells, Am. J. Physiol., 257 (1989) R889-R895. 17 Koriath, J.J., A view of cardio-cortical connections, J. A m Coll. Cardiol., 14 (1989) 528-529. 18 Light, A.R., Casale, E.J. and Menetrey, D.M., The effects of focal stimulation in nucleus raphe magnus and periaqueductal gray on intracellularly recorded neurons in spinal laminae I and II, J. Neurophysiol., 56 (1986) 555-571. 19 Maixner, W., Bossut, D.F. and Whitsel, E.A., Cardiopulmonary vagal afferent modulation of the jaw-opening reflex, J. Dent. Res., 67 (1989) 897. 20 Maixner, W. and Randich, A., Role of the right vagal nerve
Acknowledgements.This work was supported by NIDR Grants DE07509, DE08013 (W.M.) and NRSA fellowship DE05585 (D.F.B.). We appreciate the helpful comments provided by Drs. Alan Light and Alan Randich in their review of previous drafts of this manuscript.
trunk in antinociception, Brain Research, 298 (1984) 374-377. 21 Mazzella, H., Mullin, G.R. and Austt, E.G., Effect of carotid sinus stimulation on the EEG, Acta Neurol. Latinoam., 3 (1957) 361-364. 22 McAllen, R.M. and Spyer, K.M., The location of cardiac vagal preganglionic motoneurones in the medulla of the cat, J. Physiol., 258 (1976) 187-204. 23 Miller, D.A., Pendleton, R.G. and Richmond, A.T., Cardiac effects of vagal stimulation in the anaesthetized cat, Br. J. Pharmacol. Chemother., 33 (1968) 390-395. 24 Oberg, B. and Thoren, P., Circulatory responses to stimulation of medullated and non-medullated afferent in the cardiac nerve in the cat, Acta Physiol. Scand., 87 (1973) 121-132. 25 Oberg, B. and White, S., Circulatory effects of interruption and stimulation of cardiac vagal afferents, Acta Physiol. Scand., 80 (1970) 383-394. 26 Penaloza Rojas, J.H., Electrocephalographic synchronization resulting from direct current application to the vagus nerves, Exp. Neurol., 9 (1964) 367-371. 27 Randich, A. and Aicher, S.A., Medullary substrates mediating antinociception produced by electrical stimulation of the vagus, Brain Research, 445 (1988) 68-76. 28 Randich, A. and Maixner, W., Interactions between cardiovascular and pain regulatory systems, Neurosci. Biobehav. Rev., 8 (1984) 343-367. 29 Randich, A. and Maixner, W., [D-AlaZ]methionine enkephalinamide reflexively induces antinociception by activating vagal afferents. Pharmacol. Biochem. Behav., 21 (1984) 441-448. 30 Randich, A. and Maixner, W., The role of sinoaortic and cardiopulmonary baroreceptor reflex arcs in nociception and stressinduced analgesia, Ann. N.Y. Acad. Sci., 467 (1986) 385-401. 31 Randich, A., Ren, K. and Gebhart, G.F., Electrical stimulation of cervical vagal afferents. II. Central relays for behavioral antinociception and arterial blood pressure decreases, J. Neurophysiol., 64 (1990) 1115-1124. 32 Randich, A., Thurston, C.L., Ludwig, P.L., Timmerman, M.R. and Gebhart G.E, Antinociceptive and cardiovascular responses produced by intravenous morphine: the role of vagal afferents, Brain Research, 543 (1991) 256-270. 33 Ren, K., Randich, A. and Gebhart, G.F., Vagal afferent modulation of a nociceptive reflex in rats: involvement of spinal opioid and monoamine receptors, Brain Research, 446 (1988) 285-294. 34 Ren, K., Randich, A. and Gebhart, G.E, Vagal afferent modulation of spinal nociceptive transmission in the rat, J. Neurophysiol., 62 (1989) 401-415. 35 Ren, K., Randich, A. and Gebhart, G.F., Electrical stimulation of cervical vagal afferents. I. Central relays for modulation of spinal nociceptive transmission, J. Neurophysiol., 64 (1990) 1098-1114. 36 Thies, R. and Foreman, R.D., Inhibition and excitation of thoracic spinoreticular neurons by electrical stimulation of vagal afferent nerves, Exp. Neurol., 82 (1983) 1-16. 37 Thurston, C.L. and Randich, A., Quantitative characterization and spinal substrates for antinociception produced by electrical stimulation of the subdiaphragmatic vagus in rats, Pain, 44 (1991) 201-210. 38 Tourande, A. and Malmejac, S., Diversit~ des actions reflexes que declenche l'excitation du neff, C:'R. Soc. Biol., 100 (1929) 708-711. 39 Zamir, N. and Maixner, W., The relationship between cardiovascular and pain regulatory systems, Ann. N.Y. Acad. Sci., 467 (1986) 371-384.
Brain Research, 560 (1991) 55-62 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116969N
BRES 16969
Evaluation of vagal afferent modulation of the digastric reflex in cats W. Maixner,
D.F.
Bossut
and E.A.
Whitsel
Dental Research Center and the Departments of Pharmacology and Endodontics, University of North Carolina, Chapel Hill, NC 27599-7455 (U.S.A.)
(Accepted 30 April 1991) Key words: Reflex; Vagus; Vagal afferent; Baroreceptor; Pain; Nociception; Tooth pulp; Analgesia; Cardiovascular
In the present study, we have examined the relative ability of cervical, thoracic, cardiac and diaphragmatic vagal stimulation to modulate the digastic reflex produced by tooth-pulp stimulation in anesthetized cats. The right maxillary tooth pulp was stimulated and the digastric reflex was recorded from the fight digastric muscle. Cervical vagal stimulation produced a biphasic effect on the digastric reflex. The reflex was facilitated at conditioning test intervals less than 20 ms and inhibited at conditioning test intervals between 100 ms and 500 ms. Cardiac and thoracic vagal stimulation did not significantly facilitate the digastric reflex but inhibited the reflex at conditioning test intervals between 50 ms and 500 ms with maximum inhibition observed at 200 ms. In contrast, diaphragmatic vagal stimulation produced a weaker inhibition of the digastric reflex. The relative ability of different vagal segments to inhibit the digastric reflex was: thoracic = cardiac = cervical > diaphragmatic. The inhibitory effects were not related to cardiovascular responses to vagal afferent stimulation. These findings suggest cardiopulmonary vagal afferents represent an important source of vagal afferents which modulate the digastric reflex in the cat. INTRODUCTION The electrical stimulation of afferents which course in the cervical vagus produces both facilitatory and inhibitory effects on reflex responses to noxious stimuli 9A°'28' 33,34. Depending upon stimulation parameters, cervical vagal stimulation produces either a facilitation or an inhibition of the digastric reflex in the cat and reflex (i.e. tail-flick reflex) responses to noxious radiant heat in the rat 9A°'28"33. Several recent electrophysiological studies have also demonstrated that electrical stimulation of either cervical or thoracic vagal afferents produces either facilitatory, inhibitory or both effects on dorsal horn neurons in the rat, cat and m o n k e y 4'16'34"36. The anatomical origins of the vagal afferents which modulate somatic reflexes evoked by noxious stimuli have not been extensively examined. It has been suggested that vagal afferents originating from cardiopulmonary tissues represent a major source of vagal afferents which modulate nociceptive reflexes 28"3°. In support of this view, the physiological stimulation of afferents, induced by vascular volume expansion, inhibits the tailflick reflex in the rat 2°. Similarly, the pharmacological stimulation of vagal afferents, produced by the intravenous administration of non-selective opiate-receptor agonists, results in an immediate and profound inhibition of the tail-flick reflex. The antinociceptive effects pro-
duced by these two experimental procedures are attenuated by cervical vagotomy 29'32. These findings, though suggestive, do not provide conclusive evidence that cardiopulmonary vagal afferent stimulation produces antinociception because their conclusions are based on experimental manipulations of the cervical vagus which contains afferents originating from anatomical regions both rostral and caudal to cardiopulmonary tissues. It has been demonstrated recently that abdominal vagal afferents modulate reflex responses to noxious stimuli in the rat 33. In contrast, electrophysiological studies conducted on cats and monkeys have found that abdominal vagal afferent stimulation produces very weak inhibitory effects on the responses of spinal sensory neurons to noxious stimuli 4'5"16. Thus, the degree to which different sub-populations of vagal afferents influence reflex and neuronal responses to noxious stimuli remains unclear. In addition, the relative ability of vagal afferents originating from different peripheral sites to modulate polysynaptic reflexes other than the tail-flick reflex has not been systematically evaluated. In the present study, we have examined the relative ability of cervical, thoracic, cardiac and diaphragmatic vagal afferent stimulation to modulate the digastric reflex produced by tooth-pulp stimulation in cats. Since vagal stimulation produces a variety of cardiovascular responses 24'25, which may in some way contribute to the
Correspondence: W. Maixner, Rm 03 Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455, U.S.A. Fax: (1)
(919) 966-3683.
56 inhibitory effects on reflexes produced
by noxious stim-
uli, t h e r e l a t i o n s h i p b e t w e e n t h e c a r d i o v a s c u l a r r e s p o n s e s to vagal stimulation and the effects on the digastric reflex h a s a l s o b e e n e x a m i n e d . P r e l i m i n a r y r e p o r t s o f t h e s e findings have appeared
e l s e w h e r e ~9
MATERIALS AND M E T H O D S
General preparation Eighteen adult cats weighing from 1.6 to 3.9 kg (2.56 _+ 0.11 kg), were anesthetized with pentobarbital sodium (40-45 mg/kg, i.p.) and placed in a supine position. Rectal temperature was maintained between 36.5 and 37.5 °C by a thermostatically controlled electric heating blanket. The trachea was cannulated and the cat was ventilated artificially with room air. Stroke volume and respiration rate were adjusted to maintain expired CO 2 (measured with an infrared capnometer) levels between 3,5 and 4.5%. The left femoral vein and artery were cannulated and arterial blood pressure and heart rate were monitored. Deep anesthesia was produced with a-chloralose (60 mg/kg, i.v.) and maintained with an intravenous infusion of a-chloralose (3-5 mg/kg/h) or supplemental bolus doses as required. Adequacy of anesthesia was evaluated by monitoring arterial blood pressure, checking for blink reflex, and testing for hindlimb flexion reflex in response to strong paw pinch.
Tooth preparation On the facial surface of the right maxillary canine, 1-2 mm coronal to the gingival border, a small pit preparation was made in the enamel with a high speed dental drill until the dentino-enamel junction was reached. A hand held carbide bur was used to deepen the preparation while irrigating with isotonic saline until the pulpal vasculature was just visible. A male Amphenol ® pin was placed in contact with the bottom of the preparation and secured to the tooth with UV light-cured composite resin. The pin was insulated from the surrounding tissue using dental baseplate wax.
Tooth pulp (TP) stimulation Constant-current rectangular pulses (1.0 ms) were delivered from the cathodal pole to the TP once every 5 s to produce a reflex contraction of the ipsilateral digastric muscle. The cathodal pole was attached to the TP and the anodal pole was connected to the right ear. The intensity of the current delivered to the TP was monitored on a storage oscilloscope by assessing the voltage drop across a 100 f2 resistor in series with the cathodal pole. Fibers originating in the TP produced the reflex since pulpectomy abolished it at intensities up to 5× threshold.
stimulated. In 8 cats, the right cervical vagus was deshcathcd and isolated from the cervical sympathetics and carotid artery. The aortic depressor nerve was isolated from the cervical vagus when thcy appeared as two separate nerve trunks. In 3 preparations the w~gus was transected and in the remaining 5 preparations the vagus was left intact. Since no significant qualitative or quantitative differences were found between the effects of conditioning intact w~gus vs. the central ends of transected vagus, the data obtained from both preparations were combined for analysis. In a separate group of 10 cats, a right thoracotomy in the fourth intercostal space was performed to expose the right cardiac branch of the vagus and the thoracic bundle of the vagus just cranial to the cardiac branch. To facilitate exposure, the azygous vein was transected and the apical lobe of the right lung was resectcd. In 5 of the l0 cats, a right thoracotomy in the ninth intercostal space was also performed to isolate the vagus just cranial to the diaphragm. The vagus was left intact at these last three conditioning sites. All vagal conditioning sites were immersed in mineral oil warmed to 37 °C.
Vagal conditioning The vagus was stimulated with a bipolar platinum electrode with a pole-to-pole separation of 2-3 mm at the conditioning sites shown in Fig. 1. Anodal current was passed through the pole most distal to the brain to minimize anodal blockade. Current intensities were monitored as described for TP stimulation. All vagal sites were stimulated or conditioned with 333 Hz, 20 ms trains of pulses which were 1.0 ms in duration at 0.0 mA, 5.0 mA or 10.0 m A intensities. At the end of 3 experiments, the effects of conditioning the intact thoracic vagus were compared to effects of conditioning the peripheral end of the transected thoracic vagus. This was done to assess the degree to which vagal efferents contributed to the observed outcomes.
Experimental procedures First, baseline or control reflex magnitude was determined by signal-averaging l0 individual E M G recordings which were elicited at an intertrial interval of 5 s. Then, TP stimulation (i.e., test stimulus) resumed and 10 E M G curves were signal-averaged following the delivery of conditioning stimuli to the vagus at conditioning test intervals (CTIs) ranging from 5 to 1000 ms. The effects of vagal conditioning were evaluated at each intensity and CTI for each vagal conditioning site.
CERVICAL VAGUS
Electromyogram (EMG) recording The digastric reflex was monitored by transcutaneously implanting two stainless steel electrodes into the anterior belly of the right digastric muscle approximately 1 cm from each other. Differential recordings were obtained with a low-noise amplifier with a bandpass of 10 Hz to 1 kHz. All electromyographic activity usually occurred within 20 ms of TP stimulation and was displayed on a storage oscilloscope. With the aid of a computer, digitized (50 kHz) traces were displayed on-line, signal-averaged, and stored for offline data analysis.
THORACIC VAGUS
Digastric reflex threshold determination Analysis of threshold was performed frequently throughout the experiment. Pulse duration was set at 1.0 ms and pulse intensity was increased until 4 of 5 consecutive EMG responses to TP stimulation were obtained. The pulse intensity which produced four traces was defined as the threshold current. All data collected involved TP stimulation at 2 - 3 x threshold.
Vagal preparation Fig. I displays the four vagal segments that were isolated and
Fig. 1. Anatomical sites of vagal conditioning.
57 Data analysis Signal-averaged E M G ' s were rectified and quantified by integration using software developed by Modular Instruments, Inc. E M G integrals were expressed as a percentage of the baseline (%BL) E M G integral for each vagal site, intensity, and CTI. The effects of various conditioning parameters on the derived percentage values were analyzed by an appropriate analysis of variance. A repeated measures design was used to assess within-group main effects and a complete block design was used to assess between-group main effects. Duncan's multiple range test was used when appropriate. Cardiovascular data and the differences between the responses to stimulating the intact vs. the peripheral end of the thoracic vagus were analyzed with a paired t-test. In all cases, significance was assumed at an a ~
E
0.0 -0.5
I
I
I
I
1
I
I
I
|
Gins
200 ms
0.5 ~::
0.0 -O.G
I
I
1
I
I
I
I
1000 ms
0.0 -0.5 0
I 4
I 8
I 12
I 16
I ~
0
I 4
I 8
ms
I 12
I 111
I 20
ms
Fig. 3. The effects of cervical vagal conditioning on the digastric reflex induced by tooth-pulp stimulation. Each electromyographic trace represents the averaged response to 10 successive stimuli applied to the ipsilateral tooth-pulp at 2× threshold intensity applied at a rate of 0.2 Hz. The first trace (upper left corner) is the control response in the absence of cervical vagal conditioning. The subsequent traces were obtained when a train of stimuli (5 mA, 1.0 ms pulses delivered at 333 Hz for 20 ms) was delivered to the central end of the cervical vagus at various intervals (5-1000 ms) prior to the test stimulus. The test stimulus artifact has been removed.
The effects of stimulating the cardiac branch of the
of conditioning stimuli to the diaphragmatic vagus did
v a g u s at 5.0 a n d 10.0 m A a r e s h o w n in Fig. 2C. C o n d i -
n o t f a c i l i t a t e t h e d i g a s t r i c reflex at b r i e f C T I s a n d p r o -
tioning of the cardiac branch of the vagus produced a
d u c e d a w e a k b u t significant i n h i b i t i o n o f t h e d i g a s t r i c
p r o f o u n d i n h i b i t i o n o f t h e reflex b u t d i d n o t f a c i l i t a t e t h e
reflex at t h e l o n g e r C T I s . S i g n i f i c a n t i n h i b i t i o n r e l a t i v e
reflex r e l a t i v e t o s h a m c o n d i t i o n i n g . A s s e e n w i t h t h o -
t o s h a m s t i m u l a t i o n was o b s e r v e d at 100 m s ( F ( 2 , 7 ) =
racic v a g a l c o n d i t i o n i n g , l o n g e r C T I s f r o m 50 to 500 m s
4.76) a n d 500 m s ( F ( 2 , 7 ) = 4.87) C T I s . T h e i n h i b i t o r y
r e s u l t e d in a p r o f o u n d i n h i b i t i o n o f t h e reflex ( f o r 50 ms:
effects w e r e e q u i v a l e n t at b o t h t h e 5.0 a n d 10.0 m A
F ( 3 , 1 8 ) = 6.01; F o r 100 ms: F ( 3 , 1 8 ) = 8.80; f o r 200 ms:
conditioning intensities.
F ( 3 , 1 8 ) = 13.96; f o r 500 ms: F ( 3 , 1 8 ) = 6.86). T h e inh i b i t o r y effects w e r e e q u i v a l e n t at 5 a n d 10.0 m A .
A s s h o w n in Fig. 4, t h e r e l a t i v e ability o f v a r i o u s vagal b r a n c h e s to i n h i b i t t h e d i g a s t r i c reflex at t h e 200 m s
T h e e f f e c t s o f s t i m u l a t i n g t h e c e n t r a l e n d o f t h e dia-
C T I (5 m A ) v a r i e d as a f u n c t i o n o f c o n d i t i o n i n g site
p h r a g m a t i c v a g u s is s h o w n in Fig. 2 D . T h e a p p l i c a t i o n
( F ( 3 , 2 9 ) = 11.14). T h e r e l a t i v e efficacy o f i n h i b i t i o n w a s
TABLE I
The effect of vagal stimulation on blood pressure and heart rate Each value represents the mean ± 1 S.E.M.
Systolic (ram Hg)
Diastolic (mm Hg)
Heart rate (BPM)
baseline
vagal
baseline
vagal
baseline
vagal
Cervical (n = 8)
5 mA 10 mA
122 ± 10 120 + 7
135 -+ 12" 138 ± 9*
88 ± 9 86 ± 7
101 ± 10" 104 +_ g*
255 -_ 7 251 --- 5
257 +- 7 251 +- 7
Thoracic (n = 9)
5 mA 10mA
112 ± 10 112 ± 10
135 ± 7* 144 ± 7*
7l -+ 9 71 ± 9
93 -+ 7* 99 ± 7*
203 ± 15 204 ± 17
206 ÷ 14 209 -+ 15
Cardiac branch (n = 10)
5 mA 10 mA
112 ± 9 108 -+ 10
130 ± 9* 129 ± 8*
76 ± 9 73 ± 10
92 ± 9* 94 ± 8*
212 ± 19 209 ± 19
223 ± 16 224 ± 17
Diaphragmatic (n = 5)
5 mA 10 mA
94 -+ 5 97 ± 9
118 ± 18 126 ± 25
58 ± 6 63 ± 11
80 ± 18 86 -+ 23
206 ± 32 206 -+ 32
212 ± 33 215 ± 33
*, Significantly different from corresponding baseline value; Student's paired t-test (P diaphragmatic. Similar findings were o b t a i n e d when the various vagal branches were conditioned with 10 m A pulses.
A
CTI = 5
CTI = 200
200
Effects of stimulating the peripheral end of the thoracic vagus
100 iE
0
2~ Iota ,,,,
2~
B
o. f--A 0.5
-0.5
The effects of vagal stimulation on b l o o d pressure and heart rate are shown in Table I. Stimulation of either the cervical, thoracic o r cardiac vagus p r o d u c e d a pressor response in all experiments at both 5 and 10 m A intensities and p r o d u c e d variable effects on heart rate. Diaphragmatic vagal stimulation increased b l o o d pressure in 3 of 5 p r e p a r a t i o n s but, as shown in Table I, did not achieve statistical signficance. Cervical vagal stimulation increased heart rate in 5 of 8 preparations, thoracic vagal stimulation increased heart rate in 5 of 9 p r e p a r a tions, cardiac vagal stimulation increased heart rate in 5 of 10 preparations, and diaphragmatic vagal stimulation increased heart rate in 3 of 5 preparations. The observed inhibitory effects on the digastric reflex p r o d u c e d by vagal stimulation were i n d e p e n d e n t of changes in arterial b l o o d pressure and heart rate. A s shown in Fig. 5, the cardiovascular responses to thoracic vagal afferent stimulation were essentially identical at 5 ms and 200 ms CTIs, yet the digastric reflex was essentially abolished at the 200 ms CTI and was still evident at the 5 ms CTI. F o r a given p r e p a r a t i o n , cervical, thoracic or cardiac vagal nerve stimulation p r o d u c e d equivalent cardiovascular responses at each CTI while producing m a r k e d l y different effects on the digastric reflex.
I'
rm
0
4
8
IIl,,S,,,,
The effects of stimulating the intact thoracic vagus or the p e r i p h e r a l end of the transected thoracic vagus were evaluated in 3 preparations. Stimulation of the intact thoracic vagus at a 200 ms C T I inhibited the digastric reflex to 94% of the control value and increased both b l o o d pressure and heart rate in all three preparations. In contrast, stimulation of the peripheral end of the thoracic vagus at a 200 ms CTI did not alter the digastric reflex or b l o o d pressure from pre-stimulation values and increased heart rate from pre-stimulation values in 2 of 3 preparations.
,,,m~,o,~w
, 12 18 20
msec
• 0
DISCUSSION
, 4
, 8
1'2 18
20
msec
Fig. 5. Blood pressure and heart rate responses to thoracic vagal conditioning at condition test intervals of 5 ms (left panel) and 200 ms (right panel) are shown in A. Each tic mark denotes the time when a test stimulus was delivered to the tooth-pulp. The lower electromyographic traces (B) represent the composite or averaged trace obtained in response to the 10 test stimuli. Note that equivalent cardiovascular responses were associated with different effects on the digastric reflex.
Previous research has d e m o n s t r a t e d that stimulation of cervical vagal afferents modulates several nociceptive reflexes including the digastric reflex. In general it has been assumed that vagal afferents originating from card i o p u l m o n a r y structures represent the p r i m a r y population of afferents that m o d u l a t e somatic reflexes. Previous studies have p r o v i d e d only suggestive evidence that c a r d i o p u l m o n a r y vagal afferent stimulation suppresses somatic reflexes e v o k e d by noxious stimuli. F o r exam-
60 pie, Chase and co-workers ~ have proposed that cardiopulmonary vagal afferents represent the primary group of vagal afferents modulating the digastric reflex in the cat. This suggestion is based on the observation that alterations in the magnitude of the digastric reflex occur when vagal afferents with conduction velocities similar to those originating from cardiac tissues are stimulated. Similarly, based on the outcomes of a series of physiological and pharmacological studies, it has been proposed that cardiopulmonary vagai afferent stimulation impairs the nociceptive tail-flick reflex in rats. Both vascular volume expansion TM (i.e. physiological activation of cardiopulmonary baroreceptors) and the atrial administration of opiate-receptor agonists 2~'32 (i.e., pharmacological activation of cardiopulmonary baroreceptors) impair the tail-flick reflex in rats. The antinociceptive effects of these procedures are attenuated by cervical vagotomy. Although suggestive, these studies have not provided direct or definitive evidence that vagal afferents originating from cardiopulmonary structures mediate the observed inhibitory effects on nociceptive somatic reflexes. The results of the present study clearly and conclusively demonstrate that stimulation of cardiopulmonary vagal afferents inhibits the digastric reflex. Furthermore, the outcomes of the present study provide additional evidence that vagal afferents originating from cardiopuimonary structures represent the primary vagal afferent population mediating antinociceptive effects in the cat. The present findings are also consistent with previous reports that cervical vagal conditioning produces a biphasic effect on responses to noxious stimuli. For example, cervical vagal afferent stimulation produces a CTIdependent facilitation and inhibition on the digastric reflex in immobilized encephala isole cats 9'1°. CTIs less than 10 ms produce facilitation while CTIs greater than 10 ms produce inhibition of the reflex. Similarly, electrical stimulation of cervical vagal afferents produces an intensity-dependent biphasic effect on the tail-flick reflex in rats 33. Several recent electrophysiological studies have also demonstrated that electrical stimulation of either cervical or thoracic vagal afferents produces facilitatory, inhibitory, or both effects on spontaneous and evoked activity of spinothalamic, spinoreticular and non-classified dorsal horn neurons to nociceptive stimuli in the rat, cat and monkey 4'16'34"36. However, it should be noted that the observed effect of vagal afferent conditioning on spontaneous and evoked activity of spinal dorsal horn neurons is quite complex and appears to be dependent on a variety of factors such as the species examined, vagal site stimulated and stimulation p a r a m e t e r s 4" ~6"34"36. The mechanisms by which cervical vagal afferent stimulation produces facilitation of the digastric reflex is not evident. It is possible that vagal afferents originating
from the upper airways contribute t o the facilitatory effect. Alternatively, it is possible that the stimulation ot the aortic depressor nerve or sympathetic efferents, which are frequently ensheathed within the cervical vagal trunk 2, produces a facilitation of the reflex. In support of this view, aortic depressor nerve stimulation facilitates the digastric reflex in rabbits ~ and cervical sympathetic nerve stimulation facilitates the digastric reflex in cats 14. In this regard, Randich and co-workers have suggested that the facilitatory effects of cervical vagal stimulation on the nociceptive tail-flick reflex in the rat may result from the unintentional stimulation of the aortic depressor nerve 3L. The observation that diaphragmatic vagal afferent stimulation produces rather modest inhibitory effects on the digastric reflex in cats is consistent with the results from previous studies showing that diaphragmatic vagal stimulation produces relatively weak inhibitory effects on responses to noxious stimuli in both cats and monkeys 4'5'1°'~6. In the present study, the delivery of conditioning stimuli to the cervical, thoracic and cardiac branches of the vagus at intensities that clearly and profoundly inhibited the digastric reflex produced much less inhibition when applied to the diaphragmatic vagus. It should be noted, however, that a more profound inhibition of the reflex by diaphragmatic vagal conditioning may occur using stimulation parameters different from those evaluated in the present study. Thus, it is not possible to conclude that diaphragmatic vagal afferent stimulation is less able to produce antinociceptive effects than more rostral vagal branches until more extensive parametric studies have been conducted. The mechanism by which vagal afferent stimulation alters nociceptive reflexes and neural responses to noxious stimuli has not been extensively investigated. Recent studies have provided evidence that vagal afferent stimulation may produce antinociception by activating polysynaptic pain inhibitory networks within the central nervous system. Many of the central nervous system loci which receive vagal afferent input via the nucleus of the solitary tract modulate the responses of dorsal horn nociceptive neurons and mediate the modulatory effects of cervical vagal stimulation on the tail-flick reflex 27'28'33 35,3,~. The observation that maximum inhibitory effects occur at a relatively long CTI (i.e. 200 ms) is consistent with the view that vagal afferent stimulation impairs responses to noxious stimuli by engaging polysynaptic central nervous system networks. The long duration of the inhibition produced by brief vagal conditioning is consistent with the findings reported by Ammons and co-workers 5. Thoracic vagal afferent conditioning produces inhibition of A-6 and C-fiber evoked responses of thoracic spinothalamic cells receiv-
61 ing viscerosomatic convergence. Inhibition starts at a CTI of approximately 40 ms and lasts for at least 200 ms. The long duration of inhibition produced by vagal conditioning is consistent with a presynaptic inhibitory mechanism. In general presynaptic inhibition, which results from the depolarization of primary afferents, lasts for several hundred ms 11 while postsynaptic inhibition is thought to occur with a duration of 50 ms or less 12. However, we cannot conclude that the inhibitory effects produced by vagal stimulation are mediated by a presynaptic process since long duration (i.e., lasting several hundred ms) postsynaptic inhibition of nociceptive dorsal horn neurons can occur following the stimulation of either the nucleus raphe magnus or periaqueductal gray TM. The effects of vagal conditioning were not secondary to the cardiovascular or peripheral responses produced by vagal stimulation. In the present study, vagal stimulation produced equivalent increases in both blood pressure and heart rate regardless of the CTI, yet produced CTI-dependent effects on the digastric reflex (see Fig. 5). In addition, no clear relationship between the magnitude of the pressor or tachycardiac responses with the modulatory effects on the digastric reflex was observed. The pressor responses to vagal stimulation likely resulted from the stimulation of myelinated cardiac vagal afferents. In cats, high-frequency stimulation (i.e., greater than 10 Hz) of cardiac vagal afferents produces modest pressor responses while low-frequency stimulation (i.e. less than 10 Hz) of non-myelinated cardiac vagal afferents produces depressor responses 24. Since the stimulation intensities used in this study were clearly able to activate both myelinated and non-myelinated vagal afferents it is not possible to conclude that the effects of vagal conditioning are mediated by myelinated cardiac vagal afferents. However, previous research has indicated that myelinated cardiac vagal afferents with conduction velocities around 15 m/s mediate the inhibitory effects on the digastric reflex 9'1°. The modulatory effects of vagal afferent stimulation were not mediated by peripheral vagal efferents because stimulation of the peripheral end of the thoracic vagus did not influence the digastric reflex. Furthermore, no quantitative or qualitative difference in the modulatory effects of stimulating either the intact cervical vagus or the central cut end of the cervical vagus was observed and our findings are consistent with those of previous studies that have evaluated the effects of stimulating the central cut end of the cervical vagal on the digastric reflex in the cat 9A°. It is also noteworthy that high-frequency stimulation of the peripheral end of the thoracic vagus did not produce parasympathetic mediated responses (i.e. bradycardia and hypotension). This was not
an unexpected finding since: (1) preganglionic vagal efferents have a critical refractory period of 5 ms and do not entrain stimulation frequencies above 200 Hz22; (2) ganglionic blockade or transmission failure occurs with high frequency stimulation6; and (3) vagal escape occurs with high-frequency vagal stimulation 23. Collectively, these findings demonstrated that the effects of vagal stimulation on the digastric reflex cannot be attributed to peripheral mechanisms. Our findings also support the view that there are a variety of species differences related to the temporal effects of vagal stimulation on somatic reflexes. In rats, cervical vagal afferent stimulation produces an initial inhibition and late facilitation of neuronal responses of lumbar dorsal horn neurons to noxious heat 34. In the present study, and in agreement with previous findings 9" 10, the facilitatory effects produced by cervical vagal stimulation occur prior to the inhibitory effects. Species differences in the vagal afferent fiber types which modulate reflex and neuronal responses to noxious stimuli also appear to exist. In rats it has been proposed that the facilitatory effects associated with low intensity cervical vagal results from the stimulation of myelinated afferents while the inhibitory effect associated with high intensity cervical vagal stimulation is mediated by the stimulation of non-myelinated C-fibers 33'34. In cats, myelinated vagal afferents with conduction velocities around 15 m/s mediate the inhibitory effects on the digastric reflex9"1°. In further support of species differences, the stimulation of the diaphragmatic vagus impairs the tailflick response in rats 3"33'37, but fails to profoundly impair responses to noxious stimuli in cats and monkeys 4'5'16. Collectively, these tindings suggest that the modulatory effects of vagal afferent stimulation on somatic reflexes may be species-dependent and our understanding of the neuromechanisms which mediate these effects depend upon a number of factors including the species examined. The present findings provide additional evidence that stimulation of cardiopulmonary vagal afferents modulates somatomotor reflexes evoked by noxious stimuli. It has been argued that this system may play an important role in integrating the sensory and vascular responses to environmental stressors 2°'28'3°'39. In this regard, it seems quite plausible that cardiopulmonary vagal afferent pathways contribute to much more than the simple modulation of visceral and somatic reflexes but instead play a much broader role in modulating the integrative function of the central nervous system. For example, vagal afferents and carotid sinus baroreceptors produce profound changes in electrocortical activity, alter consciousness, and have been demonstrated to alter the perception of a variety of sensory stimuli 7'8"13"15"17"21'26'38. Thus, it is
62 likely that a f f e r e n t i n f o r m a t i o n o r i g i n a t i n g f r o m cardiop u l m o n a r y structures c o n t r i b u t e s to the h o m e o s t a t i c regulation of an o r g a n i s m ' s b e h a v i o r during a d a p t i v e and m a l a d a p t i v e states.
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Acknowledgements.This work was supported by NIDR Grants DE07509, DE08013 (W.M.) and NRSA fellowship DE05585 (D.F.B.). We appreciate the helpful comments provided by Drs. Alan Light and Alan Randich in their review of previous drafts of this manuscript.
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