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Convergence of nociceptive information from temporomandibular joint and tooth pulp afferents on C1 spinal neurons in the rat
Life Sciences 75 (2004) 1465 – 1478 www.elsevier.com/locate/lifescie
Convergence of nociceptive information from temporomandibular joint and tooth pulp afferents on C1 spinal neurons in the rat Toshimi Nishikawa *, Mamoru Takeda, Takeshi Tanimoto, Shigeji Matsumoto Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Received 6 November 2003; accepted 5 March 2004
Abstract The aim of the present study was to test the hypothesis that there is a convergence of afferent inputs from the temporomandibular joint (TMJ) on C1 spinal neurons responding to electrical stimulation of the tooth pulp (TP). In 14 pentobarbital anesthetized rats, the extracellular single unit activity of 31 C1 spinal neurons and the amplitude in a digastric muscle electromyogram (n = 31) increased proportionally during 1.0 –3.5 times the threshold for the jawopening reflex (JOR). Of 31 C1 spinal neurons responsive to TP afferents, 28 (approximately 90%) were also excited by electrical stimulation of the ipsilateral TMJ capsule. All neurons tested were divided into three categories of nociceptive specific, wide dynamic range and non-responsive as to their responsiveness to mechanical stimuli (pin prick and touch) of the somatic receptive field (skin of the face, neck, jaw and upper forearm) and TMJ capsule. Nineteen (68%) of 28 C1 spinal neurons received nociceptive information from C fibers of the TMJ capsule. These results suggest that there is a convergence of noxious information from the TMJ and TP afferents on the same C1 spinal neurons, which importantly contribute to pain perception from the TMJ region. D 2004 Elsevier Inc. All rights reserved. Keywords: Temporomandibular joint; C1 spinal dorsal horn; Referred pain; Tooth pulp; Rat
Introduction The temporomandibular joint (TMJ) region is innervated by branches of the trigeminal, upper cervical and vagus nerve (Klineberg, 1971; Denny-Brown and Yanagisawa, 1973; Widenfalk and Wiberg, 1990; Kido et al., 1993; Casatti et al., 1999), indicating that both sensory afferents and autonomic efferents * Corresponding author. Tel./Fax: +81-3-3261-8740. E-mail address: [email protected] (T. Nishikawa). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.014
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innervate the TMJ region. The former plays a significant role in nociception as well as in the modification of masticatory muscles (Dubner et al., 1978), and the latter acts as vasomotor control (Widenfalk et al., 1989). The rat trigeminal ganglion projects centrally to both the trigeminal nuclear complex and cervical dorsal horn (Jacquin et al., 1983; Pfaller and Arvidsson, 1988), and vagal afferent fibers project directly to the upper cervical segments of the spinal cord via a supraspinal route from the nodose ganglia (McNeill et al., 1991). In the studies with retrograde axonal tracers, the TMJ region is supplied with nerve fibers, originating mainly in the trigeminal ganglion, in addition to other sensory and sympathetic ganglia (Widenfalk and Wiberg, 1990; Uddman et al., 1999), and consisting of both myelinated (Ay) and unmyelinated (C) fibers (Yoshino et al., 1998). On the other hand, temporomandibular joint disorder (TMD) is expressed by a painful condition involving the TMJ and/or the muscles of mastication (Dworkin et al., 1992). Based on physical findings, it is considered that TMD is divided into three symptoms: muscle disorders, intracapsular disarrangements of the TMJ and degenerative arthritic changes in the TMJ (Laskin, 1979; Eversole and Machado, 1985; Friction et al., 1988; Dworkin et al., 1992). But the characterization of the afferent inputs from the TMJ has not yet been determined. Acute injury to the TMJ region results in a high density of Fos-positive neurons in the areas including the trigeminal spinal subnucleus caudalis (Vc) and upper cervical cord junction region (Vc/C2) (Hathaway et al., 1995; Bereiter and Bereiter, 2000; Bereiter, 2001). Based on evidence that the Vc/C2 region was excited by mechanical and/or chemical stimulation of the TMJ region, Takeshita et al. (2001) concluded that neurons in the lamina I–II in the Vc/C2 region play an important role in mediating pain sensation in TMD. Based on evidence that complete or partial relief from TMD occurred after receiving endodontic treatment or having tooth extraction, Wright and Gullickson (1996) suggested that acute pulpalgia acts as a significant factor in forming TMD symptoms. Because the C1 region is considered as an extension of the Vc, which receives most nociceptive inputs from the trigeminal nerve and because C1 spinal neurons that have somatic receptive fields in the facial skin are stimulated by electrical stimulation of the tooth pulp (TP) (Matsumoto et al., 1999; Takeda et al., 1999; Tanimoto et al., 2002), we hypothesized that C1 spinal neurons responding to TP stimulation are also excited by electrical stimulation of the TMJ region. To test this hypothesis, the present study was designed to examine the properties of the C1 spinal neurons in their response to mechanical and chemical stimulation of the exposed TMJ capsule, and convergence patterns of afferent information from the exposed TMJ capsule and the TP to these neurons.
Materials and methods Animal preparation The experiments were performed on 14 adult male Wistar rats (300–450 g). All experimental protocols used in this study were approved by the Animal Use and Care Committee of Nippon Dental University. Each animal was first anesthetized with sodium pentobarbital (45 mg/kg, i.p.) and maintained with additional doses of 2–3 mg/kg/h through a cannula inserted into the jugular vein, as required. The trachea was cannulated, and the rectal temperature was maintained at 37 F 0.5 jC with a radiant heater. The depth of anesthesia was checked by the lack of response to paw pinching. All wound
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margins were repeatedly covered with a local anesthetic, 2% lidocaine (Xylocain), throughout the experiment. Tooth pulp stimulation The method of TP stimulation was similar to that in previous studies (Takeda et al., 1998; Matsumoto et al., 1999; Tanimoto et al., 2002). Bipolar stimulating electrodes made from silver wires, (diameter 150 Am, enamel insulated except for the tip) were inserted into the pulp of the upper left incisors. To avoid spread of the stimulus current, the electrodes were isolated from surrounding tissues with dental resin. Temporomandibular joint stimulation The temporomandibular joint (TMJ) was exposed by removing the skin part on the temporal muscle and zygomatic arch. Bipolar stimulating electrodes made from silver balls (diameter 0.3 mm) were directly positioned onto the exposed capsule of the left TMJ. Recordings of dEMG and C1 spinal neuron activity The digastric electromyogram (dEMG) was recorded with silver electrodes (diameter 0.3 mm, interpolar distance 2 mm), and this EMG activity was used to assess the JOR. The rats were then placed in a stereotaxic apparatus, and a laminectomy was performed to expose the C1 spinal region of the spinal cord. The dura was cut and the brain surface was covered with a warm liquid paraffin oil (36– 37 jC). The single neuronal activity was recorded extracellularly from C1 spinal neurons, by means of a glass micropipette filled with 2 % pontamine sky blue in 0.5 M sodium acetate (tip resistance 5–12 MV at 1 kHz). The neuron activity was amplified (WPI, DAM80) and monitored with an oscilloscope (Nihon Kohden, VC-10). Receptive field and conduction velocity Somatic receptive fields of C1 spinal neurons that responded to TP stimulation were examined by tactile stimulation with a brush and by pinching the skin with forceps. The size of each receptive field was quantified by means of a planimeter (Tamaya, Planix, Tokyo) as described in a previous study (Takeda et al., 2000) Similarly, the effects of mechanical stimuli (pin prick and touch) of the TMJ capsule on the activity of C1 spinal neurons were also examined. Neurons that only responded to the brush were classified as the low-threshold mechanoreceptive (LTM), and those stimulated by pinch or pin prick were classified as high threshold (nociceptive specific, NS). Neurons excited by both brush and pinch or pin prick were classified as in a wide dynamic range (WDR). The responses of C1 spinal neurons to somatic field stimulation were recorded and monitored on an oscilloscope. The conduction velocity for each neuron responding to TP or TMJ stimulation was calculated by dividing the distance between the C1 region and the site of the ipsilateral TP (about 45 mm) or TMJ (about 18 mm) by the latency between the stimulus artifact and the first evoked spike. The values for conduction velocity were corrected for a 0.5 ms synaptic delay. We have further classified the C1 neurons based on the type of afferents inputs from the TP, TMJ and facial
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skin (conduction velocity and mechanical receptive field properties), as follows; a) Homogeneous type TP-TMJ convergent neurons belonged to the same afferent fiber range between the conduction velocities evoked by the TP and TMJ stimulation and heterogeneous type TP-TMJ convergent neurons did not belong to the same afferent fiber range between the conduction velocities evoked by the TP and TMJ stimulation. b) Homogeneous type of TP-TMJ convergent neurons had the same responsiveness to the mechanical stimulation from the TMJ, and facial skin and heterogeneous type TP-TMJ convergent neurons did not show the same responsiveness to the mechanical stimulation from the TMJ and the facial skin. Experimental protocol and data analysis The threshold of the JOR was determined from the dEMG amplitude evoked by TP stimulation (stimulus intensity 0.6–4.5 mA, duration 0.1–0.3 ms, single pulse, and stimulus frequency 1 Hz), and stimulus intensity was increased until three to five consecutive dEMG responses to TP stimulation were obtained. The peak-to-peak amplitudes of the dEMG in five stimulus trials were averaged. Poststimulus histograms of C1 spinal neuron activity induced by TP stimulation were made from 8 to 16 stimulus repetitions. The capsule of the ipsilateral TMJ was electrically stimulated (stimulus intensity 1.2–4.9 mA, duration 0.1–0.3 ms, single pulse, stimulus frequency 1 Hz). Trials of 8 or 16 responses were summed to construct the poststimulus histogram. Mustard oil (MO, 20%, 20 Al) was delivered from a Hamilton syringe inserted into the TMJ capsule and each injection was performed slowly for 15–30 s. The statistical significance of the effect of mustard oil on the C1 spinal neuron activity was calculated by using a paired t-test. Data are expressed as the mean F SEM. A P-value of less than 0.05 was statistically significant. Histology After the recording sessions for C1 spinal neurons responding to TMJ stimulation, the rats were deeply anesthetized. Cathodal DC currents (50 AA, 1 min) were passed through a recording micropipette and the animals were transcardially perfused with 10 % buffered formalin and their spinal cords were then removed and fixed in the same fixative solution for at least 1 week. To determine the lesion sites, the spinal cords were frozen to –30 jC, sectioned at 40 Am, mounted on glass slides and then stained with hematoxylin-eosin. Recording sites were also identified by the blue spots, and construction of electrode tracks was done by means of a combination with micromanipulator readings. Data in this study were analyzed only for animals in which recording sites were histologically confirmed.
Results Unit sample The ipsilateral TP stimulation excited a total of 31 C1 spinal neurons. All the neurons were located on the same side as the stimulation. Twenty-five recording sites were found in laminae I–II and 5 neurons were in lamina III. The remaining neuron was located in lamina IV (Fig. 1).
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Fig. 1. Locations of all 31 C1 spinal neurons responding to both tooth pulp (TP) and temporomandibular joint (TMJ) stimuli. Wide dynamic range (WDR) neurons (.), ociceptive specific (NS) neurons (E) and on-responsive (NR) neurons (4). I-X, laminae; IBN, internal basilar nucleus; Pyt, pyramidel tract; CCN, central cervical nucleus; IM, intermediomedial nucleus; LCN, lateral cervical nucleus; LSN, lateral spinal nucleus).
Effect of TP electrical stimulation on C1 spinal neuron activity and dEMG Typical examples of the effects of the ipsilateral TP stimulation, corresponding to 1 (Fig. 2A) and 3.5 (Fig. 2B) times the threshold of JOR, on C1 spinal neuron activity and dEMG are shown in Fig. 2. The number of spikes and the amplitude of dEMG increased with increasing stimulus intensity. The somatic receptive field of this neuron involved the ipsilateral cheek skin and nose (Fig. 2C). The summarized results on the responses of dEMG and C1 spinal neuron activity to TP stimulation at 1– 3.5 times the threshold for JOR are shown in Fig. 2. As the stimulus intensity was increased, both dEMG amplitude (Fig. 2C) and C1 spinal neuron activity (Fig. 2D) were increased proportionally. As shown in Fig. 2D and 2E, there was a positive correlation between the activities of C1 spinal neurons and dEMG during TP stimulation at 1–3.5T. The threshold and latency of TP stimulation-evoked dEMG were 0.5 F 0.1 mA (n = 14) and 7.0 F 0.4 ms (n = 14), respectively. The threshold and latency of TP stimulation for activation of 31 C1 spinal neurons were 1.0 F 0.1 mA and 11.2 F 0.9 ms, respectively. The average distance from the site of TP to the ipsilateral C1 segment was approximately 45 mm, and the average value for conduction velocity was 5.2 F 0.6 m/s (n = 31). Excitatory somatic receptive fields of 25 C1 spinal neurons that responded to the ipsilateral TP stimulation are shown in Fig. 3. Twenty-two neurons had small somatic fields with an area including the ipsilateral cheek skin and hair (Fig. 3A). The neurons that had intermediate-size somatic fields including the ipsilateral face, neck and jaw were not found. Somatic fields of 3 neurons were large, and their area included the ipsilateral site of the face, neck, jaw and upper forearm (Fig. 3B). Fig. 3 also shows two different types of C1 spinal neurons in their responsiveness to noxious (pinch) and non-noxious (touch) stimulation. The neuron that responded to pinch only was classified as the NS (Fig. 3C), and 7 C1 spinal neurons
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Fig. 2. Effect of tooth pulp (TP) stimulation on C1 spinal neuron and digastric electromyogram (dEMG) activities. A, Responses of the single spike of C1 spinal neuron activity (upper left panel), the dEMG (lower left panel) and poststimulus histograms of the neuronal responses to TP stimulation (E) at a 1-fold threshold (T) for jaw-opening reflex (JOR). B, Responses of three traces to TP stimulation at a 3.5-fold T for JOR. C, hatched area is the location of the somatic receptive field. D, Threshold (T)-related responses of digastric electromyogram (dEMG) to tooth pulp (TP) stimulation. E, threshold (T)-related responses of C1 spinal neuron activity to TP stimulation. Vertical bars show mean F S.E. r, correlation coefficient. *, p < 0.01 significant difference from 1T effect.
were NS neurons. The remaining 3 neurons did not respond to either pinch or touch and were classified as the non-responsive (NR). The neuron that responded to both pinch and touch was classified as the WDR, and 18 C1 spinal neurons were WDR neurons (Fig. 3D). The mean receptive field sizes of WDR and NS neurons were 1.58 F 0.62 cm2 and 0.68 F 0.12 cm2, respectively. Effect of TMJ stimulation on C1 spinal neuron activity Concerning the responsiveness of C1 spinal neurons to the TMJ stimulation, each neuron was first tested at 4.0 mA. If it responded to the TMJ stimulation, the threshold for activation was determined by reducing the stimulus intensity. Fig. 4 shows typical excitatory responses to the ipsilateral TP stimulation at 3.5 T (Fig. 4A) as well as the ipsilateral TMJ stimulation (2.7 mA) (Fig. 4B). The
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Fig. 3. Hatched areas show somatic receptive fields of the 25 C1 spinal neurons responding to tooth pulp (TP) stimulation. A, Small somatic field (n = 22). B, large somatic fields (n = 3). C and D, Typical responses of C1 spinal neuron activity to mechanical stimulation (touch and pinch)(C: nociceptive specific (NS) neuron; D: wide dynamic range (WDR) neuron).
excitatory somatic field included a small somatic area (Fig. 4C). In 28 of 31 C1 spinal neurons responding to TP stimulation, the stimulus-dependent responses to a single pulse applied to the TMJ capsule were determined. The threshold of TMJ stimulation for activation of 28 C1 spinal neurons was 2.4 F 0.2 mA. As shown in Fig. 4D, the mean discharge of C1 spinal neurons was increased by increasing the stimulus intensity (2T). TMJ stimulation evoked dEMG activity, the number of spike and the amplitude of dEMG increased with increasing the stimulus intensity (Fig. 4B, 4D). There was a positive correlation between the activities of C1 spinal neurons and dEMG amplitude during TP stimulation at 1–3.5T. As shown in Fig. 4D and 4E, there was a positive correlation between the activities of C1 spinal neurons and dEMG activity during TMJ stimulation at 1 and 2T. The mean latency of 28 C1 spinal neurons responding to the TMJ stimulation was 10.0 F 0.8 ms. The threshold and latency of TMJ-induced dEMG activity were 2.4 F 0.2 mA (n = 14) and 2.3 F 0.3 ms (n = 28), respectively. The average distance from the site of TMJ to the ipsilateral C1 segment was approximately 18 mm, and the average conduction velocity was 1.8 F 0.3 m/sec. Table 1 summarizes the values for conduction velocities of afferent fibers from the TMJ and TP. Approximately 43% of 28 C1 spinal neurons were homogenous. Furthermore, approximately
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Fig. 4. Effects of temporomandibular joint (TMJ) stimulation on C1 spinal neurons responding to tooth pulp (TP) stimulation. A, Responses of the single spike of C1 spinal neuron activity, dEMG activity (left panel) and poststimulus histograms (right panel) of the neuronal responses to TP stimulation at a 3.5-fold threshold (T) for jaw-opening reflex (JOR) (open column, stimulus artifact; solid columns, recorded responses). B, Responses of the single spike of C1 spinal neuron, dEMG activity and poststimulus histograms of the neuronal responses to TMJ stimulation (4) at a 1-fold T for evoked spike in exactly the same neuron as shown in A (open column, stimulus artifact; solid columns, recorded responses). C, Hatched area is the location of the somatic receptive field. D and E, Threshold (T)-related responses of dEMG and C1 spinal neuron activity and dEMG to TMJ stimulation. Vertical bars show the mean F S.E. r, correlation coefficient *, p < 0.01, significant difference from 1T effect.
68% of the C1 spinal neurons tested received nociceptive information from C fibers in the TMJ region. Effect of mechanical stimulation of TMJ capsule on C1 spinal neuron activity Responses of C1 spinal neuron activity to mechanical stimulation of the TMJ capsule were divided into two groups: one responded to noxious stimulation (pin prick) only (Fig. 5A) and one responded to both nonnoxious (touch) and noxious stimuli (Fig. 5B). Eighteen out of 31 C1 spinal neurons were
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Table 1 Changes in mean conduction velocity evoked by the tooth pulp (TP) and temporomandibular joint (TMJ) stimulation TP Homogeneous type Heterogeneous type
n
Fiber type
Conduction velocity (m/s)
Ay C Ay
6.6 F 0.9 1.8 F 0.1 5.0 F 0.9
9 3 19
TMJ
n
Fiber type
Conduction velocity (m/s)
Ay C C
3.3 F 0.4 0.8 F 0.2 1.2 F 0.1
9 3 16
Data show mean F S.E.; n, the number of experiments.
WDR neurons, and 7 C1 spinal neurons were NS neurons. The remaining 6 C1 spinal neurons were NR neurons. As shown in Table 2, 36% of 25 C1 spinal neurons tested were homogenous when mechanical stimuli were applied to the TMJ capsule and facial skin, and the remaining C1 spinal neurons were heterogeneous. Of the 28 TMJ-TP convergent neurons, approximately 80% of these neurons received nociceptive information.
Fig. 5. Responses of C1 spinal neuron activity to mechanical stimuli (touch and pin prick) of the temporomandibular joint (TMJ) capsule. A, C1 spinal neuron responded to pin prick only. B, C1 spinal neuron responded to both touch and pin prick. bin = 100 ms.
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Table 2 Responses of C1 spinal neuron activity to mechanical stimulation of the temporomandibular joint (TMJ) and facial skin TMJ Homogeneous type Heterogeneous type
+
Facial skin
n
Touch
Pin prick
Touch
Pinch
+ + +
+ + + + + +
+ + +
+ + + + +
3 6 4 1 4 1 6
C1 spinal neurons responded to mechanical stimulation (+) and C1 spinal neurons did not responded to mechanical stimulation ( ). n, the number of experiments.
Chemical stimulation of the TMJ capsule on C1 spinal neuron activity A typical response of C1 spinal neuron activity to chemical stimulation of the TMJ region is shown in Fig. 6A. The C1 spinal neuron activity was excited by application of MO into the TMJ capsule. Such an effect was obtained in 8 out of 11 C1 spinal neurons that responded to the TMJ stimulation. The summarized results are shown in Fig. 6B. MO injection produced a significant increase in the C1 spinal neuron activity and this effect lasted for approximately 15 min. The average values for conduction velocity in these neurons responding to MO injection were 1.6 F 0.2 m/sec and were similar to the range of conduction velocity of C fibers.
Discussion The present study provided evidence that C1 spinal neurons activated by TP stimulation were also excited by TMJ stimulation. The results indicate that there may be a convergence of noxious afferent inputs from the TMJ and TP on the same C1 spinal neurons. In the present study, the activity of 31 C1 spinal neurons and the amplitude of dEMG (n = 31) both increased in magnitude during TP stimulation at 1–3.5 times the threshold for JOR. Similar excitatory effects of TP stimulation on C1 spinal neuron activity and dEMG has been demonstrated in the rat (Matsumoto et al., 1999; Tanimoto et al., 2002). Based on evidence that the histological structures of the C1 region are analogous to the caudalis in the trigeminal spinal nucleus, which receives most of the noxious afferent inputs, it is most likely that C1 spinal neurons play a significant role in the perception of tooth pain in the rat. This was further confirmed by evidence that c-fos expression in lamina I in C1–C2 segments of the rat spinal cord appears after noxious thermal or mechanical stimulation of the TP (Coimbra and Coimbra, 1994) or that chemical (MO) stimulation of the rat nasal mucosa produces numerous Fos-immunoreactive neurons in laminae I–II of the rat C1–C2 regions (Takeda et al., 1998). Small diameter afferent fibers that consist of the mandibular division of the trigeminal nerve, rootlets of the upper cervical spinal cord and branches of the vagus nerve are known to enter the TMJ region (Klineberg, 1971; Denny-Brown and Yanagisawa, 1973; Widenfalk and Wiberg, 1990; Kido et al., 1993; Uddman et al., 1999). These fibers that innervate adjacent masticatory muscles project into
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Fig. 6. A typical example of the effect of chemical stimulation of the temporomandibular joint (TMJ) region on C1 spinal neuron activity. A, Responses of C1 spinal neuron activity to 20% mustard oil (MO, 20 Al) injected into the TMJ capsule. B, Effect of chemical stimulation of the TMJ region on the mean spike numbers of C1 spinal neurons responding to TP stimulation. Vertical bars show the mean F S.E.; n = 11. *P < 0.05, significant difference from control values. bin = 100 ms.
laminae I in the trigeminal spinal subnucleus caudalis and/or cervical cord junction (Vc/C2) region (Shigenaga et al., 1988; Arvidsson and Raappana, 1989), involving the C1 region, but the central projection of small diameter afferents that especially innervate the TMJ has not been identified. When considering collective data from Fos studies, particularly concerning the TMJ region after chemical stimulation and injury (Hathaway et al., 1995; Bereiter and Bereiter, 2000; Bereiter, 2001), it is possible to speculate that neurons in laminae I–II of the Vc/C2 region play an important role in processing the pain signal from the TMJ and surrounding masticatory muscles. Many neurons located at the caudalis and interpolaris in the trigeminal spinal nucleus receive nociceptive inputs from the TMJ and/or the masseter muscle (Mm) and have extensive convergence of afferent inputs, involving the TMJ, Mm, or the facial skin (Kojima, 1990; Ohya, 1992). In order to selectively stimulate the TMJ region only, we exposed the TMJ capsule by removing the Mm and directly stimulating the afferent terminal in the TMJ. In the present study, C1 spinal neurons that responded to TMJ stimulation were also excited by TP stimulation. Furthermore, locations of recorded C1 spinal neurons were consistent with those of c-Fos immunoreactivity neurons after TMJ injury (Bereiter and Bereiter, 2000; Bereiter et al., 2002). The C1 neurons that responded to both TMJ and TP stimuli had somatic receptive fields
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on the facial skin, and most C1 spinal neurons (8/11) were also stimulated by administration of mustard oil into the TMJ region. There is evidence that trigeminal and upper cervical afferent nerve fibers converge on the cells in the C1–C2 segments of the rat spinal cord (Pfaller and Arvidsson, 1988). When considering the fact that the face, neck, jaw, TP and phrenic nerve (PN) fibers converge on the same C1 spinal neurons (Matsumoto et al., 1999), taken together, it is more conceivable that there may be the convergence of face, neck, jaw, TP, PN and TMJ afferents on the same C1 spinal neurons in the rat. In addition, that the PN converged on C1 spinal neurons along with somatic afferents has been demonstrated in the rat (Razook et al., 1995) as well as more recently in the monkey (Chandler et al., 1998). Chandler et al. (1999) also found convergence of trigeminal input with visceral and phrenic inputs on primate C1–C2 spinothalamic tract cells. Furthermore, Chandler et al. (2000) in the monkey and Qin et al. (2001) in the rat demonstrated that stimulation of cardiac afferent fibers excites C1–C2 spinothalamic tract cells and superficial and deeper C1–C2 spinal cells, respectively. Finally, Foreman (2000) suggested that organization of the neurons of the upper cervical segments may be more than just a simple excitation of the trigeminal spinal nucleus caudalis. Presumably, the characteristics of C1 spinal neurons having various receptive fields may be responsible for the diffuse nature and spreading and referral of pain in TMD patients (Sessle et al., 1993). Concerning the effect of TMJ stimulation on C1 spinal neuron activity, the difference in the type of TMJ afferent fibers would reflect different conduction velocities. The mean conduction velocity was 1.8 F 0.3 m/sec, and the distribution consisted of both Ay (32%, 9/28) and C fibers (68%, 19/ 28). But the range of the Ah fibers was not identified in this study. The homogeneity of the conduction velocities of TMJ and TP afferents was seen in 43% of C1 spinal neurons (12/28) tested, but the remaining (57%) of C1 neurons revealed heterogeneity for the conduction velocity. In the in vitro TMJ preparation with the rat auriculo-temporal nerve (TMJ-nerve preparation), Takeuchi et al. (2001) reported that among a total of 22 single afferent fibers, responses to mechanical stimulation of the TMJ region could be recorded from 7 Ay and 15 C fibers. Concerning the convergence patterns from afferent inputs from the TMJ and facial skin, 80% of 28 TMJ-TP convergent neurons received nociceptive information from deep structures and skin. This probably implies that the convergence of information from the TP and TMJ may contribute to the mechanism of a referred pain. When we consider the fact that most C1 spinal neurons (90%, 28/31) responding to the TMJ stimulation were also excited by administration of 20 % mustard oil (20 Al) into the TMJ capsule, taken together, it can be suggested that the TMJ region is mainly innervated by small diameter afferent fibers. On the behavior of their responsiveness to mechanical stimulation, not all C1 spinal neurons recorded were not LTM neurons, which are thought to respond only to non-noxious stimulation (touch) of the facial skin and TMJ capsule, but they were classified as either WDR or NS, based on their responsiveness to touch and pin prick of the TMJ or the facial skin. Similar behavior in response to mechanical stimulation involving the somatic receptive field stimulation has been reported in C1 spinal neurons (Matsumoto et al., 1999; Tanimoto et al., 2002). The homogeneity of the mechanical behavior of the facial skin and the TMJ capsule was observed in 36% of C1 spinal neurons (9/25), but there was heterogeneity between their relationships in 64 % of C1 spinal neurons (16/25). Some C1 spinal neurons (3/28) that responded to TMJ stimulation were not excited by mechanical stimulation (touch and pin prick) of the TMJ capsule, indicating that this type of neuron may belong to the category responding to high-threshold mechanoreceptive and/or proprioceptive afferent fibers from the TMJ region.
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In this study, we provided new insights into the contribution of C1 spinal neurons to the convergent patterns of afferent nociceptive information from the exposed TMJ capsule and the TP. Our results are basically consistent with the fact that superficial neurons of the Vc/C2 region play an important role in the regulation of pain perception from the TMJ (Bereiter and Bereiter, 2000). In clinical studies, it has been reported that acute pulpalgia contributes to a factor forming TMD symptoms (Wright and Gullickson, 1996). In fact, there is evidence that complete or partial relief from TMD symptoms occurrs after receiving endodontic treatment or having tooth extraction (Wright and Gullickson, 1996). Accordingly, our results may explain this clinical evidence and suggest convergence of primary afferent inputs from the TMJ and TP onto the upper cervical spinal cord, which contributes to a referred pain associated with TMD. References Arvidsson, J., Raappana, P., 1989. An HRP study of the central projection from primary sensory neurons innervating the rat masseter muscle. Brain Res. 480, 111 – 118. Bereiter, D.A., Bereiter, D.F., 2000. Morphine and NMDA receptor antagonism reduce c-fos expression in spinal trigeminal nucleus produced by acute injury to the TMJ region. Pain 85, 65 – 77. Bereiter, D.A., 2001. Sex differences in brainstem neural activation after injury to the TMJ region. Cells Tissues Organs 169, 226 – 237. Bereiter, D.A., Bereiter, D.F., Ramos, M., 2002. Vagotomy prevents morphine-induced reduction in Fos-like immunoreactivity in trigeminal spinal nucleus produced after TMJ injury in sex-dependent manner. Pain 96, 205 – 213. Casatti, C.A., Frigo, L., Bauer, J.A., 1999. Origin of sensory and autonomic innervation of rat temporomandibular joint: a retrograde axonal tracing study with the fluorescent day fast blue. J. Dent. Res. 78, 776 – 783. Chandler, M.J., Qin, C., Yuan, Y., Foreman, R.D., 1999. Convergence of trigeminal input with visceral and phrenic inputs on primate C1 – C2 spinothalamic tract neurons. Brain Res. 829, 204 – 208. Chandler, M.J., Zhang, J., Foreman, R.D., 1998. Phrenic nerve inputs to upper cervical (C1 – C3) spinothalamic tract neurons in monkey. Brain Res. 798, 93 – 100. Chandler, M.J., Zhang, J., Qin, C., Yuan, Y., Foreman, R.D., 2000. Intrapericardiac injections of algogenic chemicals excite primate C1 – C2 spinothalamic tract neurons. Am. J. Physiol. Regl. Intg. Comp. Physiol. 279, R560 – R568. Coimbra, F., Coimbra, A., 1994. Dental noxious input reaches the subnucleus caudalis of the trigeminal complex in the rat, as shown by c-fos expression upon thermal or mechanical stimulation in the rat. Neurosci. Lett. 173, 201 – 204. Denny-Brown, D., Yanagisawa, N., 1973. The function of the descending root of the fifth nerve. Brain 96, 783 – 814. Dubner, R., Sessle, B.J., Storey, A.T., 1978. The Neural Basis of Oral and Facial Function. Plenum Press, New York. Dworkin, S.F., Friction, J.R., Hollender, L., Huggins, K.H., Le Resche, L., Lund, J., Mohl, N., Ohrbach, R., Palla, S.F., Sommers, E.E., Stohler, C., Truelove, E.L., Von Korff, M., Widmer, C.G., 1992. Research diagnostic criteria for temporomandibular disorder: review, criteria, examinations and specifications, critique. J. craniomand. Disord. Facial Pain Oral Pain 6, 301 – 355. Eversole, L.R., Machado, L., 1985. Temporomandibular joint internal derangements and associated neuromuscular disorders. J. Am. Dent. Assoc. 110, 69 – 79. Foreman, R.D., 2000. Integration of viscerosomatic sensory input at the spinal level. Prog. Brain Res. 122, 219 – 221. Friction, J.R., Kroening, R.J., Hathway, K.M., 1988. TMJ and Craniofacial Pain: Diagnosis and Management Ishiyaku Euro America, St. Louis, MO. Hathaway, C.B., Hu, J.W., Bereiter, D.A., 1995. Distribution of Fos-like immunoreactivity in the caudal brainstem of the rat following noxious chemical stimulation of the temporomandibular joint. J. Comp. Neurol. 356, 444 – 456. Jacquin, M.F., Semba, K., Egger, M.D., Rhoades, R.W., 1983. Organization of HRP-labeled trigeminal mandibular primary afferent neurons in the rat. J. Comp. Neurol. 215, 397 – 420. Kido, M.A., Kiyoshima, T., Kondo, T., Ayasaka, N., Moroi, R., Terada, Y., Tanaka, T., 1993. Distribution of substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat temporomandibular joint. J. Dental Res. 72, 592 – 598.
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Klineberg, I., 1971. Structure and function of temporomandibular joint innervation. Ann. Roy Coll. Surg. Engl. 49, 268 – 288. Kojima, Y., 1990. Convergence patterns of afferent information from the temporomandibular joint and masseter muscle in the trigeminal subnucleus caudalis. Brain Res. Bull 24, 609 – 616. Laskin, D.M., 1979. Myofascial pain-dysfunction syndrome. In: Sarner, B.G., Laskin, D.M. (Eds.), The Temporomandibular Joint. Biological Basis for Clinical Practice, CC Thomas, Springfield, IL, p. 289. Matsumoto, S., Takeda, M., Tanimoto, T., 1999. Effects of electrical stimulation of the tooth pulp and phrenic nerve fiber on C1 spinal neurons in the rat. Exp. Brain Res. 126, 351 – 358. McNeill, D.L., Chandler, M.J., Fu, Q.G., Foreman, R.D., 1991. Projection of nodose ganglion cells to the rat upper cervical spinal cord in the rat. Brain Res. Bull 27, 151 – 155. Ohya, A., 1992. Responses of trigeminal subnucleus interpolaris neurons to afferent inputs from deep oral structures. Brain Res. Bull 29, 773 – 781. Pfaller, K., Arvidsson, J., 1988. Central distribution of trigeminal and upper cervical primary afferent in the rat studied by anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin. J. Comp. Neurol. 268, 91 – 108. Qin, C., Chandler, M.J., Miller, K.E., Foreman, R.D., 2001. Responses and afferent pathways of superficial and deeper C1 – C2 spinal cells to intrapericardial algogenic chemicals in rats. J. Neurophysiol. 85, 1522 – 1532. Razook, J.C., Chandler, M.J., Foreman, R.D., 1995. Phrenic afferent input excites C1 – C2 spinal neurons in rats. Pain 63, 117 – 125. Sessle, B.J., Hu, J.W., Yu, X.M., 1993. Brainstem mechanisms of referred pain and hyperalgesia in the orofacial and temporomandibular region. In: Vecchiet, L., Albe-Fessard, D., Lindblom, U., Giamberardino, M.A. (Eds.), New Trends in Referred Pain and Hyperaalgesia, 59 – 71. Shigenaga, Y., Sera, M., Nishimori, T., Suemune, S., Nishimura, M., Yoshida, A., Tsuru, K., 1988. The central projection of masticatory afferent fibers to the trigeminal sensory nuclear complex and upper cervical spinal cord. J. Comp. Neurol. 268, 489 – 507. Takeda, M., Matsumoto, S., Tanimoto, T., 1998. C-Fos-like immunoreactivity in the upper cervical spinal dorsal horn neurons following noxious chemical stimulation of the nasal mucosa in pentobarbital-anesthetized rat. Arch. Histol. Cytol. 61, 83 – 87. Takeda, M., Tanimoto, T., Ikeda, M., Nishikawa, T., Kawanishi, N., Mohri, M., Shimizu, T., Matsumoto, S., 1999. Changes in C-Fos expression induced by noxious stimulation in the trigeminal spinal nucleus caudalis and C1 spinal neurons of rats after hyperbaric exposure. Arch. Histol. Cytol. 62, 165 – 170. Takeda, M., Tanimoto, T., Matsumoto, S., 2000. Changes in mechanical receptive field properties induced by GABAA receptor activation in the trigeminal spinal nucleus caudalis neurons in rats. Exp. Brain Res. 134, 409 – 416. Takeshita, S., Hirata, H., Bereiter, D.A., 2001. Intensity coding by TMJ-responsive neurons in superficial laminae of caudal medullary dorsal horn of the rat. J. Neurophysiol. 86, 2393 – 2404. Takeuchi, Y., Ishii, N., Toda, K., 2001. An in vitro temporomandibular joint-nerve preparation for pain study in rats. J. Neurosci. Methods 109, 123 – 128. Tanimoto, T., Takeda, M., Matsumoto, S., 2002. Suppressive effect of vagal afferents on cervical dorsal horn neurons responding to tooth pulp electrical stimulation in the rat. Exp. Brain Res. 145, 468 – 479. Uddman, R., Kato, J., Lindgren, P., Sundler, F., Edvinsson, L., 1999. Expression of calcitonin gene-related peptide-1 receptor mRNA in human tooth pulp and trigeminal ganglion. Arch. Oral Biol. 44, 1 – 6. Widenfalk, B., Wasteson, A., Ohlsen, L., 1989. Formation of chondroitin sulphate proteoglycan in cartilage regenerated from free perichondrial graft. Scand. J. Plast. Reconstr. Surg. Hand Surg. 23, 163 – 168. Wright, E.F., Gullickson, D.C., 1996. Idetifying acute pulpalgia as a factor in TMD pain. J. Am. Dent. Assoc. 127, 773 – 780. Widenfalk, B., Wiberg, M., 1990. Origin of sympathetic and sensory innervation of the tempolo-mandibular joint. A retrograde axonal tracing study in the rat. Neurosci. letter 109, 30 – 35. Yoshino, K., kawagishi, S., Amano, N., 1998. Morphological characteristics of primary sensory and post synaptic sympathetic neurons supplying the temporomandibular joint in the cat. Arch. Oral Biol. 43, 679 – 686.
Convergence of nociceptive information from temporomandibular joint and tooth pulp afferents on C1 spinal neurons in the rat Toshimi Nishikawa *, Mamoru Takeda, Takeshi Tanimoto, Shigeji Matsumoto Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Received 6 November 2003; accepted 5 March 2004
Abstract The aim of the present study was to test the hypothesis that there is a convergence of afferent inputs from the temporomandibular joint (TMJ) on C1 spinal neurons responding to electrical stimulation of the tooth pulp (TP). In 14 pentobarbital anesthetized rats, the extracellular single unit activity of 31 C1 spinal neurons and the amplitude in a digastric muscle electromyogram (n = 31) increased proportionally during 1.0 –3.5 times the threshold for the jawopening reflex (JOR). Of 31 C1 spinal neurons responsive to TP afferents, 28 (approximately 90%) were also excited by electrical stimulation of the ipsilateral TMJ capsule. All neurons tested were divided into three categories of nociceptive specific, wide dynamic range and non-responsive as to their responsiveness to mechanical stimuli (pin prick and touch) of the somatic receptive field (skin of the face, neck, jaw and upper forearm) and TMJ capsule. Nineteen (68%) of 28 C1 spinal neurons received nociceptive information from C fibers of the TMJ capsule. These results suggest that there is a convergence of noxious information from the TMJ and TP afferents on the same C1 spinal neurons, which importantly contribute to pain perception from the TMJ region. D 2004 Elsevier Inc. All rights reserved. Keywords: Temporomandibular joint; C1 spinal dorsal horn; Referred pain; Tooth pulp; Rat
Introduction The temporomandibular joint (TMJ) region is innervated by branches of the trigeminal, upper cervical and vagus nerve (Klineberg, 1971; Denny-Brown and Yanagisawa, 1973; Widenfalk and Wiberg, 1990; Kido et al., 1993; Casatti et al., 1999), indicating that both sensory afferents and autonomic efferents * Corresponding author. Tel./Fax: +81-3-3261-8740. E-mail address: [email protected] (T. Nishikawa). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.014
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innervate the TMJ region. The former plays a significant role in nociception as well as in the modification of masticatory muscles (Dubner et al., 1978), and the latter acts as vasomotor control (Widenfalk et al., 1989). The rat trigeminal ganglion projects centrally to both the trigeminal nuclear complex and cervical dorsal horn (Jacquin et al., 1983; Pfaller and Arvidsson, 1988), and vagal afferent fibers project directly to the upper cervical segments of the spinal cord via a supraspinal route from the nodose ganglia (McNeill et al., 1991). In the studies with retrograde axonal tracers, the TMJ region is supplied with nerve fibers, originating mainly in the trigeminal ganglion, in addition to other sensory and sympathetic ganglia (Widenfalk and Wiberg, 1990; Uddman et al., 1999), and consisting of both myelinated (Ay) and unmyelinated (C) fibers (Yoshino et al., 1998). On the other hand, temporomandibular joint disorder (TMD) is expressed by a painful condition involving the TMJ and/or the muscles of mastication (Dworkin et al., 1992). Based on physical findings, it is considered that TMD is divided into three symptoms: muscle disorders, intracapsular disarrangements of the TMJ and degenerative arthritic changes in the TMJ (Laskin, 1979; Eversole and Machado, 1985; Friction et al., 1988; Dworkin et al., 1992). But the characterization of the afferent inputs from the TMJ has not yet been determined. Acute injury to the TMJ region results in a high density of Fos-positive neurons in the areas including the trigeminal spinal subnucleus caudalis (Vc) and upper cervical cord junction region (Vc/C2) (Hathaway et al., 1995; Bereiter and Bereiter, 2000; Bereiter, 2001). Based on evidence that the Vc/C2 region was excited by mechanical and/or chemical stimulation of the TMJ region, Takeshita et al. (2001) concluded that neurons in the lamina I–II in the Vc/C2 region play an important role in mediating pain sensation in TMD. Based on evidence that complete or partial relief from TMD occurred after receiving endodontic treatment or having tooth extraction, Wright and Gullickson (1996) suggested that acute pulpalgia acts as a significant factor in forming TMD symptoms. Because the C1 region is considered as an extension of the Vc, which receives most nociceptive inputs from the trigeminal nerve and because C1 spinal neurons that have somatic receptive fields in the facial skin are stimulated by electrical stimulation of the tooth pulp (TP) (Matsumoto et al., 1999; Takeda et al., 1999; Tanimoto et al., 2002), we hypothesized that C1 spinal neurons responding to TP stimulation are also excited by electrical stimulation of the TMJ region. To test this hypothesis, the present study was designed to examine the properties of the C1 spinal neurons in their response to mechanical and chemical stimulation of the exposed TMJ capsule, and convergence patterns of afferent information from the exposed TMJ capsule and the TP to these neurons.
Materials and methods Animal preparation The experiments were performed on 14 adult male Wistar rats (300–450 g). All experimental protocols used in this study were approved by the Animal Use and Care Committee of Nippon Dental University. Each animal was first anesthetized with sodium pentobarbital (45 mg/kg, i.p.) and maintained with additional doses of 2–3 mg/kg/h through a cannula inserted into the jugular vein, as required. The trachea was cannulated, and the rectal temperature was maintained at 37 F 0.5 jC with a radiant heater. The depth of anesthesia was checked by the lack of response to paw pinching. All wound
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margins were repeatedly covered with a local anesthetic, 2% lidocaine (Xylocain), throughout the experiment. Tooth pulp stimulation The method of TP stimulation was similar to that in previous studies (Takeda et al., 1998; Matsumoto et al., 1999; Tanimoto et al., 2002). Bipolar stimulating electrodes made from silver wires, (diameter 150 Am, enamel insulated except for the tip) were inserted into the pulp of the upper left incisors. To avoid spread of the stimulus current, the electrodes were isolated from surrounding tissues with dental resin. Temporomandibular joint stimulation The temporomandibular joint (TMJ) was exposed by removing the skin part on the temporal muscle and zygomatic arch. Bipolar stimulating electrodes made from silver balls (diameter 0.3 mm) were directly positioned onto the exposed capsule of the left TMJ. Recordings of dEMG and C1 spinal neuron activity The digastric electromyogram (dEMG) was recorded with silver electrodes (diameter 0.3 mm, interpolar distance 2 mm), and this EMG activity was used to assess the JOR. The rats were then placed in a stereotaxic apparatus, and a laminectomy was performed to expose the C1 spinal region of the spinal cord. The dura was cut and the brain surface was covered with a warm liquid paraffin oil (36– 37 jC). The single neuronal activity was recorded extracellularly from C1 spinal neurons, by means of a glass micropipette filled with 2 % pontamine sky blue in 0.5 M sodium acetate (tip resistance 5–12 MV at 1 kHz). The neuron activity was amplified (WPI, DAM80) and monitored with an oscilloscope (Nihon Kohden, VC-10). Receptive field and conduction velocity Somatic receptive fields of C1 spinal neurons that responded to TP stimulation were examined by tactile stimulation with a brush and by pinching the skin with forceps. The size of each receptive field was quantified by means of a planimeter (Tamaya, Planix, Tokyo) as described in a previous study (Takeda et al., 2000) Similarly, the effects of mechanical stimuli (pin prick and touch) of the TMJ capsule on the activity of C1 spinal neurons were also examined. Neurons that only responded to the brush were classified as the low-threshold mechanoreceptive (LTM), and those stimulated by pinch or pin prick were classified as high threshold (nociceptive specific, NS). Neurons excited by both brush and pinch or pin prick were classified as in a wide dynamic range (WDR). The responses of C1 spinal neurons to somatic field stimulation were recorded and monitored on an oscilloscope. The conduction velocity for each neuron responding to TP or TMJ stimulation was calculated by dividing the distance between the C1 region and the site of the ipsilateral TP (about 45 mm) or TMJ (about 18 mm) by the latency between the stimulus artifact and the first evoked spike. The values for conduction velocity were corrected for a 0.5 ms synaptic delay. We have further classified the C1 neurons based on the type of afferents inputs from the TP, TMJ and facial
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skin (conduction velocity and mechanical receptive field properties), as follows; a) Homogeneous type TP-TMJ convergent neurons belonged to the same afferent fiber range between the conduction velocities evoked by the TP and TMJ stimulation and heterogeneous type TP-TMJ convergent neurons did not belong to the same afferent fiber range between the conduction velocities evoked by the TP and TMJ stimulation. b) Homogeneous type of TP-TMJ convergent neurons had the same responsiveness to the mechanical stimulation from the TMJ, and facial skin and heterogeneous type TP-TMJ convergent neurons did not show the same responsiveness to the mechanical stimulation from the TMJ and the facial skin. Experimental protocol and data analysis The threshold of the JOR was determined from the dEMG amplitude evoked by TP stimulation (stimulus intensity 0.6–4.5 mA, duration 0.1–0.3 ms, single pulse, and stimulus frequency 1 Hz), and stimulus intensity was increased until three to five consecutive dEMG responses to TP stimulation were obtained. The peak-to-peak amplitudes of the dEMG in five stimulus trials were averaged. Poststimulus histograms of C1 spinal neuron activity induced by TP stimulation were made from 8 to 16 stimulus repetitions. The capsule of the ipsilateral TMJ was electrically stimulated (stimulus intensity 1.2–4.9 mA, duration 0.1–0.3 ms, single pulse, stimulus frequency 1 Hz). Trials of 8 or 16 responses were summed to construct the poststimulus histogram. Mustard oil (MO, 20%, 20 Al) was delivered from a Hamilton syringe inserted into the TMJ capsule and each injection was performed slowly for 15–30 s. The statistical significance of the effect of mustard oil on the C1 spinal neuron activity was calculated by using a paired t-test. Data are expressed as the mean F SEM. A P-value of less than 0.05 was statistically significant. Histology After the recording sessions for C1 spinal neurons responding to TMJ stimulation, the rats were deeply anesthetized. Cathodal DC currents (50 AA, 1 min) were passed through a recording micropipette and the animals were transcardially perfused with 10 % buffered formalin and their spinal cords were then removed and fixed in the same fixative solution for at least 1 week. To determine the lesion sites, the spinal cords were frozen to –30 jC, sectioned at 40 Am, mounted on glass slides and then stained with hematoxylin-eosin. Recording sites were also identified by the blue spots, and construction of electrode tracks was done by means of a combination with micromanipulator readings. Data in this study were analyzed only for animals in which recording sites were histologically confirmed.
Results Unit sample The ipsilateral TP stimulation excited a total of 31 C1 spinal neurons. All the neurons were located on the same side as the stimulation. Twenty-five recording sites were found in laminae I–II and 5 neurons were in lamina III. The remaining neuron was located in lamina IV (Fig. 1).
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Fig. 1. Locations of all 31 C1 spinal neurons responding to both tooth pulp (TP) and temporomandibular joint (TMJ) stimuli. Wide dynamic range (WDR) neurons (.), ociceptive specific (NS) neurons (E) and on-responsive (NR) neurons (4). I-X, laminae; IBN, internal basilar nucleus; Pyt, pyramidel tract; CCN, central cervical nucleus; IM, intermediomedial nucleus; LCN, lateral cervical nucleus; LSN, lateral spinal nucleus).
Effect of TP electrical stimulation on C1 spinal neuron activity and dEMG Typical examples of the effects of the ipsilateral TP stimulation, corresponding to 1 (Fig. 2A) and 3.5 (Fig. 2B) times the threshold of JOR, on C1 spinal neuron activity and dEMG are shown in Fig. 2. The number of spikes and the amplitude of dEMG increased with increasing stimulus intensity. The somatic receptive field of this neuron involved the ipsilateral cheek skin and nose (Fig. 2C). The summarized results on the responses of dEMG and C1 spinal neuron activity to TP stimulation at 1– 3.5 times the threshold for JOR are shown in Fig. 2. As the stimulus intensity was increased, both dEMG amplitude (Fig. 2C) and C1 spinal neuron activity (Fig. 2D) were increased proportionally. As shown in Fig. 2D and 2E, there was a positive correlation between the activities of C1 spinal neurons and dEMG during TP stimulation at 1–3.5T. The threshold and latency of TP stimulation-evoked dEMG were 0.5 F 0.1 mA (n = 14) and 7.0 F 0.4 ms (n = 14), respectively. The threshold and latency of TP stimulation for activation of 31 C1 spinal neurons were 1.0 F 0.1 mA and 11.2 F 0.9 ms, respectively. The average distance from the site of TP to the ipsilateral C1 segment was approximately 45 mm, and the average value for conduction velocity was 5.2 F 0.6 m/s (n = 31). Excitatory somatic receptive fields of 25 C1 spinal neurons that responded to the ipsilateral TP stimulation are shown in Fig. 3. Twenty-two neurons had small somatic fields with an area including the ipsilateral cheek skin and hair (Fig. 3A). The neurons that had intermediate-size somatic fields including the ipsilateral face, neck and jaw were not found. Somatic fields of 3 neurons were large, and their area included the ipsilateral site of the face, neck, jaw and upper forearm (Fig. 3B). Fig. 3 also shows two different types of C1 spinal neurons in their responsiveness to noxious (pinch) and non-noxious (touch) stimulation. The neuron that responded to pinch only was classified as the NS (Fig. 3C), and 7 C1 spinal neurons
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Fig. 2. Effect of tooth pulp (TP) stimulation on C1 spinal neuron and digastric electromyogram (dEMG) activities. A, Responses of the single spike of C1 spinal neuron activity (upper left panel), the dEMG (lower left panel) and poststimulus histograms of the neuronal responses to TP stimulation (E) at a 1-fold threshold (T) for jaw-opening reflex (JOR). B, Responses of three traces to TP stimulation at a 3.5-fold T for JOR. C, hatched area is the location of the somatic receptive field. D, Threshold (T)-related responses of digastric electromyogram (dEMG) to tooth pulp (TP) stimulation. E, threshold (T)-related responses of C1 spinal neuron activity to TP stimulation. Vertical bars show mean F S.E. r, correlation coefficient. *, p < 0.01 significant difference from 1T effect.
were NS neurons. The remaining 3 neurons did not respond to either pinch or touch and were classified as the non-responsive (NR). The neuron that responded to both pinch and touch was classified as the WDR, and 18 C1 spinal neurons were WDR neurons (Fig. 3D). The mean receptive field sizes of WDR and NS neurons were 1.58 F 0.62 cm2 and 0.68 F 0.12 cm2, respectively. Effect of TMJ stimulation on C1 spinal neuron activity Concerning the responsiveness of C1 spinal neurons to the TMJ stimulation, each neuron was first tested at 4.0 mA. If it responded to the TMJ stimulation, the threshold for activation was determined by reducing the stimulus intensity. Fig. 4 shows typical excitatory responses to the ipsilateral TP stimulation at 3.5 T (Fig. 4A) as well as the ipsilateral TMJ stimulation (2.7 mA) (Fig. 4B). The
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Fig. 3. Hatched areas show somatic receptive fields of the 25 C1 spinal neurons responding to tooth pulp (TP) stimulation. A, Small somatic field (n = 22). B, large somatic fields (n = 3). C and D, Typical responses of C1 spinal neuron activity to mechanical stimulation (touch and pinch)(C: nociceptive specific (NS) neuron; D: wide dynamic range (WDR) neuron).
excitatory somatic field included a small somatic area (Fig. 4C). In 28 of 31 C1 spinal neurons responding to TP stimulation, the stimulus-dependent responses to a single pulse applied to the TMJ capsule were determined. The threshold of TMJ stimulation for activation of 28 C1 spinal neurons was 2.4 F 0.2 mA. As shown in Fig. 4D, the mean discharge of C1 spinal neurons was increased by increasing the stimulus intensity (2T). TMJ stimulation evoked dEMG activity, the number of spike and the amplitude of dEMG increased with increasing the stimulus intensity (Fig. 4B, 4D). There was a positive correlation between the activities of C1 spinal neurons and dEMG amplitude during TP stimulation at 1–3.5T. As shown in Fig. 4D and 4E, there was a positive correlation between the activities of C1 spinal neurons and dEMG activity during TMJ stimulation at 1 and 2T. The mean latency of 28 C1 spinal neurons responding to the TMJ stimulation was 10.0 F 0.8 ms. The threshold and latency of TMJ-induced dEMG activity were 2.4 F 0.2 mA (n = 14) and 2.3 F 0.3 ms (n = 28), respectively. The average distance from the site of TMJ to the ipsilateral C1 segment was approximately 18 mm, and the average conduction velocity was 1.8 F 0.3 m/sec. Table 1 summarizes the values for conduction velocities of afferent fibers from the TMJ and TP. Approximately 43% of 28 C1 spinal neurons were homogenous. Furthermore, approximately
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Fig. 4. Effects of temporomandibular joint (TMJ) stimulation on C1 spinal neurons responding to tooth pulp (TP) stimulation. A, Responses of the single spike of C1 spinal neuron activity, dEMG activity (left panel) and poststimulus histograms (right panel) of the neuronal responses to TP stimulation at a 3.5-fold threshold (T) for jaw-opening reflex (JOR) (open column, stimulus artifact; solid columns, recorded responses). B, Responses of the single spike of C1 spinal neuron, dEMG activity and poststimulus histograms of the neuronal responses to TMJ stimulation (4) at a 1-fold T for evoked spike in exactly the same neuron as shown in A (open column, stimulus artifact; solid columns, recorded responses). C, Hatched area is the location of the somatic receptive field. D and E, Threshold (T)-related responses of dEMG and C1 spinal neuron activity and dEMG to TMJ stimulation. Vertical bars show the mean F S.E. r, correlation coefficient *, p < 0.01, significant difference from 1T effect.
68% of the C1 spinal neurons tested received nociceptive information from C fibers in the TMJ region. Effect of mechanical stimulation of TMJ capsule on C1 spinal neuron activity Responses of C1 spinal neuron activity to mechanical stimulation of the TMJ capsule were divided into two groups: one responded to noxious stimulation (pin prick) only (Fig. 5A) and one responded to both nonnoxious (touch) and noxious stimuli (Fig. 5B). Eighteen out of 31 C1 spinal neurons were
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Table 1 Changes in mean conduction velocity evoked by the tooth pulp (TP) and temporomandibular joint (TMJ) stimulation TP Homogeneous type Heterogeneous type
n
Fiber type
Conduction velocity (m/s)
Ay C Ay
6.6 F 0.9 1.8 F 0.1 5.0 F 0.9
9 3 19
TMJ
n
Fiber type
Conduction velocity (m/s)
Ay C C
3.3 F 0.4 0.8 F 0.2 1.2 F 0.1
9 3 16
Data show mean F S.E.; n, the number of experiments.
WDR neurons, and 7 C1 spinal neurons were NS neurons. The remaining 6 C1 spinal neurons were NR neurons. As shown in Table 2, 36% of 25 C1 spinal neurons tested were homogenous when mechanical stimuli were applied to the TMJ capsule and facial skin, and the remaining C1 spinal neurons were heterogeneous. Of the 28 TMJ-TP convergent neurons, approximately 80% of these neurons received nociceptive information.
Fig. 5. Responses of C1 spinal neuron activity to mechanical stimuli (touch and pin prick) of the temporomandibular joint (TMJ) capsule. A, C1 spinal neuron responded to pin prick only. B, C1 spinal neuron responded to both touch and pin prick. bin = 100 ms.
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Table 2 Responses of C1 spinal neuron activity to mechanical stimulation of the temporomandibular joint (TMJ) and facial skin TMJ Homogeneous type Heterogeneous type
+
Facial skin
n
Touch
Pin prick
Touch
Pinch
+ + +
+ + + + + +
+ + +
+ + + + +
3 6 4 1 4 1 6
C1 spinal neurons responded to mechanical stimulation (+) and C1 spinal neurons did not responded to mechanical stimulation ( ). n, the number of experiments.
Chemical stimulation of the TMJ capsule on C1 spinal neuron activity A typical response of C1 spinal neuron activity to chemical stimulation of the TMJ region is shown in Fig. 6A. The C1 spinal neuron activity was excited by application of MO into the TMJ capsule. Such an effect was obtained in 8 out of 11 C1 spinal neurons that responded to the TMJ stimulation. The summarized results are shown in Fig. 6B. MO injection produced a significant increase in the C1 spinal neuron activity and this effect lasted for approximately 15 min. The average values for conduction velocity in these neurons responding to MO injection were 1.6 F 0.2 m/sec and were similar to the range of conduction velocity of C fibers.
Discussion The present study provided evidence that C1 spinal neurons activated by TP stimulation were also excited by TMJ stimulation. The results indicate that there may be a convergence of noxious afferent inputs from the TMJ and TP on the same C1 spinal neurons. In the present study, the activity of 31 C1 spinal neurons and the amplitude of dEMG (n = 31) both increased in magnitude during TP stimulation at 1–3.5 times the threshold for JOR. Similar excitatory effects of TP stimulation on C1 spinal neuron activity and dEMG has been demonstrated in the rat (Matsumoto et al., 1999; Tanimoto et al., 2002). Based on evidence that the histological structures of the C1 region are analogous to the caudalis in the trigeminal spinal nucleus, which receives most of the noxious afferent inputs, it is most likely that C1 spinal neurons play a significant role in the perception of tooth pain in the rat. This was further confirmed by evidence that c-fos expression in lamina I in C1–C2 segments of the rat spinal cord appears after noxious thermal or mechanical stimulation of the TP (Coimbra and Coimbra, 1994) or that chemical (MO) stimulation of the rat nasal mucosa produces numerous Fos-immunoreactive neurons in laminae I–II of the rat C1–C2 regions (Takeda et al., 1998). Small diameter afferent fibers that consist of the mandibular division of the trigeminal nerve, rootlets of the upper cervical spinal cord and branches of the vagus nerve are known to enter the TMJ region (Klineberg, 1971; Denny-Brown and Yanagisawa, 1973; Widenfalk and Wiberg, 1990; Kido et al., 1993; Uddman et al., 1999). These fibers that innervate adjacent masticatory muscles project into
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Fig. 6. A typical example of the effect of chemical stimulation of the temporomandibular joint (TMJ) region on C1 spinal neuron activity. A, Responses of C1 spinal neuron activity to 20% mustard oil (MO, 20 Al) injected into the TMJ capsule. B, Effect of chemical stimulation of the TMJ region on the mean spike numbers of C1 spinal neurons responding to TP stimulation. Vertical bars show the mean F S.E.; n = 11. *P < 0.05, significant difference from control values. bin = 100 ms.
laminae I in the trigeminal spinal subnucleus caudalis and/or cervical cord junction (Vc/C2) region (Shigenaga et al., 1988; Arvidsson and Raappana, 1989), involving the C1 region, but the central projection of small diameter afferents that especially innervate the TMJ has not been identified. When considering collective data from Fos studies, particularly concerning the TMJ region after chemical stimulation and injury (Hathaway et al., 1995; Bereiter and Bereiter, 2000; Bereiter, 2001), it is possible to speculate that neurons in laminae I–II of the Vc/C2 region play an important role in processing the pain signal from the TMJ and surrounding masticatory muscles. Many neurons located at the caudalis and interpolaris in the trigeminal spinal nucleus receive nociceptive inputs from the TMJ and/or the masseter muscle (Mm) and have extensive convergence of afferent inputs, involving the TMJ, Mm, or the facial skin (Kojima, 1990; Ohya, 1992). In order to selectively stimulate the TMJ region only, we exposed the TMJ capsule by removing the Mm and directly stimulating the afferent terminal in the TMJ. In the present study, C1 spinal neurons that responded to TMJ stimulation were also excited by TP stimulation. Furthermore, locations of recorded C1 spinal neurons were consistent with those of c-Fos immunoreactivity neurons after TMJ injury (Bereiter and Bereiter, 2000; Bereiter et al., 2002). The C1 neurons that responded to both TMJ and TP stimuli had somatic receptive fields
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on the facial skin, and most C1 spinal neurons (8/11) were also stimulated by administration of mustard oil into the TMJ region. There is evidence that trigeminal and upper cervical afferent nerve fibers converge on the cells in the C1–C2 segments of the rat spinal cord (Pfaller and Arvidsson, 1988). When considering the fact that the face, neck, jaw, TP and phrenic nerve (PN) fibers converge on the same C1 spinal neurons (Matsumoto et al., 1999), taken together, it is more conceivable that there may be the convergence of face, neck, jaw, TP, PN and TMJ afferents on the same C1 spinal neurons in the rat. In addition, that the PN converged on C1 spinal neurons along with somatic afferents has been demonstrated in the rat (Razook et al., 1995) as well as more recently in the monkey (Chandler et al., 1998). Chandler et al. (1999) also found convergence of trigeminal input with visceral and phrenic inputs on primate C1–C2 spinothalamic tract cells. Furthermore, Chandler et al. (2000) in the monkey and Qin et al. (2001) in the rat demonstrated that stimulation of cardiac afferent fibers excites C1–C2 spinothalamic tract cells and superficial and deeper C1–C2 spinal cells, respectively. Finally, Foreman (2000) suggested that organization of the neurons of the upper cervical segments may be more than just a simple excitation of the trigeminal spinal nucleus caudalis. Presumably, the characteristics of C1 spinal neurons having various receptive fields may be responsible for the diffuse nature and spreading and referral of pain in TMD patients (Sessle et al., 1993). Concerning the effect of TMJ stimulation on C1 spinal neuron activity, the difference in the type of TMJ afferent fibers would reflect different conduction velocities. The mean conduction velocity was 1.8 F 0.3 m/sec, and the distribution consisted of both Ay (32%, 9/28) and C fibers (68%, 19/ 28). But the range of the Ah fibers was not identified in this study. The homogeneity of the conduction velocities of TMJ and TP afferents was seen in 43% of C1 spinal neurons (12/28) tested, but the remaining (57%) of C1 neurons revealed heterogeneity for the conduction velocity. In the in vitro TMJ preparation with the rat auriculo-temporal nerve (TMJ-nerve preparation), Takeuchi et al. (2001) reported that among a total of 22 single afferent fibers, responses to mechanical stimulation of the TMJ region could be recorded from 7 Ay and 15 C fibers. Concerning the convergence patterns from afferent inputs from the TMJ and facial skin, 80% of 28 TMJ-TP convergent neurons received nociceptive information from deep structures and skin. This probably implies that the convergence of information from the TP and TMJ may contribute to the mechanism of a referred pain. When we consider the fact that most C1 spinal neurons (90%, 28/31) responding to the TMJ stimulation were also excited by administration of 20 % mustard oil (20 Al) into the TMJ capsule, taken together, it can be suggested that the TMJ region is mainly innervated by small diameter afferent fibers. On the behavior of their responsiveness to mechanical stimulation, not all C1 spinal neurons recorded were not LTM neurons, which are thought to respond only to non-noxious stimulation (touch) of the facial skin and TMJ capsule, but they were classified as either WDR or NS, based on their responsiveness to touch and pin prick of the TMJ or the facial skin. Similar behavior in response to mechanical stimulation involving the somatic receptive field stimulation has been reported in C1 spinal neurons (Matsumoto et al., 1999; Tanimoto et al., 2002). The homogeneity of the mechanical behavior of the facial skin and the TMJ capsule was observed in 36% of C1 spinal neurons (9/25), but there was heterogeneity between their relationships in 64 % of C1 spinal neurons (16/25). Some C1 spinal neurons (3/28) that responded to TMJ stimulation were not excited by mechanical stimulation (touch and pin prick) of the TMJ capsule, indicating that this type of neuron may belong to the category responding to high-threshold mechanoreceptive and/or proprioceptive afferent fibers from the TMJ region.
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In this study, we provided new insights into the contribution of C1 spinal neurons to the convergent patterns of afferent nociceptive information from the exposed TMJ capsule and the TP. Our results are basically consistent with the fact that superficial neurons of the Vc/C2 region play an important role in the regulation of pain perception from the TMJ (Bereiter and Bereiter, 2000). In clinical studies, it has been reported that acute pulpalgia contributes to a factor forming TMD symptoms (Wright and Gullickson, 1996). In fact, there is evidence that complete or partial relief from TMD symptoms occurrs after receiving endodontic treatment or having tooth extraction (Wright and Gullickson, 1996). Accordingly, our results may explain this clinical evidence and suggest convergence of primary afferent inputs from the TMJ and TP onto the upper cervical spinal cord, which contributes to a referred pain associated with TMD. References Arvidsson, J., Raappana, P., 1989. An HRP study of the central projection from primary sensory neurons innervating the rat masseter muscle. Brain Res. 480, 111 – 118. Bereiter, D.A., Bereiter, D.F., 2000. Morphine and NMDA receptor antagonism reduce c-fos expression in spinal trigeminal nucleus produced by acute injury to the TMJ region. Pain 85, 65 – 77. Bereiter, D.A., 2001. Sex differences in brainstem neural activation after injury to the TMJ region. Cells Tissues Organs 169, 226 – 237. Bereiter, D.A., Bereiter, D.F., Ramos, M., 2002. Vagotomy prevents morphine-induced reduction in Fos-like immunoreactivity in trigeminal spinal nucleus produced after TMJ injury in sex-dependent manner. Pain 96, 205 – 213. Casatti, C.A., Frigo, L., Bauer, J.A., 1999. Origin of sensory and autonomic innervation of rat temporomandibular joint: a retrograde axonal tracing study with the fluorescent day fast blue. J. Dent. Res. 78, 776 – 783. Chandler, M.J., Qin, C., Yuan, Y., Foreman, R.D., 1999. Convergence of trigeminal input with visceral and phrenic inputs on primate C1 – C2 spinothalamic tract neurons. Brain Res. 829, 204 – 208. Chandler, M.J., Zhang, J., Foreman, R.D., 1998. Phrenic nerve inputs to upper cervical (C1 – C3) spinothalamic tract neurons in monkey. Brain Res. 798, 93 – 100. Chandler, M.J., Zhang, J., Qin, C., Yuan, Y., Foreman, R.D., 2000. Intrapericardiac injections of algogenic chemicals excite primate C1 – C2 spinothalamic tract neurons. Am. J. Physiol. Regl. Intg. Comp. Physiol. 279, R560 – R568. Coimbra, F., Coimbra, A., 1994. Dental noxious input reaches the subnucleus caudalis of the trigeminal complex in the rat, as shown by c-fos expression upon thermal or mechanical stimulation in the rat. Neurosci. Lett. 173, 201 – 204. Denny-Brown, D., Yanagisawa, N., 1973. The function of the descending root of the fifth nerve. Brain 96, 783 – 814. Dubner, R., Sessle, B.J., Storey, A.T., 1978. The Neural Basis of Oral and Facial Function. Plenum Press, New York. Dworkin, S.F., Friction, J.R., Hollender, L., Huggins, K.H., Le Resche, L., Lund, J., Mohl, N., Ohrbach, R., Palla, S.F., Sommers, E.E., Stohler, C., Truelove, E.L., Von Korff, M., Widmer, C.G., 1992. Research diagnostic criteria for temporomandibular disorder: review, criteria, examinations and specifications, critique. J. craniomand. Disord. Facial Pain Oral Pain 6, 301 – 355. Eversole, L.R., Machado, L., 1985. Temporomandibular joint internal derangements and associated neuromuscular disorders. J. Am. Dent. Assoc. 110, 69 – 79. Foreman, R.D., 2000. Integration of viscerosomatic sensory input at the spinal level. Prog. Brain Res. 122, 219 – 221. Friction, J.R., Kroening, R.J., Hathway, K.M., 1988. TMJ and Craniofacial Pain: Diagnosis and Management Ishiyaku Euro America, St. Louis, MO. Hathaway, C.B., Hu, J.W., Bereiter, D.A., 1995. Distribution of Fos-like immunoreactivity in the caudal brainstem of the rat following noxious chemical stimulation of the temporomandibular joint. J. Comp. Neurol. 356, 444 – 456. Jacquin, M.F., Semba, K., Egger, M.D., Rhoades, R.W., 1983. Organization of HRP-labeled trigeminal mandibular primary afferent neurons in the rat. J. Comp. Neurol. 215, 397 – 420. Kido, M.A., Kiyoshima, T., Kondo, T., Ayasaka, N., Moroi, R., Terada, Y., Tanaka, T., 1993. Distribution of substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat temporomandibular joint. J. Dental Res. 72, 592 – 598.
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