Intensity Coding by TMJ-Responsive Neurons in Superficial Laminae of Caudal Medullary Dorsal Horn of the Rat

S. Takeshita,1 H. Hirata,1 and D. A. Bereiter1,2

 1Department of Surgery and  2Department of Neuroscience, Brown Medical School, Rhode Island Hospital, Providence, Rhode Island 02903


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Takeshita, S., H. Hirata, and D. A. Bereiter. Intensity Coding by TMJ-Responsive Neurons in Superficial Laminae of Caudal Medullary Dorsal Horn of the Rat. J. Neurophysiol. 86: 2393-2404, 2001. Temporomandibular disorders (TMD) represent a family of recurrent conditions that often cause pain in the temporomandibular joint (TMJ) region and muscles of mastication. To determine if TMJ-responsive neurons encoded the intensity of pro-inflammatory chemical signals, dose-effect relationships were assessed after direct injection bradykinin into the joint space and compared with responses after injection of glutamate or saline. Neurons were recorded from superficial laminae of the trigeminal subnucleus caudalis/upper cervical cord junction region (Vc/C2) and identified by palpation of the TMJ region in barbiturate-anesthetized male rats. The majority (62 of 84) of units received convergent input from facial skin, while 26% were driven only by deep input from the TMJ region. Conduction-velocity based on the latency to firing after electrical stimulation of the TMJ region indicated 64% of units were driven by A-delta fiber input only. Bradykinin (0.1-10 µM) excited 69% of neurons tested, and 70% (19 of 27) of these units were activated by the lowest dose (0.1 µM). Glutamate (50-200 mM) excited 27% of units; however, when tested after bradykinin, 58% of units were activated by glutamate. Some TMJ units (17%) were excited by saline injection alone and not enhanced further by bradykinin or glutamate. Most (88%) TMJ units were activated by injection of the small fiber excitant, mustard oil (20% solution), into the TMJ region. Units responsive to bradykinin or glutamate were not restricted to particular classes [e.g., wide dynamic range (WDR), nociceptive specific (NS), deep only]. A small percentage of TMJ units (~15%) were activated antidromically from the contralateral posterior thalamus. In parallel studies using c-fos immunocytochemistry, bradykinin (1 µM) injection into the TMJ region produced a greater number of Fos-positive neurons at the Vc/C2 region than glutamate (200 mM) or saline. These results revealed two broad classes of TMJ units that encoded the intensity of pro-inflammatory chemical stimuli applied to the TMJ region, units that received convergent nociceptive input from facial skin (i.e., WDR and NS units) and units that responded only to deep input from the TMJ region. On the basis of encoding properties and efferent projection status, it is concluded that activation of TMJ units within the superficial laminae at the Vc/C2 region contribute to the diffuse and spreading nature of TMD pain sensation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Temporomandibular disorders (TMD) represent a group of recurrent chronic conditions that often present with diffuse pain in the temporomandibular joint (TMJ) region, spreading of pain to adjacent facial and neck regions (Denucci et al. 1996; Dworkin et al. 1990) and greater sensitivity to other experimentally induced pains (Maixner et al. 1995, 1998). Although articular pain is a common clinical complaint (Kidd et al. 1996; LeResche 1997; Schaible and Grubb 1993), the central neural basis for TMD and other forms of joint pain is less well understood and has received less attention than cutaneous pain. Furthermore, current understanding of the neural basis for articular pain has relied mainly on the knee or ankle joint as models, while fewer studies have used models specifically related to TMJ injury.

The TMJ region and overlying facial skin is supplied by branches of the trigeminal, upper cervical, and vagus nerves (Casatti et al. 1999; Denny-Brown and Yanagisawa 1973; Kido et al. 1993; Klineberg 1971; Uddman et al. 1998; Widenfalk and Wiberg 1990). The central projections of these sensory nerves suggest common regions of termination in the lower brain stem and upper cervical spinal cord (Jacquin et al. 1983; McNeill et al. 1991; Pfaller and Arvidsson 1988). The organization of overlapping peripheral fields from multiple sensory nerves and convergence of cutaneous and deep TMJ inputs onto common central neurons has been proposed as the basis for the diffuse nature and spreading and referral of pain in TMD patients (Sessle et al. 1993). Previously, we used c-fos immunocytochemistry to determine which brain stem regions were activated by acute injury to the TMJ region (Bereiter and Bereiter 2000; Hathaway et al. 1995). These studies indicated that the highest density of Fos-positive neurons was produced in the superficial laminae at the caudal level of trigeminal subnucleus caudalis (Vc) as it merges with the upper cervical dorsal horn (Vc/C2 region). The number of Fos-positive neurons produced in the Vc/C2 region was proportional to the intensity of the inflammatory stimulus (Bereiter 2001) and reduced by pretreatment with morphine (Bereiter and Bereiter 2000). Selective destruction of the Vc/C2 region, but not of more rostral areas of the trigeminal sensory complex, blocked the increase in activity in masseter muscle caused by inflammation of the TMJ region (Hu et al. 1997). Collectively, these studies support the hypothesis that the Vc/C2 region plays a significant role in the initial stages of integration of peripheral signals that cause TMD pain.

Despite considerable indirect evidence for spinal lamina I as a critical site for processing signals that evoke muscle and joint pain (Craig 1996; Mense 1993; Schaible and Grubb 1993), the encoding properties of neurons in superficial laminae activated by input from deep tissues, particularly craniofacial tissues, are not well defined. Broton et al. (1988) recorded from TMJ-responsive neurons in deep laminae of rostral Vc in the cat and reported that most received convergent cutaneous input from facial skin and responded to one or more algesic chemicals, including bradykinin, injected into the joint space. Kojima (1990) used electrical and mechanical stimulation of the exposed TMJ capsule and masseter muscle and noted a high degree of convergence onto Vc cells in the rat, while Ohya (1992) used similar methods to report convergence from the TMJ region and masseter muscle onto trigeminal subnucleus interpolaris (Vi) neurons. Other studies have induced inflammation in the TMJ region and adjacent muscles and reported enlargement of cutaneous receptive field size in Vc neurons (Hu et al. 1992; Iwata et al. 1999); however, the properties of units responding specifically to TMJ input were not determined.

In the present study, single units were recorded extracellularly from laminae I-II at the Vc/C2 region and identified by their response to mechanical stimulation of the TMJ region. This approach was adapted from previous studies in which stable recordings of cornea-responsive lamina I units at the Vc/C2 region could be held for several hours (Hirata et al. 2000; Meng et al. 1997). Because it is assumed that at least low levels of inflammation of articular tissue accompany TMD pain (Denucci et al. 1996), emphasis was placed on determining the sensitivity of TMJ units to the pro-inflammatory agent, bradykinin (Bhoola et al. 1992; Calixto et al. 2000; Dray and Perkins 1993), by direct injections into the TMJ capsule. TMJ units also were tested for responsiveness to glutamate since this amino acid has been implicated in deep craniofacial (Cairns et al. 1998, 2001; Yu et al. 1996) and knee joint pain (Lawand et al. 1997, 2000).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The experimental protocols were approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital and conformed to the established guidelines set by the National Institute of Health guide for the care and use of laboratory animals (Publication No. 86-23, revised 1985).

Animal preparation

Adult male rats (Sprague-Dawley, Harlan, 303-493 g) were anesthetized initially with pentobarbital sodium (70 mg/kg ip). The right femoral artery (blood pressure monitor) and jugular vein (anesthesia and drug infusions) were catheterized, and after tracheostomy, animals were respired artificially with oxygen-enriched room air. Anesthesia was maintained by a continuous infusion of methohexital sodium (35-40 mg · kg-1 · h-1) and switched to a mixture of methohexital sodium (30-40 mg · kg-1 · h-1) and the paralytic agent, gallamine triethiodide (15-30 mg · kg-1 · h-1), after completion of all surgical procedures and just prior to the electrophysiological recording session. The animal was placed in a stereotaxic frame, and the C1 and a portion of the C2 vertebrae were removed to expose the upper cervical dorsal horn. The brain stem surface was bathed in warm mineral oil. A portion of parietal bone was removed from the right side of the skull, contralateral to the recording site, for placement of antidromic stimulating electrodes in the sensory thalamus. The left temporalis muscle was reflected partially to expose the connective tissue overlying the dorsal surface of the posterior mandibular condyle. Expiratory end-tidal CO2 (3.5-4.5%) and mean arterial pressure (MAP, 100-120 mmHg) were monitored throughout all experiments and remained within normal range. Body temperature was maintained at 38°C with a heating blanket and thermal probe.

Electrophysiology

The caudal portion of trigeminal subnucleus caudalis (Vc) and the upper cervical (C1-C2) spinal cord, 4-7 mm caudal to the obex, was searched for TMJ-responsive units. A tangential approach (~43° off vertical, 60° off midline) was used to record single units extracellularly with tungsten microelectrodes (9 MOmega , FHC, Bowdoinham, ME) within 500 µm after surface penetration. Unit activity was amplified, discriminated, then stored and analyzed off-line on an Apple G3 computer using a DAQ interface board and LabVIEW software (National Instruments), as described previously (Hirata et al. 1999).

Single units were identified initially by gentle mechanical probing (palpation with cotton-tipped applicator) of the posterior TMJ region. All units included for further analysis also were excited by deep mechanical indentation of the TMJ region applied through the overlying skin with a metallic dental burnisher (~1-mm round tip) and by probing the dorsal aspect of the condyle region and temporalis muscles with a wooden dowel. The force exerted by the metal burnisher was not measured; however, application of a similar force to the back of the experimenter's hand evoked a sense of deep pressure but not pain. TMJ units that received convergent input from a cutaneous receptive field (RF) were classified further as wide dynamic range (WDR), nociceptive specific (NS), or low threshold mechanoreceptive (LTM) cells. WDR cells were excited by brush or indentation of the skin surface with low-threshold von Frey filaments and showed a greater response to press or pinch with an arterial clip. NS units were activated by press or pinch of the skin but not by brushing, while cells classified as LTM were excited only by brushing the skin. Units activated by deep indentation of the TMJ region but displayed no apparent cutaneous RF were classified as deep only cells. Electrical stimuli (0.1- to 5-mA, 0.2- to 2.0-ms, square-wave pulses) were applied to the TMJ region by a dorsal approach to estimate the type of fiber input. A bipolar electrode (FHC; 2-mm separation) was inserted ~2 mm deep into the tissue surrounding the posterior aspect of the mandibular condyle after retracting the temporalis muscle. The latency of the response to electrical stimulation was measured from the stimulus onset to the first spike seen at two times threshold intensity in which threshold current was defined as the minimal current necessary to evoke responses after three of five stimulus pulses.

Experimental protocol

The properties of a single TMJ-responsive unit were characterized in each animal. First, the entire surface on the face and neck was explored for convergent cutaneous inputs; however, intraoral regions were not tested systematically. Jaw movement was not used to assess possible extrafusal muscle fiber input because this stimulus often caused instability in unit recording. After determining the mechanical properties of the cutaneous RF, a guide cannula (26 gauge) was inserted into the TMJ region (~3 mm deep) by a dorsal approach directed at the posterior edge of the mandibular condyle located under the zygomatic arch. Test solutions were delivered from a Hamilton syringe attached by polyethylene tubing to an inner cannula (33 gauge) that protruded ~0.5 mm from the end of the guide cannula. Different solutions (e.g., phosphate buffered saline, glutamate, bradykinin, or mustard oil) were injected from separate inner cannula-PE tubing assemblies to prevent drug mixing. Each injection was made slowly over 30 s (total volume = 20 µl) with an interstimulus interval of 30 min to minimize tachyphylaxis, especially for bradykinin. Generally, four to eight injections could be made while recording from each unit. The protocol for chemical stimulation involved injection of PBS (pH 7.4) followed by three doses of either L-glutamate (50, 100, and 200 mM, pH = 7.1, monosodium salt, Sigma) or bradykinin (BK, 0.1, 1, and 10 µM, pH 7.3, acetate salt, Sigma) using a cumulative-dose design. In several experiments, glutamate was injected after the series of BK solutions (n = 12). Mustard oil (allyl isothiocyanate, 20% solution) was injected into the TMJ region at the end of the experiment after other chemical tests were completed (n = 32). In several early experiments, only single doses of glutamate (200 mM) or BK (1 or 10 µM) were injected after PBS, and these results were not included in further statistical analyses. In four cases, the selective B2 antagonist, HOE 140, 10 µg in 20 µl of PBS (Sigma), was injected into the TMJ region 10 min prior to BK injection. To assess possible effects of volume injection alone, PBS was injected repeatedly at 30-min intervals into the TMJ joint space (n = 6). In several cases (n = 11), lidocaine (4%, 300 µl) was injected into the center of the cutaneous receptive field to validate the search protocol used to identify TMJ units that received convergent input from facial skin (see Figs. 1 and 2).



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Fig. 1. Mechanical response properties of a temporomandibular joint (TMJ) unit classified as nociceptive specific and the effect of lidocaine injection into the cutaneous field. A: responses to mechanical stimuli applied to the convergent cutaneous receptive field (RF) and deep structures. B: lidocaine (4%, 300 µl) injection into the cutaneous RF blocked activity caused by skin input, while activity caused by deep inputs were reduced but still present. C: cutaneous (shaded region) and deep (solid) RF areas. D: recording site in laminae I-II at the caudal level of trigeminal subnucleus caudalis as it merges with the upper cervical dorsal horn (Vc/C2 region). Number at lower right indicates distance from obex (in mm). BR, brush; C, mechanical probing of dorsal condyle surface; D, deep probe of TMJ region through the overlying skin; PI, pinch of facial skin; PR, press of facial skin.



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Fig. 2. Response properties of a TMJ unit classified as nociceptive specific to mechanical and chemical stimuli and the effects of lidocaine injection into the cutaneous field. A: responses to mechanical stimuli applied to the convergent cutaneous RF and deep structures before and after lidocaine (4%, 300 µl) injection. Note that cutaneous inputs were blocked by lidocaine while the deep probe stimulation of the TMJ region persisted. B: unit activity evoked by BK was reduced but not blocked by lidocaine injection into the cutaneous RF. C: oscillographic records of unit activity to saline, bradykinin, and glutamate injections into the TMJ region. D: cutaneous (shaded region) and deep (solid) RF areas. E: recording site. Glu, glutamate; PBS, phosphate-buffered saline.

Antidromic stimulation

An array of two concentric bipolar stimulating electrodes (Rhodes Medical Instruments, SNE-100), separated rostrocaudally by 2 mm, were positioned in the contralateral ventral posteromedial nucleus (VPM), and posterior nuclear group (PO) of the thalamus. The approximate stereotaxic coordinates in mm for VPM were AP -2.5 from bregma, ML 2.5, DV 4-9 from the cortical surface; for PO, AP = -4.5, ML 2.5, DV 4-9. Antidromic activation was defined by a constant latency (<0.1-ms jitter), high-frequency following (0.1-ms pulse, 200-300 Hz, 20-ms train duration) and collision with an orthodromically driven spike within a critical time interval (Lipski 1981). The maximum allowable current used to define an antidromic response was 500 µA. Brain loci for antidromic responses were marked by passing current (30 µA, 30 s) through the center electrode.

Data analysis

Neural recording and blood pressure data were acquired and displayed by LabVIEW as peristimulus time histograms (PSTHs) of spikes per 1-s bins, exported to a spreadsheet and analyzed off-line. Spontaneous or background activity (spikes/s) was calculated by averaging the spike counts during the 1-min epoch preceding each stimulus. The response magnitude (Rmag) to mechanical and chemical stimuli was defined as the spike count per 1-s bins that exceeded the mean plus two times the standard deviation (SD) of background for 1 min preceding each stimulus. The latency of the evoked response was defined as earliest time after stimulus onset for which three consecutive 1-s bins exceeded the mean + 2 SD of background activity (i.e., Rmag). Response duration was defined as the interval from the initial occurrence of a positive Rmag (initial latency) until the value of three consecutive bins no longer exceeded the mean + 2 SD of the background. This approach has been described previously for caudal Vc units excited by corneal stimuli (Hirata et al. 1999, 2000). Units that failed to show a positive Rmag within 100 s after stimulus onset were considered as not responsive to that condition. To assess responses to glutamate or BK injections into the TMJ region, units were defined as responsive if the total Rmag (spike count × response duration) exceeded that seen after injection of PBS by >50%. The threshold concentration of glutamate or BK that evoked a significant response was defined as the lowest concentration that produced responses of >50% after PBS. Responses to mechanical and chemical stimuli were assessed statistically by ANOVA (ANOVA) corrected for repeated measures (Winer 1971) and individual comparisons were made by Newman-Keuls after ANOVA. chi 2 analysis was used to determine if different classes of neurons (e.g., WDR, NS, deep only) were differentially responsive to glutamate or BK injections into the TMJ region.

Histology

At the end of the experiment, Sudan black dye (20 µl) was injected into TMJ region through the guide cannula to verify placement in the joint space. The animal was given a bolus dose of methohexital sodium (60 mg/kg iv) and perfused through the heart with 10% formalin containing potassium ferrocyanide to identify antidromic stimulation sites by the Prussian Blue reaction. Recovered sites were drawn on a standard series of brain outlines adapted from the atlas of Paxinos and Watson (1986). The recording site was marked electrolytically (<5 µA, 20 s) as seen in Fig. 7A.

C-fos immunocytochemistry

In parallel experiments, male rats (238-430 g, Sprague-Dawley, Harlan) were anesthetized with pentobarbital sodium (65 mg/kg ip), and catheters were placed in the jugular vein (drugs) and the femoral artery (arterial pressure). Surgical incisions were infiltrated with 2% lidocaine jelly. Heart rate was monitored continuously by a standard 3-lead ECG and body temperature was kept at 38°C with a heating blanket monitored from a rectal probe. A single microinjection (25 µl) of PBS (pH 7.4, n = 4), glutamate (200 mM, n = 4), or BK (1 µM, n = 3) was made into the left TMJ region through the skin after palpation of the joint region. An additional group served as surgical controls and did not receive injections into the TMJ region (n = 3). At 1.5 h following TMJ injury, 2% Evan's Blue was injected intravenously to confirm the site of injury (Haas et al. 1992).

Two hours after TMJ injection animals were given a bolus injection of pentobarbital (60 mg/kg iv) and perfused through the heart with 100 ml heparinized saline, followed by 250 ml cold fixative (4% paraformaldehyde, 0.02% picric acid in 0.1 M phosphate buffer, pH 7.3-7.4). The lower brain stem and upper cervical spinal cord was removed and postfixed for 1 h. Transverse sections (50 µm) were cut on a vibratome and collected in cold 0.01 M PBS. Sections were incubated successively in 5% normal donkey serum (30 min), affinity-purified rabbit polyclonal anti-Fos antibody (Ab-2, Oncogene Science; 1: 15,000, 40 h at 4°C), biotinylated donkey anti-rabbit antibodies (Chemicon; 1: 300, 105 min) and avidin-biotin-peroxidase complex (Vector; 60 min). Fos-positive neuronal nuclei were visualized by incubation in a nickel-cobalt diaminobenzidine solution activated by 0.01% hydrogen peroxide. After rinsing in PBS, sections were mounted on slides, air-dried, and coverslipped with mounting medium. Fos-positive neurons were distinguished as homogenous gray-black elements with a regular border under bright-field illumination. Specific staining was abolished by omission of primary antiserum. Immunostaining runs included the brain sections from animals that received different drug treatments.

C-FOS DATA ANALYSIS. The rostrocaudal location of each section was noted and categorized at 500-µm intervals. In each animal 20-25 sections were counted at ×100 magnification without prior knowledge of the drug treatment over the approximate distance of 4.5-7 mm caudal to the obex. This area, which includes the most caudal portion of Vc and the upper cervical spinal segments (Vc/C2 region), produced a high-density of Fos-positive neurons after injection of the inflammatory agent mustard oil into the TMJ region (Bereiter and Bereiter 2000; Hathaway et al. 1995). Separate cell counts were made for Fos-positive neurons in the superficial laminae (I-II) and deeper laminae (III-V; see Fig. 7B for example). Average cell counts were assessed across treatments and different laminae ipsilateral and contralateral to the TMJ injection by two-way ANOVA. Individual comparisons across treatments were made using the Newman-Keuls test after ANOVA (Winer 1971).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General properties

A total of 84 neurons were activated by indentation of deep tissues in the TMJ region and recorded from the superficial laminae of the Vc/C2 region in 75 rats (see example in Fig. 7A). Experiments were designed to record from only a single unit in each animal; however, in a few cases, TMJ units were isolated and characterized for mechanical inputs but were lost prior to testing with injections of chemical agents into the TMJ region. A majority of TMJ units (74%, n = 62) also received excitatory input from facial skin. Units with a convergent cutaneous RF were classified as WDR (34%, n = 29), NS (32%, n = 28), or LTM (6%, n = 5) neurons. The remaining 22 neurons (26%) had no apparent cutaneous RF and were classified as deep only units. Fifty-four of 62 units with input from skin had a cutaneous RF in facial regions supplied by mandibular and/or maxillary branches, while 8 units also had a RF in the ophthalmic region. Typically, the cutaneous RF was located anterior to and contiguous with the deep field (see examples in Figs. 1, 4, and 6); however, in eight cases, the cutaneous RF did not overlie the deep TMJ-related RF (see Fig. 2D). Although no effort was made to quantify the size of the cutaneous RF, the examples shown in Figs. 1 and 2 were representative of the finding that the cutaneous RF of TMJ units was large and often covered 10-30% of the ipsilateral face. No TMJ units received input from contralateral facial skin; however, this was not examined systematically. To verify that unit responses to deep stimulation of the TMJ region were not due solely to activation of adjacent or overlying skin, 11 units with a convergent cutaneous RF were tested for deep input before and after local blockade of the overlying skin by 4% lidocaine injection. Although the Rmag to deep input was reduced after lidocaine (31.2 ± 14.1%, mean ± SE), responses to deep stimulation (Figs. 1 and 2) and BK injection into the TMJ region (Fig. 2) were evident in all units after lidocaine. Responses to gentle probing of the dorsal surface of the mandibular condyle remained vigorous before and after lidocaine despite complete blockade of the responses to noxious cutaneous stimuli (Fig. 1, A and B).

Table 1 summarizes the general properties for those units tested for chemical sensitivity by injections of BK or glutamate into the TMJ region. Nearly all TMJ units tested with BK or glutamate (64 of 65) had a resting discharge and the average spontaneous firing rates of chemically responsive and unresponsive units were similar. The force threshold necessary to activate units by deep indentation of skin overlying the TMJ region, by calibrated von Frey monofilaments, also was similar for units responsive or unresponsive to BK or glutamate injection. However, the force threshold to activate the deep RF, independent of chemical responsiveness, was significantly (P < 0.05) higher for deep only units (44.05 ± 22.84 g, n = 17) compared with units with convergent input from facial skin (16.78 ± 2.73 g, n = 48).


                              
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Table 1. General properties of TMJ units classified on the basis of responses to injection of bradykinin or glutamate into the TMJ region

A bipolar stimulating electrode was inserted into the TMJ region to estimate the type of fiber input that relayed information from the TMJ to the Vc/C2 region. Twenty-one of 33 (63.6%) TMJ units driven by electrical stimulation of the posterior mandibular condyle region had a latency [5.4 ± 0.3 (SE) ms, range, 3.0-9.0 ms] consistent with A-delta fiber input, while 12 of 33 units (36.4%) also displayed activity at C-fiber-like latencies. No unit received C-fiber-only input. C-fiber-like conduction velocity was defined as <2.0 m/s based on a measured distance of 20 mm from the dorsal posterior edge of the mandibular condyle to the C2 rootlets and allowing 1 ms for synaptic transmission (Hu et al. 1992). The conduction velocities for 7 deep only units versus 13 WDR or NS units driven by A-delta input alone were similar and averaged 4.5 ± 0.5 and 3.8 ± 0.3 ms, respectively. In 21 cases, TMJ units also were tested for projections to the contralateral sensory thalamus. Three of 21 units were activated antidromically from a site within the PO group and none from the more anterior electrode in the VPM. The average latency and threshold current for the 3 units with effective sites in PO were 4.5 ± 1.8 ms (range = 2.1-8.0 ms) and 155 ± 143 µA (range = 10-440 µA), respectively.

Responses to saline

Sixty-five TMJ units were tested for responses to PBS followed by cumulative doses of BK (n = 39) or glutamate (n = 26) into the joint space. chi 2 analysis of the sampling distribution revealed a greater number of NS units were included among cells tested with BK compared with those tested with glutamate (P < 0.02, chi 2 = 11.01, df = 3). Of the cells tested with BK, 34 of 39 (87.1%) displayed a total Rmag of >10 spikes/stimulus to the initial injection of PBS (see examples in Figs. 2 and 6). By contrast, only 10 of 26 (38.5%) units tested with glutamate had a similar response to the initial injection of PBS. chi 2 analysis indicated that the neurons tested for BK sensitivity were more likely to respond to PBS than the units tested for glutamate (chi 2 = 16.92, df = 1, P < 0.001).

Responses to BK

Twenty-seven of 39 neurons were responsive to BK (BK+ units), defined as a total Rmag (i.e., spikes per stimulus > mean + 2 SD of background) that exceeded the response to PBS by >= 50%. The minimal dose of BK sufficient to activate 19 of 27 units was 0.1 µM, while 6 units were activated by 1 µM BK. Two additional BK+ units were tested only with 10 µM BK. BK-responsive units typically displayed a greater response to 0.1 or 1 µM BK than to 10 µM (16 of 25), whereas in seven cases, unit activity showed a progressive increase with higher concentrations of BK. BK-responsive units were found among all cell classes (deep only, n = 4; WDR, n = 6; NS, n = 14; and LTM, n = 3). Figure 3 compares total Rmag, response duration and response latency of BK+ and BK- units tested by injection of PBS followed by each of the three cumulative doses of BK. These results indicated that BK+ units encoded BK concentration, expressed as an increase in total Rmag (top) or response duration (middle), and a decrease in response latency (bottom) with increasing concentrations of BK. Unexpectedly, BK- units displayed a higher total Rmag (P < 0.01) and longer response duration to PBS than BK+ units. Although BK- units had numerically smaller responses to BK than to PBS, these differences were not significant nor did BK- units show consistent responses to increasing concentrations of BK. In four cases, BK+ units were re-tested for responses to BK (10 µM) after local injection of the selective B2 antagonist, HOE 140, into the joint space. In two of these cases, HOE reduced the response to BK by >20% (not shown).



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Fig. 3. Summary of dose-effect responses of TMJ units classified as BK+ or BK- on the basis of the responses to bradykinin. Top: total Rmag (spikes/stimulus > mean +2 SD of background). Middle: response duration defined as the total response time in which the Rmag exceeded the background activity. Bottom: response latency defined as time from onset of injection until 3 consecutive 1-s bins displayed a positive Rmag value. *P < 0.05, **P < 0.01 vs. response to PBS; b = P < 0.01 BK+ vs. BK-.

Responses to glutamate

Seven of 26 TMJ units were activated by glutamate injection into the TMJ region (GLU+ units) as defined by an increase in total Rmag. Three of seven units were excited by the lowest dose of (50 mM) and four of seven units were activated by 100 mM glutamate. The majority (5 of 7) of GLU+ units were classified as deep only, one was WDR and one was NS-like. An example of a GLU+ unit is shown in Fig. 4 (A and B) in which activity increased with higher concentrations of glutamate; however, this was not a consistent finding. Five of seven GLU+ units had a maximum response to 100 mM and a smaller response to 200 mM. Although the average total Rmag for BK+ and GLU+ units increased numerically with higher concentrations of BK and glutamate, respectively, only the increase among BK+ units was significantly different from that seen after the initial injection of PBS alone (Fig. 5). The average response duration and response latency for GLU+ units also was not altered significantly by different concentrations of glutamate (not shown). Repeated injection of PBS alone did not alter significantly total Rmag (Fig. 5), indicating that volume injection (20 µl per stimulus) alone had little effect on unit activity.



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Fig. 4. Example of a TMJ unit classified as nociceptive specific and responsive to glutamate injections into the TMJ region. A: oscillographic records of responses to PBS and glutamate. B: peristimulus time histograms of responses to PBS and increasing concentrations of glutamate shown in A. C: responses to mechanical stimuli applied to cutaneous and deep RF areas. D: cutaneous (shaded region) and deep (solid) receptive field areas. E: recording site in Vc/C2 region. See Figs. 1 and 2 for other abbreviations.



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Fig. 5. Summary of total Rmag values for units tested with repeated injection of PBS alone compared with units classified as BK+ or GLU+ on the basis of BK or glutamate injections into the TMJ region. Each injection was delivered in 20 µl volume and 30 min elapsed between injections. *P < 0.05, **P < 0.01 vs. response to PBS. open circle , PBS;  BK; , glutamate.

To determine if prior testing with BK altered the proportion of cells responsive to glutamate 12 BK+ units were tested subsequently for sensitivity to glutamate. Seven of 12 BK+ units had a significant response to glutamate (Rmag >50% of response to PBS) as shown by the example in Fig. 6. chi 2 analysis revealed that the frequency of occurrence of GLU+ units when tested after BK was greater than would be expected if tested without prior injection of BK (chi 2 = 7.15, df = 1, P < 0.01). By comparison, only one of seven BK- units displayed a significant Rmag to glutamate when tested after BK. The maximum Rmag values, independent of dose, seen in response to BK, glutamate or glutamate after BK were not different statistically and averaged 519 ± 92, 172 ± 50, and 361 ± 131 spikes per stimulus, respectively. At the end of the experiment a number of units were tested by injection of the small fiber excitant, mustard oil (20% solution) into the joint space. As seen in Table 1, 88% of TMJ units responded to mustard oil independent of their responsiveness to BK or glutamate.



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Fig. 6. Example of TMJ unit classified as nociceptive specific tested for responses to BK and then glutamate. A: peristimulus time histograms of responses to mechanical (BR, PR, and PI) stimulation of cutaneous RF and probing of deep field in the TMJ region (D). B: unit responses (above background) to PBS, BK, and glutamate. Note large response to glutamate when injected after BK. C: cutaneous () and deep () RF areas.

Recording sites

The recording sites for all cells tested for BK and glutamate were located within 250 µm of the dorsal brain stem surface within the superficial laminae of the Vc/C2 region, and most were in the medial half of the dorsal horn (see example in Fig. 7A). There were no apparent differences in the distribution of sites for units tested with BK compared with those tested with glutamate. Also, no differences were seen in the recording site locations along the rostrocaudal or mediolateral planes for BK+ versus BK- units or for GLU+ versus GLU- units (not shown).



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Fig. 7. Examples of recording site (A) and c-fos-positive (B) neuronal nuclei in laminae I-II produced after 1 µM BK injection into the TMJ region. Calibration = 250 µm.

C-fos immunocytochemistry

The number of Fos-positive neurons was quantified at the Vc/C2 region after injection of PBS, glutamate (200 mM), or BK (1 µM) into the TMJ region. BK evoked a significantly (P < 0.01) greater number of Fos-positive neurons than PBS or glutamate; however, both PBS and glutamate increased the number of Fos-positive cells compared with unstimulated controls (P < 0.01, Fig. 8). The majority of Fos-positive neurons were produced within laminae I-II and only a few scattered neurons (<5 cells per section) were found in deeper laminae (Fig. 7B) regardless of which drug was injected into the joint space.



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Fig. 8. Average number of c-fos positive neurons produced in laminae I-II at the Vc/C2 region. , ipsilateral; , contralateral to TMJ injection. b, P < 0.01 vs. no stimulus group; dagger , P < 0.01 vs. other treatments. n = 3-5 animals per treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study determined the RF properties of TMJ units recorded in the superficial laminae of the Vc/C2 region with emphasis on the encoding of stimulus intensity from chemical as well as mechanical signals. All units were recorded within 250 µm of the brain stem surface and activated by deep pressure applied to the posterior mandibular condyle region in or near the TMJ. Classification based on the responses to mechanical and chemical test stimuli revealed distinct subpopulations of TMJ-responsive units. Tests for mechanical properties indicated most TMJ units (74%) received convergent input from large areas of facial skin, encoded noxious levels of intensity (i.e., WDR or NS), and in some cases projected to contralateral posterior thalamus. However, in contrast to spinal units in deep dorsal horn with knee or ankle joint input (see Schaible and Grubb 1993), a significant number of TMJ units in superficial laminae (26% of total) were excited only by stimulation of deep probing of the TMJ region. A high percentage of all TMJ units (69%) were activated by injection of BK into the joint space independent of mechanical RF properties. Evidence for chemical selectivity was seen between BK+ and BK- units because BK+ units were more likely to respond to glutamate, while BK- units had greater responses to saline injection.

Technical considerations

The search strategy to identify TMJ units required intermittent application of deep pressure stimuli through the skin overlying the TMJ region. In units with convergent cutaneous and deep inputs, activity evoked by this approach could have resulted from activation of skin receptors alone, especially for units in which the cutaneous RF was overlying the TMJ region. However, several findings argued against this possibility. First, in those cases tested (n = 11) lidocaine injection into the cutaneous RF blocked activity evoked by skin stimulation, while responses to deep mechanical (Fig. 1 and 2) or chemical stimuli (Fig. 2) were reduced but never inhibited completely. Second, in all but eight cases, deep pressure stimulation through the skin evoked greater Rmag values than noxious stimulation of skin alone. Third, direct stimulation by gentle probing of the connective tissue overlying the dorsal surface of the mandibular condyle caused a vigorous response in all units classified as TMJ responsive.

The time needed to identify a single TMJ unit varied considerably from 30 min to 4 h. If repeated application of deep pressure to the TMJ region through the skin altered the responses of TMJ units, then units found later in the recording session might be expected to have different properties than those found at the onset; however, this did not appear to be the case. When grouped according to the time required to search for and then isolate each unit (early = <2 h; late = 2-4 h), the proportion of cells with deep only versus those with convergent cutaneous input was similar (P > 0.05, chi 2 test). Also, the proportion of TMJ units responsive to BK was not different for cells recorded early or late in the recording session. GLU+ units also were found early as well as late in the recording session. Although nearly all TMJ units displayed background activity, cells isolated early had resting discharge rates similar to those found late in the recording session (5.3 ± 1.4 vs. 5.6 ± 1.3 spikes/s, respectively). These results suggested that repeated mechanical testing did not affect the proportion of units found among different cell classes and did not cause persistent changes in discharge rates suggestive of enhanced peripheral input. There was no apparent edema or damage to the skin overlying the TMJ region during the course of the experiment.

RF properties of TMJ units

Current understanding of the neural basis for joint pain derives mainly from studies using ankle or knee joint models (see Schaible and Grubb 1993). Sensory fibers innervating the ankle or knee are mainly small caliber, excited by mechanical and chemical stimuli and terminate most heavily in laminae I and V-VI of the spinal dorsal horn. Similarly, the TMJ region is supplied mainly by small diameter afferent fibers (Kido et al. 1993, 1995; Uddman et al. 1998). Although the central projections of afferent fibers that specifically innervate the TMJ have not been reported, those that innervate adjacent masticatory muscles terminate heavily in lamina I at the Vc/C2 region (Arvidsson and Raappana 1989; Shigenaga et al. 1988). These findings are supported by results from c-fos studies (Bereiter 2001; Bereiter and Bereiter 2000; Hathaway et al. 1995) and implicate neurons in laminae I-II of the Vc/C2 region as critical for the initial processing of pain signals arising from the TMJ and surrounding masticatory muscles.

A dominant role for lamina I neurons in the various aspects of pain (Craig 1996) is supported by evidence that pain is the only sensation normally evoked by joint receptors other than position sense (Schaible and Grubb 1993). Indeed, the majority of TMJ units recorded from laminae I-II received convergent input from facial skin and nearly all were classified as nociceptive (i.e., WDR or NS type). Similarly, nearly all TMJ units recorded from deep laminae of Vc (Broton et al. 1988; Kojima 1990) or deep dorsal horn units with knee joint input (Schaible et al. 1987) had a convergent cutaneous RF and were classified as WDR and NS-like. In contrast to previous studies, we found an additional population of TMJ units (deep only, 26% of total) in laminae I-II with no discernable input from facial skin despite having undergone prior surgery to expose the dorsal condyle region. Prior surgical exposure of leg muscles was reported to increase the proportion of deep dorsal horn units in lumbar cord with convergent input from muscle and skin, while the proportion of units with muscle only input was reduced (Hoheisel and Mense 1989). After extensive surgery to expose the TMJ capsule in the cat (Broton et al. 1988) or rat (Kojima 1990) by reflecting the temporalis and masseter muscles, all TMJ units recorded mainly from deep laminae in Vc received convergent input from facial skin or intraoral regions. Although some units may have received inputs from noncutaneous tissues not explored in this study (e.g., intraoral cavity), it remains possible that a distinctive feature of the brain stem circuit for TMD pain includes second-order neurons in lamina I at the Vc/C2 region that process inputs exclusively from deep receptors in the jaw joint region. It is not known if a similar population of lamina I cells exists in spinal cord that receive only deep input from knee and ankle nociceptors since those studies have recorded mainly from deeper laminae (Schaible and Grubb 1993; Schaible et al. 1987).

Laminae I-II units activated by TMJ input also displayed RF properties that differed from those of cornea units, despite a partial overlap of recording sites in the caudal portion of Vc. For example, all cornea units recorded from laminae I-II in caudal Vc had a small cutaneous RF classified as WDR or NS that typically (>95% of cells) was contiguous with the corneal surface (Hirata et al. 1999, 2000; Meng et al. 1997). By contrast, 26% of TMJ units received no apparent convergent input from facial skin and of those with cutaneous input the RF often encompassed a large skin area distant from the deep field (see Fig. 2). Interestingly, in one case, a TMJ unit was activated by convergent input from the cornea, nasal cavity, and facial skin that did not overlay the deep field.

Chemical encoding by TMJ units

Injured tissues release a variety of chemical agents, including BK, into the extracellular fluid that can activate sensory nerve endings (Levine and Reichling 1999). BK is a potent algesic agent that directly excites sensory nerve fibers in skin (Lang et al. 1990), muscle (Mense 1993) and joints (Grubb et al. 1991; Kanaka et al. 1985; Schaible and Grubb 1993). Endogenous release of BK may contribute to TMD pain since BK levels are elevated in synovial fluid within minutes after inflammation in animal models (Swift et al. 1998). The effectiveness of BK as an excitant for TMJ afferents was supported by the high density of Fos-positive neurons produced in laminae I-II at the Vc/C2 region after 1 µM BK. The influence of BK on TMJ unit activity was rapid and could not be predicted from the mechanical RF properties of the cell, since a similar proportion of deep only units and those with a convergent cutaneous RF responded to BK (60-70% of total) and had similar Rmag values. The sensitivity of TMJ units to BK agreed well with previous studies in which BK was injected into the ankle (Grubb et al. 1991) or knee (Kanaka et al. 1985). After close arterial injection the minimal dose of BK needed to evoke activity in ankle (1-10 µg) (Grubb et al. 1991) or knee joint afferents (0.26-2.6 µg) (Kanaka et al. 1985) was slightly higher than we observed after direct injection into the TMJ capsule (0.002-0.2 µg). In an in vitro skin-nerve preparation, BK bath concentrations as low as 0.1 µM reliably evoked activity in afferent fibers (Lang et al. 1990). Because 19 of 27 TMJ units were excited by the lowest concentration of BK (0.1 µM), it is likely that the threshold dose of BK sufficient to activate some TMJ units may be lower than that used here. Also, the response duration (15-60 s) and latency (7-20 s) evoked in TMJ units after BK were similar to responses seen for afferents innervating the ankle (Grubb et al. 1991). These results indicated that TMJ afferents encoded BK concentration, and thus the intensity of inflammation and were as sensitive to BK as nociceptors in other peripheral tissues.

Several studies have suggested a role for glutamate in articular pain. Inflammation of the knee enhanced glutamate immunoreactivity in the medial articular nerve in monkey (Westlund et al. 1992), while in rats glutamate levels increased in the knee joint after inflammation (Lawand et al. 2000) and injection of glutamate into the knee joint increased pain behavior (Lawand et al. 1997). Pretreatment with glutamate receptor antagonists reduced the activity in masseter and digastric muscles caused by mustard oil injection into the TMJ region (Cairns et al. 1998; Yu et al. 1996) and reduced the number of Fos-positive neurons in superficial laminae of the Vc/C2 region (Bereiter and Bereiter 2000). Recently, injection of glutamate into the TMJ region excited 27 of 34 trigeminal primary afferents fibers identified by mechanical probing of the jaw joint region (Cairns et al. 2001). Injection of glutamate into the plantar skin increased pain behavior to thermal stimuli (Jackson et al. 1988) and enhanced the thermal-evoked responses of >90% of mechanoheat A-delta and C-fibers in an in vitro preparation; however, <50% of afferents responded to glutamate alone (Du et al. 2001). The present results suggest that glutamate makes a greater contribution to pain signaling after injury or inflammation than under naïve conditions. Glutamate injection into the joint space excited ~27% of TMJ units tested; however, when tested after BK, a higher percentage of TMJ units were responsive to glutamate (58% of cells tested). The maximum total Rmag values of TMJ units after BK (~500 spikes/stimulus), glutamate alone (~170 spikes/stimulus), and glutamate after BK (~350 spikes/stimulus) were not significantly different. This suggested that BK and glutamate did not act by an additive mechanism to excite a common population of TMJ afferents. Also, the number of Fos-positive neurons produced by BK was nearly three times greater than that seen after glutamate or saline injection into the TMJ region. A small number of TMJ units were excited by saline alone and not enhanced further by BK or glutamate. Although saline units may have been responsive only to the mechanical component of the injection, several of these units were activated by mustard oil injection into the TMJ capsule, suggesting that they could have been responsive to other naturally occurring inflammatory agents not tested in this study.

Neural circuits for TMD pain

The central neural circuitry that mediates TMD pain is not known. The present study examined the properties of TMJ units located in laminae I-II of the Vc/C2 region, since previous c-fos studies revealed that acute TMJ injury produced the greatest number of Fos-positive neurons in this region (Bereiter 2001; Bereiter and Bereiter 2000; Hathaway et al. 1995). However, a distinctive feature of the trigeminal system is that craniofacial tissues are represented somatotopically at multiple levels of the trigeminal brain stem complex (Bereiter et al. 2000). Indeed, c-fos studies also revealed a high density of Fos-positive cells produced bilaterally in the dorsal paratrigeminal area and the trigeminal subnucleus interpolaris/caudalis transition region after TMJ injury, regions separated by several millimeters from the caudal Vc/C2 region in the rat brain stem. It is not yet known if TMJ units in the dorsal paratrigeminal area and the trigeminal subnucleus interpolaris/caudalis transition region have properties similar to those in laminae I-II at the Vc/C2 region. TMJ units in laminae I-II of the Vc/C2 region encoded the intensity of mechanical and chemical stimuli and at least some units projected to the sensory thalamus. One possible function for TMJ-responsive units in superficial laminae of Vc/C2 may be to mediate the sensory-discriminative aspects of TMD pain. A second feature of the trigeminal sensory complex is the extensive longitudinal fiber system that connects rostral and caudal portions of the trigeminal spinal nucleus (Jacquin et al. 1990). Previously, we reported that cornea units in laminae I-II of the caudal Vc likely projected to more rostral portions of the trigeminal brain stem complex to modulate activity in those regions (Hirata et al. 2000). Thus an additional role for TMJ units in the Vc/C2 region may be to relay activity to rostral portions of the trigeminal brain stem complex that mediate other aspects of TMD pain. The notion that the Vc/C2 region acts as an important relay for TMD pain circuitry was supported by the finding that selective lesion of caudal Vc but not more rostral regions of Vc, prevented the increase in EMG activity caused by acute TMJ injury (Hu et al. 1997). In future studies, it also will be important to determine if the properties of TMJ units in laminae I-II at the Vc/C2 region are similar in males and females because it is well recognized that chronic TMD pain in humans is most prevalent in females (LeResche 1997). Recently, we reported that acute TMJ injury produced ~65% more Fos-positive neurons in laminae I-II at the Vc/C2 region of pro-estrus females compared with males (Bereiter 2001). In conclusion, several distinct subpopulations of neurons in the superficial laminae at the Vc/C2 region were excited by mechanical and/or chemical stimulation of the TMJ region. Based on response properties and projection status, it is likely that neurons in laminae I-II at the Vc/C2 region play a critical role in mediating pain sensation in TMD.


    ACKNOWLEDGMENTS

The authors thank D. F. Bereiter and A. Benetti for excellent technical support and Dr. J. W. Hu (University of Toronto) for helpful advice in preparing the manuscript.

This study was supported by National Institute of Dental and Craniofacial Research Grant RO1 DE-12758.


    FOOTNOTES

Address for reprint requests: D. A. Bereiter, Depts. of Surgery and Neuroscience, Brown Medical School, Rhode Island Hospital, 222 Nursing Arts Bldg., Providence, RI 02903 (E-mail: DBereiter{at}lifespan.org).

Received 12 March 2001; accepted in final form 31 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society