Faculty of Dentistry, The University of Toronto, Toronto, Ontario M5G 1G6, Canada
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ABSTRACT |
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Cairns, Brian E.,
Barry J. Sessle, and
James W. Hu.
Characteristics of Glutamate-Evoked Temporomandibular Joint
Afferent Activity in the Rat.
J. Neurophysiol. 85: 2446-2454, 2001.
Injection of glutamate into the rat
temporomandibular joint (TMJ) capsule can reflexly induce a prolonged
increase in the electromyographic (EMG) activity of the jaw muscles,
however, the characteristics of TMJ afferents activated by glutamate
have not been investigated. In the present study, we examined the
effect of glutamate injection into the TMJ capsule on jaw muscle EMG activity and the extracellularly recorded activity of single trigeminal afferents that had receptive fields in the TMJ tissue and
antidromically identified projections to the brain stem subnucleus
caudalis (Vc) in rats of both sexes. Glutamate (0.05-1.0 M, 10 µl)
injection into the TMJ capsule evoked EMG activity in a dose-related
manner; however, at concentrations of 0.5 and 1.0 M, glutamate-evoked digastric muscle responses were greater in female than in male rats. In
experiments where jaw muscle EMG and afferent activity were recorded
simultaneously, glutamate (0.5 M, 10 µl) injection into the TMJ
capsule evoked activity in the jaw muscles as well as in 27 (26 A
and 1 C-fiber afferent) of 34 trigeminal afferents that could be
activated by blunt mechanical stimulation of the TMJ tissue. In these
experiments, glutamate-evoked jaw muscle activity was significantly
increased for 6 min after the glutamate injection, whereas afferent
activity was significantly increased only during the first minute after
the glutamate injection. The glutamate-evoked afferent activity was
inversely related to conduction velocity and, in afferents with
conduction velocities <10 m/s, was significantly greater in female
(n = 6) than in male (n = 10) rats.
These results suggest that glutamate excites putative nociceptive
afferents within the TMJ to a greater degree in female than in male
rats. This sex-related difference in afferent discharge may, in part,
underlie sex-related differences in glutamate-evoked jaw muscle EMG activity.
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INTRODUCTION |
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We have
previously suggested that glutamate may play a role in peripheral
mechanisms of nociception within the temporomandibular joint (TMJ)
(Cairns et al. 1998; Yu et al. 1996
).
Injection of the inflammatory irritant and small-fiber excitant mustard
oil into the rat TMJ capsule results in a characteristic reflexly induced increase in the electromyographic (EMG) activity of both the
digastric (jaw opener) and masseter (jaw closer) muscles (Bakke et al. 1998
; Yu et al. 1994
,
1995
). Mustard oil-evoked EMG activity can be
attenuated by peripheral application of the
N-methyl-D-aspartate (NMDA) receptor antagonist
MK-801 (Yu et al. 1996
). Injection of glutamate into the
rat TMJ capsule induces a similar prolonged co-activation of the jaw
muscles through activation of peripheral NMDA and non-NMDA receptors
(Cairns et al. 1998
). In contrast, injection of the
amino acid neurotransmitters
-aminobutyric acid (GABA) or glycine
into the TMJ capsule does not evoke activity in the jaw muscles
(Cai et al. 1999
; Cairns et al.
1999b
).
The rat TMJ is innervated by small myelinated and unmyelinated
afferents that project, via the trigeminal ganglion, to the trigeminal
brain stem sensory nuclear complex (Casatti et al. 1999;
Chen and Turner 1992
; Kido et al.
1995
; Widenfalk and Wiberg 1990
; Yoshino
et al. 1998
). The subnucleus caudalis (Vc) appears to be an
important target for these afferents, since inactivation of the Vc
attenuates jaw muscle activity reflexly evoked by injection of mustard
oil or glutamate into the TMJ capsule (Cairns et al. 1998
; Hu et al. 1997
; Tsai et al.
1999
). Injection of either mustard oil or glutamate into the
TMJ capsule also evokes activity in Vc neurons, which are thought to
relay nociceptive information from the craniofacial region to higher
centers (Broton et al. 1988
; Hathaway et al.
1995
; Kishimoto et al. 1999
;
Kojima 1990
; Sessle 1999
; Sessle
and Hu 1991
; Zhou et al. 1999
). These findings are consistent with anatomical evidence that primary afferents from the
TMJ, like those from other deep craniofacial tissues, project to the Vc
(Capra 1987
; Capra and Wax 1989
;
Nishimori et al. 1986
; Shigenaga et al.
1988
). However, the characteristics of afferents
activated by injection of glutamate into the TMJ capsule of rats have
not been investigated.
Recently, we have reported that male and female rats differ in their
response to injection of glutamate into the TMJ capsule (Cairns
et al. 1999b). Specifically, at concentrations of >0.25 M,
injection of glutamate into the TMJ capsule reflexly evokes greater jaw
muscle responses in female as compared with male rats. However, since
glutamate-evoked reflex jaw muscle activity reflects the integration of
primary afferent drives with the activity of central neurons
intercalated in the TMJ reflex pathway, it remains to be determined
whether there is a sex-related difference in glutamate-evoked TMJ
afferent discharge.
In the present study we have employed a method that allows the activity of trigeminal primary afferents with TMJ mechanoreceptive fields and projections to the Vc to be recorded in intact, lightly anesthetized rats. This methodology also allows the simultaneous recording of jaw muscle EMG activity. We have used this methodology to investigate the type and discharge characteristics of TMJ afferents excited by glutamate injection into the TMJ capsule and compared these features with the glutamate-evoked EMG activity. Further, we have explored the possibility that there is a sex-related difference in the glutamate-evoked activity of TMJ afferents.
A portion of this data has been previously presented in abstract form
(Cairns et al. 1999b, 2000
).
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METHODS |
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Surgical preparation
To construct dose-response graphs, adult male (n = 36) and female (n = 72) Sprague-Dawley rats were
prepared for acute, in vivo recording of digastric muscle EMG activity
(Cairns et al. 1998). To investigate TMJ afferent
activity, additional adult male (n = 13) and female
(n = 14) Sprague-Dawley rats were prepared for acute in
vivo recording of trigeminal primary afferent activity and EMG activity
under surgical anesthesia (O2: 0.3-0.4 l/min; N2O: 0.6-0.7 l/min; halothane: 1.5-2%)
(Cairns et al. 1998
, 1999a
,b
). In all
experiments, a tracheal cannula was inserted and artificial ventilation
initiated. Bipolar electrodes fashioned out of 40-gauge teflon-coated
single strand stainless steel wire were inserted into the ipsilateral
digastric and in the trigeminal afferent experiments, also in the
ipsilateral masseter muscle, and were used to monitor jaw muscle EMG
activity. The rat's head was then placed in a stereotaxic frame, and
the skin over the dorsal surface of the skull was reflected. For the
EMG experiments, two screws were inserted into the parietal bone and
attached to a vertical support bar with dental acrylic as a support for
the head.
For the trigeminal afferent experiments, a trephination was made on the left side of the skull to allow a microelectrode to be lowered through the brain and into the trigeminal ganglion. A second incision was made, and the skin and muscle overlying the brain stem and upper cervical spinal cord were removed; a C1 laminectomy was performed, and the dura overlying the brain stem/cervical spinal cord was removed to facilitate placement of a stimulating electrode in contact with the caudal brain stem (Vc or dorsal horn of the upper cervical spinal cord).
In female rats, a vaginal lavage was performed, and the epidermal cells
were examined under a microscope (Frye et al. 1992; Martinez-Gomez et al. 1994
). This examination revealed
that in the EMG experiments, 11 were in estrus, 25 in metestrus, and 36 in diestrus. Of the female rats used for the afferent recording experiments, two were in estrus, seven in metestrus, and four in diestrus.
After completion of all surgical procedures, the halothane level was slowly reduced (1-1.3%) until noxious pressure applied to the hind paw could induce a weak flexion reflex of the hind limb to ensure that an adequate level of anesthesia was maintained for the duration of the experiment. Heart rate and body core temperature were continuously monitored throughout the whole experiment and kept within the physiological range of 330-430/min and 37-37.5°C, respectively. All surgeries and procedures were approved by the University of Toronto Animal Care Committee in accordance with the regulations of the Ontario Animal Research Act (Canada).
Stimulation and recording techniques
EMG activity was recorded from the ipsilateral digastric and
masseter muscles (Cairns et al. 1998,
1999a
). To permit simultaneous recordings of single
trigeminal primary afferent unit activity, a parylene-coated tungsten
microelectrode (2 M
, A-M Systems, Carlsborg, WA) was slowly
lowered into the brain under stereotaxic control (3.5-4 mm anterior to
the interaural line, 3-4 mm lateral to the midline) until unit
discharge was observed in response to light brush stimuli applied to
the craniofacial region (Fig. 1).
Trigeminal afferent units were usually found 7-8 mm below the cortical
surface, and postmortem examination revealed electrode tract marks on
the surface of the trigeminal ganglion. Mechanical search stimuli were
then applied via a blunt probe (1 mm diam) over the TMJ at an intensity
(~100 g) sufficient to evoke jaw muscle EMG activity while slowly
lowering the electrode in an attempt to identify trigeminal afferents
with deep receptive fields. When a unit was found that appeared to
respond to this blunt mechanical stimuli of the TMJ that was considered
to be noxious, the skin overlying the mechanoreceptive field was pulled
gently away from contact with the TMJ, and brush, pinch, and pressure
stimuli were applied directly to the skin surface. If the unit did not
respond to any of these cutaneous stimuli, then the mechanoreceptive
field was considered to lie within the TMJ. The mandible was then moved once left, right, up, down, forward, and backward through its complete
range of motion to investigate whether the units were activated by jaw
movement.
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In initial experiments, electrical stimuli (1-10 mA, 0.5 ms, 0.33 Hz)
were then applied to the TMJ tissues (articular disk, capsule, and
associated ligaments) in an attempt to determine conduction time, and
thus allow for estimation of conduction velocity. However, this
approach proved unreliable. Since there is physiological and anatomical
evidence for the projection of small-diameter TMJ primary afferents to
the Vc (Broton et al. 1988; Capra 1987
;
Hathaway et al. 1995
; Hu et al. 1997
;
Sessle and Hu 1991
), constant-current electrical stimuli
(50-µs biphasic pulse, range 10-80 µA, 0.5 Hz) were applied to a
stimulating electrode (2 M
, parylene-coated tungsten electrode, A-M
Systems) lowered into the caudal brain stem (1-1.5 mm lateral to the
midline, 0-5 mm caudal to the obex, depth 0-0.5 mm below the brain
stem surface; Fig. 1). For each recorded TMJ afferent, the stimulating
electrode was moved mediolaterally (0.1-mm steps) and rostrocaudally
(0.5-mm steps) in the caudal brain stem to determine whether electrical
stimulation could evoke an antidromic action potential with an
invariant latency (<0.2 ms variability) and the ability to follow
high-frequency electrical stimuli (
100 Hz) (Cairns et al.
1996
; Dostrovsky et al. 1981
; Hu and
Sessle 1988
; Hu et al. 1978
). The initial
electrical stimuli were applied 5 mm caudal to the obex. If stimulation
at this location did not evoke an antidromic action potential, the
stimulating electrode was moved rostrally toward the obex until either
an antidromic action potential was evoked or electrical stimuli had been applied unsuccessfully up to the level of the obex. Antidromic action potentials were collided with orthodromic action potentials evoked by mechanical stimulation of the TMJ tissue, to confirm the
projection of the TMJ afferent to the caudal brain stem. At the end of
the experiment, the distance between the stimulating and recording
electrodes was measured with a ruler, and divided by the latency of the
antidromically evoked response of an afferent to give an estimation of
conduction velocity (CV) of the recorded afferent.
After the above characterization of each afferent was completed, the tip of a catheter, consisting of a 27-gauge needle connected by polyethylene tubing to a Hamilton syringe (50 µL), was carefully inserted into the TMJ joint space and was used to inject drug solutions. It was observed that insertion of the catheter needle into the TMJ capsule evoked a spike discharge in all TMJ afferents under study.
In all experiments involving simultaneous recordings of afferent and EMG activity, baseline primary afferent and EMG activity was recorded for 10 min prior to injection of any substance into the TMJ capsule. Glutamate (0.5 M, 10 µl, pH 7.0) was then injected slowly (over 5 s) into the TMJ capsule, and the resulting primary afferent and jaw muscle EMG activity was monitored for 30 min after the injection. Units that did not respond to application of glutamate were excluded from further analysis.
We have previously found that a repeated injection of glutamate into
the TMJ capsule at 30-min intervals evokes jaw muscle EMG activity of
similar magnitude (Cairns et al. 1998,
1999a
). To investigate the effect of repeated
glutamate injection on TMJ afferent activity, in three experiments a
second injection of glutamate was made 30 min after the initial
injection of glutamate into the TMJ capsule. When compared with
glutamate, injection of GABA (0.5 M) into the TMJ capsule does not
evoke significant jaw muscle EMG activity (Cairns et al.
1999a
). To compare the effect of GABA and glutamate on TMJ
afferent activity, in four experiments GABA (0.5 M, 10 µl, pH 7.0)
was injected 30 min after the initial injection of glutamate into the
TMJ capsule. As a positive control for the GABA experiments, in a
single experiment the nonselective excitant KCl (2.0 M, 10 µl, pH
7.0) was injected into the TMJ capsule 30 min after glutamate.
At the end of each experiment, rats were killed with the agent T61 (Hoechst, Regina, Canada). Electrolytic lesions were first made in the brain stem of some rats by applying a monopolar, monophasic current pulse of 20 µA for 20 s. The brain and brain stem were removed, and it was also confirmed that microelectrode tracts were visible on the surface of the trigeminal ganglion. Thin sections of the brain stem and upper cervical spinal cord (100 µm) were cut with a vibratome and viewed under a microscope.
Data analysis
To construct EMG dose-response curves, EMG activity was recorded
from the ispilateral digastric muscle, amplified (gain: ×500; bandwidth 30-1,000 Hz), and fed into a computer equipped with a CED
1401 Plus board and analysis software (Spike 2; Cambridge Electronics)
(Cairns et al. 1998, 1999a
). Recorded EMG
activity was stored electronically and analyzed off-line. Saline (165 mM, 10 µl) or one of five doses of glutamate (0.05 M, 0.1 M, 0.25 M,
0.5 M, or 1.0 M, 10 µl saline, pH ~7; Research Biochemicals International, Natick, MA) was injected into the TMJ capsule to determine the dose-response relationship in male (n = 6 per dose) and female (n = 12 per dose) rats.
The activity of identified primary afferents and jaw muscles was amplified (afferent gain: ×100; EMG activity gain: ×500; bandwidth 30-1,000 Hz) and fed into a computer equipped with a CED 1401 Plus board and analysis software (Spike 2; Cambridge Electronics). Peristimulus time histograms (PSTH; 1-min bins) were constructed from the recorded primary afferent discharge. Mean baseline afferent discharge was calculated by averaging the first 10 bins prior to injection of glutamate. Mean baseline afferent activity was subtracted from each bin of the PSTH to yield residual afferent discharge. The area under the glutamate-evoked response curve (AUC; spike/min) was calculated by the summation of the residual afferent discharge after glutamate injection.
Recorded EMG activity data were rectified off-line and EMG activity
area (1-min bins) calculated. Mean baseline EMG activity was calculated
by averaging the first 10 bins prior to injection of glutamate. Mean
baseline EMG activity was subtracted from each EMG activity area bin to
yield residual area bins. The AUC (µV/min) was calculated by
summation of the residual EMG activity area bins (Cairns et al.
1998, 1999b
).
Statistical analysis
Most of the collected data were not normally distributed, and thus all data are reported as a median with the interquartile range indicated in square brackets. Paired comparisons were made with the Mann-Whitney rank sum test; multiple comparisons were made with a Friedman repeated measures ANOVA on Ranks and post hoc Dunnett's method as appropriate.
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RESULTS |
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Glutamate-evoked EMG dose-response relationship
To determine whether male and female rats differ in their response to glutamate injected into the TMJ capsule, dose-response curves of glutamate-evoked digastric muscle activity were constructed for male (n = 36) and female (n = 72) rats. The median AUC evoked by application of saline to the TMJ region was similar in male (0 [0-51] µV/min) and female rats (0 [0-100] µV/min; P > 0.05, Mann-Whitney rank sum test; Fig. 2). In both sexes, injection of glutamate into the TMJ capsule in concentrations of 0.25, 0.5, and 1.0 M evoked muscle activity that was significantly greater than that evoked by saline (P < 0.05, ANOVA on ranks, Dunn's method). The AUC in female rats was greater than the AUC of male rats when 0.5 or 1.0 M glutamate was injected into the TMJ capsule. The 0.5-M concentration was subsequently used for the TMJ afferent recording experiments.
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Afferent characteristics
Single-unit recording experiments were undertaken to examine how TMJ afferents respond to glutamate injections and to investigate whether sex differences in glutamate-evoked EMG activity might be due, in part, to a peripheral mechanism. A total of 34 trigeminal afferents responded to mechanical stimuli applied via a blunt probe to the TMJ tissues at an intensity sufficient to evoke jaw muscle EMG activity (Fig. 3). None of these afferents responded to mechanical stimulation (brush, pinch, pressure) of the skin overlying the TMJ. Ten of these afferents also responded to movements of the mandible. Insertion of the catheter needle used to inject glutamate into the TMJ capsule evoked a spike discharge in all afferents.
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Injection of glutamate into the TMJ capsule evoked jaw muscle EMG
activity in all rats and activity in 27 of 34 afferents. Only five of
these glutamate-sensitive afferents also responded to movements of the
mandible. The majority of glutamate-sensitive afferents had CVs in the
A afferent range (n = 26; CV: 6.5 [4.5-11.8] m/s), although one of the afferents had a CV in the C-fiber afferent range (CV: 1.3 m/s; Fig. 4A).
The median CV of the 7 afferents that did not respond to glutamate (14 [11.3-17.3] m/s) was greater than the median CV of the 27 glutamate-sensitive afferents (5.9 [4.2-11.0] m/s, P < 0.05 Mann-Whitney test). Of the seven glutamate-insensitive afferents, five responded to movements of the mandible. Based on the
distance from the obex where electrical stimuli were applied, in
concert with histological reconstructions of selected stimulation sites, all glutamate-sensitive afferents were found to project to
either the caudal Vc or the dorsal horn of the cervical spinal cord
(Fig. 4B). The majority of glutamate-sensitive afferents were antidromically activated by electrical stimuli applied 2-3 mm
caudal to the obex, a region that includes the caudal Vc, the Vc/C1
transition area, and C1 dorsal horn.
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In afferents with CVs of 2.5-10 m/s (slow A afferents,
n = 16), injection of glutamate into the TMJ capsule
generally evoked a prolonged (30-1,800 s) discharge of action
potentials (Fig. 5A). In
contrast, the glutamate-evoked activity of afferents with CVs >10 m/s
(fast A
afferents, n = 10) consisted of only brief (5-20 s) action potential discharges (Fig. 5B). Overall,
there was an inverse relationship between the CV and the AUC of these A
afferents (Fig. 5C). The median glutamate-evoked AUC
calculated for slow A
afferents (103 [20.0-450.5] spikes/min) was
significantly greater than that calculated for fast A
afferents
(10.0 [4.0-50.0] spikes/min, P < 0.05, Mann-Whitney
test).
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Relationship to glutamate-evoked EMG activity
The median glutamate-evoked action potential spike discharge of
the different afferent subgroups, as well as the associated glutamate-evoked jaw muscle EMG activity, is illustrated in Fig. 6. A significant increase in the activity
of fast and slow A afferents, relative to baseline, occurred only
within the first minute after injection of glutamate to the TMJ capsule
(repeated measures ANOVA on Ranks, Dunnett's method, P < 0.05). In the examples of fast and slow A
afferents shown in the
insets of Fig. 5, it can be seen that the action potential
discharge evoked by glutamate preceded increases in jaw muscle EMG
activity by ~4 s.
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In all experiments, glutamate-evoked TMJ afferent activity began before
glutamate-evoked EMG activity. In contrast to the short-duration of
glutamate-evoked A afferent activity (1 min), EMG activity in the
jaw muscles was significantly increased relative to baseline for a
total of 6 min (ANOVA on Ranks, Dunnett's method, P < 0.05). Interestingly, injection of glutamate into the TMJ capsule
evoked a burst-pause action potential discharge pattern in the single
C-fiber afferent that outlasted the EMG response (Fig. 6).
Comparison of male and female rats
Of the 27 glutamate-sensitive afferents, 13 (10 slow, 3 fast) A
afferents were recorded in male rats, and 13 (6 slow, 7 fast) A
afferents as well as the single C-fiber afferent were recorded in
female rats. The median weight of the male rats (335 [320-360] g)
was significantly greater than the median weight of female rats (280 [265-325] g; P < 0.05 rank sum test). However,
there was no significant relationship between weight and
glutamate-evoked afferent response in either males (r = 0.02, P > 0.05, Spearman rank order correlation) or
females (r = 0.062; P > 0.05, Spearman rank order correlation). The median AUC of slow, but not fast, A
afferents was significantly greater in female than male rats (Table
1). This difference was manifested by
both a greater peak response and greater duration of glutamate-evoked
slow A
afferent discharge (Fig. 7).
Glutamate-evoked jaw muscle activity was significantly elevated above
initial baseline levels for 8 min in female rats but for only 1 min in
male rats (Fig. 7). There was no significant difference in
glutamate-evoked afferent activity between female rats in different
stages of the estrus cycle (ANOVA on ranks).
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Comparison of glutamate, GABA, and KCl
In eight A afferents, a second injection of glutamate
(n = 3), GABA (n = 4), or KCl
(n = 1) was made 30 min after the initial injection of
glutamate into the TMJ capsule. The AUC evoked by a second injection of
glutamate into the TMJ capsule (33 [26.0-532.5] spikes/min) was
comparable to the AUC evoked by the initial injection of glutamate (41 [25.0-482.5] spikes/min; Fig. 8). In
contrast, the AUC evoked by GABA (0.5 [0-2.0] spikes/min) was less
than that evoked by glutamate (81 [5.3-281.0] spikes/min). In a
single experiment, the AUC evoked by KCl (293 spikes/min) was roughly half that evoked by the initial injection of glutamate (581 spikes/min).
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DISCUSSION |
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Injection of glutamate into the TMJ capsule evoked activity in
~80% of TMJ afferents. Since all TMJ afferents were, by definition, mechanoreceptors, it could be argued that the glutamate-evoked afferent
activity observed in the present study occurred indirectly as a result
of glutamate-evoked jaw muscle activity. However, this is unlikely for
the following reasons: 1) not all afferents responded to
glutamate injection into the TMJ capsule even though this always evoked
jaw muscle activity; 2) when glutamate did evoke afferent
activity, this preceded reflex jaw muscle EMG activity by several
seconds; and 3) there was a marked difference in the duration of glutamate-evoked afferent activity as compared with jaw
muscle EMG activity. The possibility that glutamate-evoked responses
may have been due solely to distention of the joint or to the osmotic
strength of the solution appears unlikely, since injection of the same
concentration of GABA evoked much less TMJ afferent activity than
glutamate (Fig. 8). This finding is consistent with our previous
results that injection of GABA into the TMJ capsule evokes
significantly less jaw muscle activity than glutamate (Cairns et
al. 1999a). Therefore we conclude that the observed increases
in afferent activity were due to a direct action of glutamate on the
TMJ afferents.
In the present study, the rat TMJ was left intact (except for the
insertion of a catheter) to avoid extensive injury to the TMJ tissues,
which in our experience often prevents jaw muscle activity from being
evoked by injection of algesic substances into the TMJ capsule.
However, this methodology does not allow for direct, electrical
stimulation of TMJ tissues. Instead, mechanical search stimuli were
applied to the TMJ tissues to identify TMJ afferents, which greatly
limited the number and type of TMJ afferents that could be identified
in the present study. As a result, the majority of glutamate-sensitive
TMJ afferents identified by this method were A afferents, although
one glutamate-sensitive C-fiber afferent was also identified. All these
glutamate-sensitive TMJ afferents projected to the Vc, where anatomical
evidence has indicated that there is a selective projection of
small-diameter afferents from deep craniofacial tissues (Capra
1987
; Capra and Wax 1989
; Nishimori et
al. 1986
; Shigenaga et al. 1988
).
Moreover, trigeminal brain stem neurons activated by noxious TMJ tissue
stimulation have previously been identified in the Vc (Broton et
al. 1988
; Hathaway et al. 1995
; Sessle
and Hu 1991
), and chemical or surgical disruption of the Vc
eliminates the TMJ-jaw muscle nociceptive reflex (Cairns et al.
1998
; Hu et al. 1997
; Tsai et al.
l999). Thus these findings point to the possibility that
glutamate injection into the TMJ capsule may activate putative
nociceptive afferents.
The lack of A afferents identified in the present study appears
consistent with anatomical evidence that the rat TMJ is principally innervated by small-diameter myelinated and unmyelinated afferents (Kido et al. 1995
). With regard to the paucity of
C-fiber afferents identified in this study, we propose that our use of
mechanical search stimuli on the intact TMJ tissues may have limited
our ability to sample TMJ C-fiber afferents. In the knee joint, a subpopulation of C-fiber afferents are insensitive to intense mechanical stimuli, although they do respond to algesic chemicals, e.g., KCl (Kanaka et al. 1985
; Schaible and
Schmidt 1985
, 1988
; see review by
Michaelis et al. 1996
; Schaible and Grubb
1993
). Many of these afferents become sensitive to mechanical
stimulation only after experimentally induced joint inflammation; a
process that increases joint glutamate levels (Lawand et al.
1997
, 2000
; Schaible and Grubb
1993
; Schaible and Schmidt 1985
,
1988
; Westlund et al. 1992
). Further, in
the goat, C-fiber afferents that respond to noxious rotation of the jaw
have been recently described; however, the methodology employed in this
study required extensive surgical dissection of the TMJ be undertaken
prior to afferent recording (Loughner et al. 1997
). If
many of the C-fiber afferents innervating the rat TMJ are also
mechanically "silent" and thus not activated by mechanical stimuli
unless sensitized, then the methodology employed in the current study
would have failed to identify many of the C-fiber afferents innervating
the TMJ.
The results of the present study indicate that the glutamate-evoked
activity of A afferents precedes but has a markedly shorter duration
than glutamate-evoked jaw muscle EMG activity. In vitro investigations
of the effects of glutamate on primary afferent neurons in spinal
dorsal root, trigeminal mesencephalic nucleus, and trigeminal ganglion
neurons as well as corneal afferents have found that glutamate-evoked
depolarizing responses are transient, lasting only seconds, and are
followed by a variable period of desensitization (Jones et al.
1997
; Lovinger and Weight 1988
; MacIver
and Tanelian 1993
; Pelkey and Marshall 1998
;
Puil and Spigelman 1988
). One possible explanation for
the disparity between the duration of glutamate-evoked afferent and EMG
responses in the present study may be that a brief activation of slowly
conducting TMJ afferents by glutamate is sufficient to induce a period
of prolonged increase in the excitability of brain stem neurons
intercalated in the TMJ-jaw muscle reflex pathway. For example, a
prolonged enhancement in the excitability of Vc neurons occurs after
acute or chronic inflammation of deep craniofacial tissues, including the TMJ tissues (Chiang et al. 1998
; Hu et al.
1992
; Iwata et al. 1999
; Ren and Dubner
1999
; Yu et al. 1993
). Further, the
duration of activity evoked in Vc nociceptive neurons and the jaw
muscles by application of algesic chemicals to the TMJ tissues are
similar (Broton and Sessle 1988
; Broton et al.
1988
; Hu et al. 1992
; Yu et al.
1995
). It is therefore possible that the difference in the
duration of afferent and EMG activity evoked by glutamate may reflect,
at least in part, a brain stem process of central sensitization
(Hu et al. 1992
, 1997
; Sessle
1999
; Sessle and Hu 1991
) induced by the brief
glutamate-evoked TMJ afferent barrage.
The results of the present study indicate that injection of glutamate
into the TMJ capsule produces a dose-dependent reflex increase in jaw
muscle EMG activity in both sexes but evokes greater muscle activity in
female rats than in male rats. Further, glutamate evoked greater
activity in slow (<10 m/s) A afferents in female than in male rats.
These results suggest that sex-related differences in glutamate-evoked
TMJ afferent activity may, in part, underlie the observation of
enhanced jaw muscle responses evoked in female rats by injection of
glutamate into the TMJ capsule. Previous research has also indicated
that the activation threshold of slowly conducting TMJ afferents to
noxious mechanical rotation of the jaw was lower in female than male
goats; however, this difference was attributed to sex-related
differences in the biomechanical properties of the TMJ tissues
(Loughner et al. 1997
). Importantly, activation of
peripheral excitatory amino acid receptors, rather than joint
distention, was found to be responsible for glutamate-evoked jaw muscle
activity (Cairns et al. 1998
). Further, in the present study, observed increases in afferent activity appear due to a direct
action of glutamate on the TMJ afferents. Therefore it is conceivable
that differences between males and females in the sensitivity of
peripheral excitatory amino acid receptors could underlie the observed
sex-related differences in glutamate-evoked jaw muscle activity. A
possible mechanism for sex-related differences in glutamate-evoked
afferent activity could involve either increased expression of
excitatory amino acid receptors or enhancement of receptor function,
both of which have been shown to occur secondary to increased levels of
the female sex hormone estrogen (Bi et al. 2000
;
Gazzaley et al. 1996
). Further, such peripherally based sex-related differences might also contribute to the differences in
glutamate-evoked masseter muscle pain between men and women (B. E. Cairns, J. W. Hu, L. Arendt-Nielsen, B. J. Sessle, and P. Svensson, unpublished data) and to the well-documented gender differences in many types of craniofacial pain conditions
(Carlsson and LeResche 1995
; Dao and
LeResche 2000
).
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ACKNOWLEDGMENTS |
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The authors recognize the invaluable help of K. MacLeod, S. Carter, and Y. Sim. We also thank Dr. D. Bereiter for input on earlier drafts of the manuscript.
This research was supported by National Institute of Dental Research Grant DE-11995. B. E. Cairns was supported by a Fellowship from the Canadian Arthritis Society and the Medical Research Council of Canada.
Present address of B. E. Cairns: Dept. of Anesthesia, Children's Hospital/Harvard Medical School, Boston, MA 02115.
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FOOTNOTES |
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Address for reprint requests: J. W. Hu, Faculty of Dentistry, The University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6, Canada (E-mail: james.hu{at}utoronto.ca).
Received 26 September 2000; accepted in final form 6 March 2001.
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REFERENCES |
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