Department of Oral Physiology, Faculty of Dentistry, University of Toronto, Toronto M5G 1G6 Canada
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ABSTRACT |
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Cairns, Brian E., Barry J. Sessle, and James W. Hu. Activation of peripheral GABAA receptors inhibits temporomandibular joint-evoked jaw muscle activity. We have previously shown that injection of mustard oil or glutamate into rat temporomandibular joint (TMJ) tissues, an experimental model of acute TMJ injury, can reflexly induce a prolonged increase in the activity of both digastric (jaw-opener) and masseter (jaw-closer) muscles. In this study, GABA was applied to the TMJ region by itself or in combination with glutamate, and the magnitude of evoked jaw muscle electromyographic (EMG) activity was measured. Application of GABA alone to the TMJ region did not evoke significant jaw muscle EMG activity when compared with normal saline controls. In contrast, co-application of GABA and glutamate into the TMJ region decreased the magnitude of glutamate-evoked EMG activity. This GABA-mediated inhibition of glutamate-evoked EMG activity followed an inverse dose-response relationship with an estimated median inhibitory dose (ID50) of 0.17 ± 0.05 (SE) µmol and 0.031 ± 0.006 µmol for the digastric and masseter muscles, respectively. Co-administration of the GABAA receptor antagonist bicuculline (0.05 µmol) but not the GABAB receptor antagonist phaclofen (0.05 or 0.15 µmol) reversed the suppressive actions of GABA, indicating that this action of GABA may be mediated by peripheral GABAA receptors located within the TMJ region. Our results suggest that activation of peripheral GABAA receptors located within the TMJ region could act to decrease the transmission of nociceptive information.
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INTRODUCTION |
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Activation of either peripheral excitatory amino
acid (EAA) or GABAA receptors located in cutaneous tissues
of the tail of neonatal rats evokes a nociceptive reflex (Ault
and Hildebrand 1993, 1994
). Injection of glutamate into the
temporomandibular joint (TMJ) region also reflexly induces a prolonged
increase in the electromyographic (EMG) activity of both digastric and masseter muscles in adult rats (Cairns et al. 1998
).
This effect of glutamate is mediated through activation of peripheral
EAA receptors located within the TMJ region because co-application of
both N-methyl-D-aspartate (NMDA)- and
non-NMDA-receptor antagonists significantly reduces the magnitude of
glutamate-evoked jaw muscle EMG activity. However, it is not clear
whether there are also peripheral GABA receptors within the TMJ region
or if application of GABA to the TMJ region of adult rats can evoke jaw
muscle activity in a manner that is analogous to glutamate.
Consequently, this study was undertaken to investigate the effects of
GABA application to the TMJ region on jaw muscle EMG activity.
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METHODS |
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Surgical preparation
Forty-eight male Sprague-Dawley rats (250-450 g) were prepared
for acute recording of jaw muscle EMG activity as previously described
(Cairns et al. 1998). Briefly, under surgical anesthesia (O2: 0.3-0.4 l/min; N2O: 0.6-0.7 l/min;
halothane: 1.5-2%) a tracheal cannula was inserted, and the left
femoral vein was cannulated, bipolar electrodes were inserted
bilaterally into the digastric and masseter muscles, and a catheter was
carefully inserted into the TMJ region and used to apply different
chemicals. After surgery, the level of halothane was maintained at
0.8-1% for the duration of the experiment. 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). At the end of each experiment, rats were
euthanized with T61 (Hoechst).
Drug solutions
The following chemicals were employed: glutamate, GABA, the
GABAA receptor antagonist bicuculline and the
GABAB receptor antagonist phaclofen (Research Biochemicals
International; Natick, MA). All drugs were dissolved in isotonic
saline, and the resulting solutions were adjusted to a pH of ~7.
Solutions (total volume per injection: 10 µL) were injected
bilaterally into the TMJ capsule (Cairns et al. 1998).
Intravenous administration of GABA (5 µmol in 0.1 ml normal saline)
also was made through the femoral vein catheter.
Stimulation and recording
EMG activity was 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). EMG activity was
recorded continuously for the duration of each experiment. Baseline EMG
activity was observed for 10 min, and then glutamate (2.5 µmol) was
injected into the TMJ region over 5 s. We previously demonstrated
that application of this dose of glutamate to the TMJ region evokes EMG
activity in both the ipsilateral digastric and masseter muscles by a
reflex pathway through the trigeminal subnucleus caudalis
(Cairns et al. 1998). Thirty minutes after the initial
glutamate injection either glutamate (2.5 µmol) alone, GABA (0.005, 0.05, 0.5 or 5 µmol) alone, GABA in combination with glutamate, or
GABA in combination with glutamate and either the selective GABA
receptor antagonists bicuculline (0.05 µmol) or phaclofen (0.05 or
0.15 µmol) were injected into the TMJ region. All drugs were at room
temperature immediately before injection. Osmolarity was adjusted by
the addition of sodium chloride.
Data analysis
Recorded EMG data were rectified off-line, and EMG area bins (µV-min) were calculated. Mean baseline EMG activity was subtracted from each EMG area bin to yield residual EMG area bins. The area under the EMG response curve (AUC) was calculated by summing all residual EMG area bins greater than two times the SD of the mean baseline. The relative EMG response was then calculated by normalizing the AUC for the second application (AUC2) to the AUC evoked by glutamate (AUC1). For each dosing regimen, a mean relative EMG response was determined from the average of five experiments conducted in three rats.
To calculate ID50, nonlinear regression analysis was
performed on the dose-response curve for GABA-mediated suppression of glutamate-evoked EMG activity according to the equation
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RESULTS |
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Glutamate (2.5 µmol), GABA, or normal saline was injected into
the TMJ region 30 min after an initial injection of glutamate (2.5 µmol) was applied. Repeated application of glutamate at an interval
of 30 min evoked EMG responses in both ipsilateral jaw muscles (Fig.
1A) that were of comparable
magnitude, in agreement with previous findings (Cairns et al.
1998). In contrast, the EMG activity evoked by GABA at all the
doses applied (5, 0.5, 0.05, and 0.005 µmol) was significantly less
than that evoked by glutamate and similar to that evoked by normal
saline (P < 0.05 Kruskal-Wallis one-way ANOVA on
Ranks, Dunn's method, Fig. 1, B and C).
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The jaw muscle activity evoked by application of glutamate alone to the TMJ region was compared with that evoked by co-application of glutamate and GABA (5, 0.5, 0.05, or 0.005 µmol) or after systemic administration of GABA (5 µmol). Systemic administration of GABA (5 µmol) had no effect on glutamate-evoked EMG activity (Fig. 2A), but local application of GABA to the TMJ region suppressed glutamate-evoked EMG activity in a dose-dependent manner with an estimated mean (±SE) ID50 of 0.17 ± 0.05 µmol and 0.031 ± 0.006 µmol for the digastric and masseter muscles, respectively (Figs. 2 and 3). The ID50 for the digastric muscle was significantly larger than the ID50 for the masseter muscle (P < 0.05, Student's t-test).
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Co-application of bicuculline (0.05 µmol) but not phaclofen (0.05 or 0.15 µmol) partially attenuated GABA-mediated suppression of glutamate-evoked EMG activity (Fig. 2, C and D). Co-application of bicuculline also resulted in a rightward shift of the dose-response curves of GABA-mediated suppression for both the digastric and masseter muscles; the estimated ID50 values increased to 7.5 ± 1.5 µmol and 1.5 ± 0.9 µmol for the digastric and masseter muscles, respectively (P < 0.05, Student's t-test; Fig. 3).
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DISCUSSION |
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Before this study it was not known whether peripheral GABA
receptors exist within the TMJ region of adult rats or if activation of
these receptors evokes jaw muscle activity analogous to that evoked by
glutamate. In this study it was shown that when compared with glutamate
local application of GABA to the TMJ region was not effective in
evoking jaw muscle activity. However, TMJ application of GABA was found
to inhibit glutamate-evoked jaw muscle EMG activity, and this
GABA-mediated inhibition was reversed by co-application of the
GABAA receptor antagonist bicuculline but not by the
GABAB receptor antagonist phaclofen. These results suggest
that GABAA receptors are located within the TMJ region and
that their activation appears to attenuate the nociceptive reflex
response that can be evoked from this region. In contrast, GABA
application to the tail skin of the neonatal rat isolated spinal
cord-tail preparation evokes a nociceptive reflex (Ault and
Hildebrand 1994). This difference may reflect the use of an in
vitro neonatal as opposed to an in vivo adult rat preparation and/or
differences between cutaneous versus deep tissues.
Previous reports indicated that application of GABA to peripheral
nerves has two effects. 1) It depolarizes certain nerve fibers, and 2) it concomitantly suppresses the conduction of
sensory information along small-diameter (A, C) primary afferent
fibers that can convey nociceptive information from the periphery to the CNS (Brown and Marsh 1978
; Bhisitkul et al.
1987
; Morris et al. 1983
). GABA-induced
depolarization of peripheral nerve fibers is attenuated by
GABAA receptor antagonists and mediated through the opening
of chloride channels (Brown and Marsh 1978
;
Desarmenien et al. 1984
; Deschenes et al.
1976
; Gallagher et al. 1978
; Morris et
al. 1983
). GABA-mediated activation of chloride channels may also act as a shunt, allowing current to flow out across the axon membrane instead along the nerve fiber and thus impede action potential
conduction (Wall 1995
).
The rat TMJ region is innervated almost exclusively by A and C
fibers (Kido et al. 1995
), and there is recent evidence
that suggests that GABA may block action potential conduction through small-diameter trigeminal primary afferents (Soja et al.
1998
). We speculate that activation of peripheral
GABAA channels located on the unmyelinated terminal
segments of TMJ afferent fibers results in a current shunt that
interferes with the conduction of action potentials (Brown and
Marsh 1978
; Wall 1995
).
GABA is distributed widely in nonneural tissues and may also be
synthesized and released by a subpopulation of trigeminal primary
afferent fibers, providing evidence in favor of a modulatory role for
GABA in the peripheral nervous system (Szabat et al. 1992; Tanaka 1985
). Our findings also raise the
possibility that activation of peripheral GABAA receptors
within the TMJ region may result in a local analgesic effect, and so
further study of these mechanisms may prove to have clinical
significance in pain management.
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ACKNOWLEDGMENTS |
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The authors thank K. MacLeod for electronic services.
This research was supported by National Insitutes of Health Grant DE-11995. B. E. Cairns was supported by a Fellowship from the Canadian Arthritis Society and the Medical Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests: J. W. Hu, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6 Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 October 1998; accepted in final form 17 December 1998.
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REFERENCES |
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