P2X and NMDA receptor involvement in temporomandibular joint-evoked reflex activity in rat jaw muscles

Abstract

We have previously shown that injection of the excitatory amino glutamate into the rat temporomandibular joint (TMJ) evokes reflex activity in both anterior digastric (DIG) and masseter (MASS) muscles that can be attenuated by prior TMJ injection of an N-methyl-d-aspartate (NMDA) receptor antagonist. The aim of the present study was to test if jaw muscle activity could also be evoked by P2X receptor agonist injection into the rat TMJ region and if the reflex activity could be modulated by TMJ injection of P2X receptor antagonist or NMDA receptor antagonist. The selective P2X subtype agonist alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-me ATP) and vehicle (phosphate-buffered saline) or the selective P2X antagonist, 2'-(or-3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) or the selective NMDA antagonist (+/-)-d-2-amino-5-phosphonovalerate(APV) were injected into the rat TMJ region. Electromyographic (EMG) reflex activity was recorded in both DIG and MASS muscles. Compared with the baseline EMG activity, alpha,beta-me-ATP injection into the TMJ (but not its systemic administration) following pre-injection of the vehicle significantly increased the magnitude and the duration of ipsilateral DIG and MASS EMG activity in a dose-dependent manner. The alpha,beta-me-ATP-evoked responses could be antagonized by pre-injection of TNP-ATP into the same TMJ site but contralateral TMJ injection of TNP-ATP proved ineffective. Furthermore, the alpha,beta-me-ATP-evoked responses could also be antagonized by APV injected into the same TMJ site but not by its systemic injection. These results indicate the interaction of peripheral purinergic as well as glutamatergic receptor mechanisms in the processing of TMJ nociceptive afferent inputs that evoke reflex activity in jaw muscles.

Fig. 1

The effect of vehicle (PBS) and three doses of the P2X receptor agonist α,β-me-ATP on the ipsilateral (Ipsi.) DIG (in A) and MASS (in B) integrated EMG activity (calculated as AUC) at different time points; examples of representative raw EMG traces are shown above the graphs in A and B. In A and B, 10 min after the EMG recording, a pre-load of PBS was injected into the TMJ region and then α,β-me-ATP was injected at 15 min. *, +: represent the time points when AUC values are 2 S.D. above the baseline for 10 mM and 100 mM of α,β-me-ATP, respectively. Note the co-activation of both DIG and MASS. In C and D, AUC values of ipsilateral DIG and MASS EMG activity evoked by different doses of α,β-me-ATP are shown in box-plots. Below the x-axis, the top row of labeling refers to the first injection (Pre-load, at 10 min) and the bottom row indicates the second injection (Load, at 15 min). * represents p<0.05, ANOVA on rank and Dunn tests. At the far right of C and D are shown the desensitizing effect when two consecutive α,β-me-ATP injections were made into the same TMJ site **: p<0.01, Mann-Whitney U-tests).

Fig. 2

The effect of TNP-ATP on the α,β-me-ATP -evoked ipsilateral (Ipsi.) DIG (in A) and MASS (in B) integrated EMG activity at different time points. Ten min after the EMG recording, a pre-load of PBS, 1 μM or 100 nM TNP-ATP was injected into TMJ region and then 10 or 100 mM α,β-me-ATP was injected at 15 min. +, *: represent the time points when AUC values are 2 S.D. above the baseline between PBS vs 10 mM α,β-me-ATP or 1 μM TNP-ATP vs 10 mM α,β-me-ATP, respectively. DIG (in C) and MASS (in D) EMG activity evoked by the lower dose (10 mM) of the α,β-me-ATP was significantly modulated (Pre-load) by the higher dose of the TNP-ATP (1 μM) as shown in the box-plots. Below the x-axis, the top row of labeling refers to the first injection (Pre-load, at 10 min) and the bottom row to the second injection (Load, at 15 min). * represents p<0.05, ** represents p<0.01, ANOVA on rank and Dunn tests.

Fig. 3

The effect of APV on the α,β-me-ATP-evoked ipsilateral (Ipsi.) DIG (in A) and MASS (in B) integrated EMG activity (calculated as AUC, μV*min) at different time points. Ten minutes after the EMG recording, a pre-load of PBS, 0.05 or 0.5 μM APV was injected into the TMJ region and α,β-me-ATP was injected at 15 min. +, *, #: represent the time points when AUC values are 2 S.D. above the baseline for PBS, 0.05 μM or 0.5 μM APV and 10 mM α,β-me-ATP. Ipsilateral EMG activity in DIG (in C) but not in MASS (in D) evoked by 10 mM α,β-me-ATP was significantly modulated by the higher dose of APV (0.5 μM) as shown in the box-plots. On the x-axis labeling, the top row refers to the first injection (Pre-Load, at 10 min) and the bottom row to the second injection (Load, at 15 min). * represents p<0.05, ANOVA on rank and Dunn tests.