Estrogen and inflammation increase the excitability of rat temporomandibular joint afferent neurons

Abstract

Several painful conditions, including temporomandibular disorders (TMD), are more prevalent and more severe in women than in men. Although the physiological basis for this sex difference remains to be determined, it is likely that estrogen is an underlying factor. The present study was performed to test the hypotheses that estrogen increases the excitability of rat temporomandibular joint (TMJ) afferents and exacerbates the inflammation-induced sensitization of these sensory neurons. Retrogradely labeled TMJ neurons from ovariectomized rats and ovariectomized rats receiving chronic estrogen replacement were studied using whole cell patch-clamp techniques three days after injecting the TMJ with either saline or Complete Freund's Adjuvant to induce inflammation. Excitability was assessed with depolarizing current injection to determine action potential threshold, rheobase, and the response to suprathreshold stimuli. Spontaneous activity was also assessed. Both inflammation and estrogen increased the excitability of TMJ neurons as reflected by decreases in action potential threshold and rheobase and increases in the incidence of spontaneous activity. The effects were additive with neurons from rats receiving both estrogen and inflammation being the most excitable. The increases in excitability were associated with changes in passive properties and action potential waveform, suggesting that estrogen and inflammation affect the expression and/or properties of ion channels in TMJ neurons. Importantly, the influence of estrogen on both baseline and inflammation-induced changes in TMJ neuronal excitability may help explain the profound sex difference observed in TMD as well as suggest a novel target for the treatment of this pain condition.

Figure 1. Excitability of trigeminal ganglion neurons innervating the TMJ

Excitability was measured in DiI labeled TG neurons with depolarizing current injection. Panels A–C were recorded from a 50.5 pF TMJ neuron from a VS rat that had a resting membrane potential of −60 mV. Panels D–F are from a 50.0 pF TMJ neuron from an EC rat that had a resting membrane potential of −54 mV. The neuron from the EC rat is much more excitable than that from the VS rat. A and D: Action potential threshold and rheobase were determined with a 500 ms depolarizing current injection. Threshold (dotted line) is the most depolarized membrane potential achieved without firing an action potential (−24.74 mV in A; −44.67 mV in D). Rheobase is the minimum amount of depolarizing current sufficient to evoke an action potential (300 pA in A; 10 pA in D). The current trace in A contains three stimuli of 280, 290, and 300 pA. B,C,E,and F: The response to suprathreshold stimuli was measured as the number of action potentials evoked by 500 ms depolarizing current injections equal to 2× (B and E) and 3× rheobase (C and F).

Figure 2. N52 binding and capsaicin sensitivity

TMJ neurons are easily identified following injection of DiI into the TMJ. A. DiI-labeled neurons appear red under epifluorescence illumination (Arrow, Left Panel). Sections were probed with the monoclonal antibody against neurofiliment 200 (N52) in order to assess the percentage of TMJ neurons that are myelinated (arrow heads, middle panel). The neuron shown is unmyelinated. B. Cell body size histogram of the total population of TG neurons (n = 360, grey bars), N52+ neurons (n = 80, hatched blue bars) and that of labeled TMJ neurons (n = 103, pink hatched bars). C. Typical response to a bolus application of capsaicin (500 nM) from a TMJ neuron, considered CAP+ that was harvested from a VC rat. CAP− neurons (D) were easily distinguishable from CAP+ neurons in that they typically demonstrated little change in membrane potential in response to capsaicin. Scale bar in C is the same for D.

Figure 3. Examples of spontaneous activity

A. An example of low frequency (0.7 Hz) irregular spontaneous activity observed in a DiI labeled neuron (RMP = −54 mV, membrane capacitance = 44 pF) from an ES rat that was typical of the activity observed in VS, VC and ES rats. B. An example of higher frequency (2.5 Hz) regular activity observed in a DiI labeled neuron (RMP = −45 mV, membrane capacitance = 41 pF) from an EC rat that was typical a number of spontaneously active neurons from EC rats. Scale bar in A is the same in B.

Figure 4. Estrogen and inflammation increase the excitability of TMJ afferents

Action potential threshold, rheobase, and the response to suprathreshold stimuli were measured in TG neurons as described in Figure 1 and Methods. A: Estrogen and inflammation decreased action potential threshold. Values are means ± SEM. The number in parentheses reflects the number of neurons studied in each group. The number in A is the same for B and C. B: Estrogen and inflammation decreased rheobase, plotted here as rheobase normalized to cell capacitance (Normalized Rheobase). C: There was little influence of either estrogen or inflammation on the slope of the stimulus-response function. The inset in each panel reflects the results of statistical analyses of data (two-way ANOVA with Holm-Sidak post-hoc tests). E a significant main effect due to estrogen, and C a significant main effect due to inflammation (CFA). * p < 0.05, ** p < 0.01.

Figure 5. Inflammation increases the capacitance of TMJ afferents

Capacitance was used as a measure of the cell body size of TG neurons. Two-way ANOVA revealed a statistically significant (p < 0.01) influence of inflammation on cell body capacitance. In order to illustrate this influence, neurons from saline (gray bars) and CFA (black bars) treated animals were pooled and plotted as a percent of the total number of neurons studied in each group (105 and 119, respectively). The median cell body capacitance of neurons from saline treated animals was 38 (with 28 and 62 as 25^th and 75^th percentiles), while that of neurons from CFA treated animals was 50 (with 37 and 65 as 25^th and 75^th percentiles).