Ionic mechanisms underlying inflammatory mediator-induced sensitization of dural afferents

Abstract

Migraineurs experience debilitating headaches that result from neurogenic inflammation of the dura and subsequent sensitization of dural afferents. Given the importance of inflammatory mediator (IM)-induced dural afferent sensitization to this pain syndrome, the present study was designed to identify ionic mechanisms underlying this process. Trigeminal ganglion neurons from adult female Sprague Dawley rats were acutely dissociated 10-14 d after application of retrograde tracer DiI onto the dura. Modulation of ion channels and changes in excitability were measured in the absence and presence of IMs (in mum: 1 prostaglandin, 10 bradykinin, and 1 histamine) using whole-cell and perforated-patch recordings. Fura-2 was used to assess changes in intracellular Ca(2+). IMs modulated a number of currents in dural afferents, including those both expected and/or previously described [i.e., an increase in tetrodotoxin-resistant voltage-gated Na(+) current (TTX-R I(Na)) and a decrease in voltage-gated Ca(2+) current] as well currents never before described in sensory neurons (i.e., a decrease in a Ca(2+)-dependent K(+) current and an increase in a Cl(-) current), and produced a sustained elevation in intracellular Ca(2+). Although several of these currents, in particular TTX-R I(Na), appear to contribute to the sensitization of dural afferents, the Cl(-) current is the primary mechanism underlying this process. Activation of this current plays a dominant role in the sensitization of dural afferents because of the combination of the density and biophysical properties of TTX-R I(Na), and the high level of intracellular Cl(-) in these neurons. These results suggest novel targets for the development of antimigraine agents.

Figure 1.

Inflammatory mediators increase TTX-R Na⁺ currents in dural afferents. Na⁺ currents were recorded in dural afferents (n = 13) before and after IM application. A, Two types of Na⁺ currents were detected in dural afferents. A high-threshold slowly activating and slowly inactivating TTX-R Na⁺ current and a low-threshold rapidly activating and inactivating TTX-S Na⁺ current. B, Example of the voltage dependence of inactivation and activation when data were fitted with a Boltzmann equation. C, After IM application (Post), there was a significant increase in maximal TTX-R Na⁺ current density in dural afferents relative to the baseline current density (Pre). Calibration: 2 nA, 2 ms, *p < 0.05. D, However, there was no significant difference in TTX-S Na⁺ current density (p > 0.05). Error bars indicate SE.

Figure 2.

Inflammatory mediators decrease a Ca²⁺-dependent K⁺ current in dural afferents. K⁺ currents were elicited from dural afferents (n = 14) before and after IM application. In the presence of 50 μm Cd²⁺, to block voltage-gated Ca²⁺ currents, IMs did not reduce K⁺ current density at any potential (A). Furthermore, IMs did not significantly decrease the maximal conductance in the presence of Cd²⁺ (B) and produced no shift in the voltage dependence of activation (C). D, In the absence of Cd²⁺, IMs reduced K⁺ current density at positive potentials. IMs also significantly decreased the maximal conductance (E). However, there was no shift in the voltage dependence of activation of K⁺ currents (F). I–V and G–V data were corrected for voltage errors associated with uncompensated series resistance. A, Inset, Example of K⁺ currents elicited in dural afferents with the voltage protocol shown beneath current traces. *p < 0.05. Error bars indicate SE.

Figure 3.

Inflammatory mediators increase Cl⁻ currents in dural afferents. A, IM application produced an inward current at −60 mV. B, When current was evoked from a series of holding potentials ranging between −60 and +10 mV (n = 7), the IM-induced current reversed direction at approximately −30 mV (140 mm Cl_out; filled circles). After lowering the concentration of Cl_out (36 mm; open circles; n = 6) to produce an equimolar concentration of Cl⁻ in and out, the reversal potential for the IM-induced holding current reversed at 0 mV. C, There was also a significant increase in the holding current recorded at −60 mV when the driving force for Cl⁻ was increased by lowering Cl_out from 140 to 36 mm. The increase in holding current was reversed by application of 100 μm NFA (n = 5). *Significant difference between post-IM with 140 and 36 mm Cl_out and between 140 mm Cl_out and NFA, where p < 0.05. Error bars indicate SE.

Figure 4.

Inflammatory mediator-induced increases in Cl⁻ currents are dependent on intracellular Ca²⁺. A, IM-induced Cl⁻ currents were recorded while substituting intracellular EGTA (11 mm) with the more rapid Ca²⁺ chelator, BAPTA (10 mm). The IM-induced increase in Cl⁻ current was significantly attenuated by 10 mm BAPTA (n = 7). Additionally, when Ca²⁺ was buffered to 622 nm with 1.2 mm EGTA in the presence of Cd²⁺ to block influx through Ca²⁺ channels, IMs still produced an increase in inward current at −60 mV (n = 6). B, To further elucidate the mechanisms of Ca²⁺-dependent activation, the IM-induced Cl⁻ current was measured in the presence of ionic solutions in which Cs⁺ was used to replace intracellular K⁺ and choline was used to replace extracellular Na⁺, leaving Cl⁻ and Ca²⁺ unchanged. Currents were elicited with a two-pulse protocol, the first to activate Ca²⁺ currents and the second to measure the voltage dependence of activation of Cl⁻ currents by IMs using digital subtraction. C, IM-induced difference currents (n = 8) displayed outward rectification and a downward deflection in the I–V curve as the membrane potential was stepped closer to the reversal potential for Ca²⁺. These currents reversed direction at approximately −30 mV. D, Cl⁻ currents were examined again with Ca²⁺ buffered to 500 nm with 1.2 mm EGTA in the presence of Cd²⁺ to block influx through Ca²⁺ channels. Under these conditions, IM-induced Cl⁻ currents displayed a linear I–V curve (n = 7). Asterisk (*) denotes significant difference between exposures, where p < 0.05. Error bars indicate SE.

Figure 5.

Inflammatory mediators inhibit voltage-gated Ca²⁺ currents in dural afferents. A, IM-induced changes in isolated Ca²⁺ currents were recorded before and then after IM application (n = 5). B, IM application significantly decreased peak I_Ca density. C, Instantaneous I–V curves were plotted from tail currents. IMs produced no change in the voltage dependence of activation. *Significant difference between pre-IM and post-IM, where p < 0.05. Error bars indicate SE.

Figure 6.

Inflammatory mediator-induced increases in intracellular Ca²⁺. A, Dural afferents (arrow) and nonlabeled afferents (arrowhead) were identified with epifluorescence illumination. B, C, Fura-2 fluorescence ratio was low before IM application in both afferent populations (B) and dramatically increases in dural afferents and a subpopulation of nonlabeled neurons after IM application (C). The IM-induced change in the fluorescence ratio was determined by subtracting the baseline ratio from the peak value. The decay of the IM-induced Ca²⁺ transient was analyzed as time to 50% decay of the peak (T₅₀). D, Dural afferents displayed a larger increase in intracellular Ca²⁺ with a slower decay compared with nonlabeled afferents that responded to IMs. E, F, Of the dural (9) and nonlabeled afferents (41) that responded, the increase in fluorescence was significantly greater in dural afferents than nonlabeled afferents (E), and the T₅₀ was significantly larger in dural afferents than nonlabeled afferents (F). *Significant difference between groups, where p < 0.05. Scale bar: (in A) A–C, 50 μm. Error bars indicate SE.

Figure 7.

The Cl⁻ equilibrium potential (E_Cl) is depolarized in dural afferents. To determine the reversal potential for I_IM-Cl, currents were recorded in response to a ramp voltage protocol from +50 to −100 mV using gramicidin perforated patch to prevent dialysis of intracellular Cl⁻ before and after IM application (n = 6). To isolate the current, extracellular TEA was used to block K⁺ currents and choline was used to substitute extracellular Na⁺. A, Traces demonstrate currents recorded under these conditions before (black trace) and then after (gray trace) IM application. B, I_IM-Cl obtained with digital subtraction was similar to those recorded in Figure 4C. Additionally, I_IM-Cl reversed at −27.73 ± 2.6 mV (inset), which was close to the action potential threshold previously recorded in dural afferents. Error bars indicate SE.

Figure 8.

IM-induced sensitization of dural afferents is blocked by niflumic acid. The impact of I_IM-Cl activation was determined with changes in excitability measured in current clamp in the absence and presence of 100 μm NFA using gramicidin perforated patch to maintain a physiologically relevant E_Cl. Aa, In the absence of NFA, application of IMs resulted in the sensitization of dural afferents typified by a decrease in rheobase and a leftward shift in the stimulus response function. A typical example of this sensitization is show in Aa, where the protocol used to stimulate the neuron with depolarizing current injection before (baseline) and after application of IMs is shown beneath the voltage traces. Traces evoked at 1, 2, and 3× rheobase are shown, with 1.5 and 2.5× rheobase omitted for clarity. To enable detection of a leftward shift in the stimulus response function, rheobase was determined both before and after application of IMs so as to appropriately scale the suprathreshold current injection. Ab, Voltage traces were evoked in a second neuron before (baseline) and again after the application of NFA (100 μm), IMs alone, and then the combination of IMs with NFA. Traces evoked after the first wash of NFA have been omitted for clarity. Rheobase determined for each stimulation series is indicated below the voltage traces at 1, 2, and 3× rheobase. The resting membrane potential for the neuron in Aa was −67 mV, whereas that in Ab was −64 mV. Pooled data from seven neurons studied as in Ab are plotted in B–E. B, Blocking Cl⁻ channels with NFA alone reduced baseline excitability as indicated by an increase in rheobase. This effect returned to baseline levels with a 2 min wash. IMs produced a significant decrease in rheobase compared with wash, which was reversed with subsequent NFA application. C, In contrast to the inhibitory NFA effects on rheobase, NFA did not have baseline effects on AP threshold nor was it able to reverse the IM-induced hyperpolarization of AP threshold. D, NFA significantly decreased the slope of the SRF, which returned to baseline levels after wash. IMs produced an increase in slope, which was reversed with NFA application. E, Consistent with inhibition of a depolarizing current active at rest, NFA alone hyperpolarized the resting membrane potential and reversed the IM-induced depolarization. *Significant difference between exposures, where p < 0.05. Error bars indicate SE.

Figure 9.

IM-induced sensitization of dural afferent is blocked with a hyperpolarizing shift in E_Cl. IM-induced changes in dural afferent excitability was measured in the presence of 10 mm intracellular Cl⁻ to hyperpolarize the reversal potential for Cl⁻ from −34 to −68 mV (n = 15). A, Changing the E_Cl to −68 mV produced an IM-induced increase in rheobase in 5 of 15 dural afferents, produced no effect in 6 of 15 dural afferents, and decreased rheobase in only 4 of 15 dural afferents. B, The net effect of IMs in the presence of low intracellular Cl⁻ was a 15.35 ± 26.9% increase in rheobase, which was significantly different from that observed with an E_Cl of −34 mV. C, In contrast to these results, IMs were still able to significantly hyperpolarize AP threshold with an E_Cl of −68 mV. D, However, with an E_Cl of −68 mV, IMs did not shift the response to suprathreshold stimulation. Asterisk (*) denotes significant difference between exposures, where p < 0.05. Error bars indicate SE.

Figure 10.

IM-induced sensitization of dural afferent is blocked by BAPTA. Increases in excitability were examined after substituting 11 mm EGTA for 10 mm BAPTA (n = 8) to determine whether, similar to I_IM-Cl, IM-induced sensitization was sensitive to rapid Ca²⁺ chelation. A, In the presence of 10 mm BAPTA, four of eight cells exhibited a reduction in rheobase. B, On average, there was a reduction in rheobase in the presence of IMs; however, consistent with a role for intracellular Ca²⁺, this reduction was significantly less than the reduction observed in the presence of 11 mm EGTA. C, The 10 mm BAPTA prevented the IM-induced hyperpolarization of AP threshold. D, Additionally, although there was a left shift in the SRF, there was no change in the slope. *Significant difference between pre-IM and post-IM, where p < 0.05. Error bars indicate SE.