Depolarization-Dependent C-Raf Signaling Promotes Hyperexcitability and Reduces Opioid Sensitivity of Isolated Nociceptors after Spinal Cord Injury

Abstract

Chronic pain caused by spinal cord injury (SCI) is notoriously resistant to treatment, particularly by opioids. After SCI, DRG neurons show hyperactivity and chronic depolarization of resting membrane potential (RMP) that is maintained by cAMP signaling through PKA and EPAC. Importantly, SCI also reduces the negative regulation by Gαi of adenylyl cyclase and its production of cAMP, independent of alterations in G protein-coupled receptors and/or G proteins. Opioid reduction of pain depends on coupling of opioid receptors to Gαi/o family members. Combining high-content imaging and cluster analysis, we show that in male rats SCI decreases opioid responsiveness in vitro within a specific subset of small-diameter nociceptors that bind isolectin B4. This SCI effect is mimicked in nociceptors from naive animals by a modest 5 min depolarization of RMP (15 mm K⁺; -45 mV), reducing inhibition of cAMP signaling by μ-opioid receptor agonists DAMGO and morphine. Disinhibition and activation of C-Raf by depolarization-dependent phosphorylation are central to these effects. Expression of an activated C-Raf reduces sensitivity of adenylyl cyclase to opioids in nonexcitable HEK293 cells, whereas inhibition of C-Raf or treatment with the hyperpolarizing drug retigabine restores opioid responsiveness and blocks spontaneous activity of nociceptors after SCI. Inhibition of ERK downstream of C-Raf also blocks SCI-induced hyperexcitability and depolarization, without direct effects on opioid responsiveness. Thus, depolarization-dependent C-Raf and downstream ERK activity maintain a depolarized RMP and nociceptor hyperactivity after SCI, providing a self-reinforcing mechanism to persistently promote nociceptor hyperexcitability and limit the therapeutic effectiveness of opioids.SIGNIFICANCE STATEMENT Chronic pain induced by spinal cord injury (SCI) is often permanent and debilitating, and usually refractory to treatment with analgesics, including opioids. SCI-induced pain in a rat model has been shown to depend on persistent hyperactivity in primary nociceptors (injury-detecting sensory neurons), associated with a decrease in the sensitivity of adenylyl cyclase production of cAMP to inhibitory Gαi proteins in DRGs. This study shows that SCI and one consequence of SCI (chronic depolarization of resting membrane potential) decrease sensitivity to opioid-mediated inhibition of cAMP and promote hyperactivity of nociceptors by enhancing C-Raf activity. ERK activation downstream of C-Raf is necessary for maintaining ongoing depolarization and hyperactivity, demonstrating an unexpected positive feedback loop to persistently promote pain.

Keywords: MAP kinase; adenylyl cyclase; chronic pain; nociceptive; opiate; resting membrane potential.

Figure 1.

SCI reduces the effect of MOR agonist DAMGO on cAMP signaling. A, Phosphorylation of PKA-RII (S99) is used as a surrogate measurement of cAMP production in response to Fsk activation of AC and inhibition by the Gαi/o-coupled MOR agonist, DAMGO. B, DAMGO inhibition of Fsk responses in Control group (Control, white boxes, pooled Naive and Sham groups) and SCI group (Fsk 3 μm, DAMGO 0.3 μm, 5 min). PKA-pRII levels are normalized to the baseline of the unstimulated condition in each case. Control, n = 9; SCI, n = 7, compared via two-way ANOVA, followed by Sidak's multiple comparisons test. Significant p values are indicated on the graph. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. C, Dose–response curves for DAMGO inhibition of Fsk responses (Fsk 3 μm) in Control group (black line) and SCI (red) DRG cultures. PKA-pRII data were normalized to the Fsk response in the absence of DAMGO after baseline (BL) subtraction [Y_Norm= (Y – BL)/(Fsk_Max – BL)]. IC₅₀: Control = 0.047 μm (n = 17-22); SCI = 0.15 μm (n = 15-17). Difference between groups tested by two-way ANOVA, followed by Sidak's test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are mean ± SEM. D, IC₅₀ values from individual DAMGO dose–response curves between Control and SCI cultures; p = 2 × 10⁻⁵, Mann–Whitney test. Control, n = 20; SCI, n = 16. In the control column, Blue empty circles represent IC₅₀ values from Sham controls; black circles represent naive controls. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. Detailed statistical information is provided in Table 1.

Figure 2.

Cluster analysis of DRG neurons. A, 3D representation of DRG neurons in culture (7638 neurons) using area (X), CGRP (Y), and IB4 (Z) values as spatial coordinates. Different colors represent the seven different clusters identified by k-medians cluster analysis and plotted based on X, Y, Z coordinates. Neurons from two different IB4⁺ clusters are represented as a single group. B, Representative experiment showing the neuronal subgroup specificity of PKA-pRII responses to Fsk in cultured neurons from a naive animal. Dot color saturation is proportional to the intensity of PKA-pRII fluorescent values: Darker colors represent stronger PKA-pRII signals (n > 2000 neurons per condition) C, Representative examples of DRG neurons arranged according to the identified clusters in A. GGRP (white), IB4 (red), and soma size (indicated by green outline of cell membrane based on PGP9.5). Clusters include N (neurons negative for IB4 and CGRP, with small soma size), CGRP, IB-CG (weak staining for both IB4 and CGRP), IB4, and L and XL (large and extra-large soma size). Scale bar, 25 μm. D, Relative mean cluster size in the total neuronal population for naive and SCI cultures. Colors correspond to clusters in A and C. Light and dark green represent weak and strong IB4 staining clusters, respectively. No significant differences in relative cluster size were found between Naive and SCI groups (p > 0.99, n = 3 per group, two-way ANOVA, followed by Sidak's test). E, F, Fsk responses and DAMGO effects in specific neuronal clusters for naive (E) and SCI (F) cultures. n = 3 per group; DAMGO effects per cluster tested via paired t test. The % DAMGO inhibition over the control Fsk response is indicated for each group. ^#Red represents significance difference in DAMGO effects comparing IB-CG and IB4⁺ clusters from naive (E) and SCI (F) (n = 3, unpaired t test). Detailed statistical information is provided in Table 1.

Figure 3.

Acute depolarization activates ERK and reduces DAMGO effects on cAMP. A, RMP for Naive (black) and SCI (red) small- to medium-sized DRG neurons were measured on successive perfusions (30 s) of increasing extracellular K⁺ concentration, [K⁺]_e. RMP at each [K⁺]_e was measured when steady state was reached (n = 11). *p = 0.039; **p = 0.0081; two-way ANOVA with Sidak's test. B, C, Concentration–response curves for pERK (B) and PKA-pRII (C) to increased [K⁺]_e. Neurons were exposed to media with indicated [K⁺]_e for 5 min. B, Naive, n = 5-7; SCI, n = 5 per data point, C, Naive, n = 5; SCI, n = 5-7 per data point. Two-way ANOVA followed by Sidak's test. D, pERK responses to 18 mm [K⁺]_e (5 min) for IB4⁺ and IB4^– neuronal subpopulations in Naive and SCI DRG neurons: Naive, SCI, n = 3, p = 0.047, two-way ANOVA followed by Sidak's test. E, K⁺-induced depolarization decreases DAMGO responses in Naive DRG neurons. Neurons were exposed to either control media (5 mm [K⁺]_e) or 15 mm [K⁺]_e media during Fsk ± DAMGO (Dm) stimulation (Fsk = 3 μm, DAMGO = 1 μm, 5 min); n = 6. Control, p = 0.041; Fsk+DAMGO, p = 0.0015, two-way ANOVA, followed by Sidak's test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. F, Dose–response curves of DAMGO inhibition of Fsk responses in Naive DRG neurons under control conditions (black) or 15 mm [K⁺]_e (blue). IC₅₀ Control, 0.051 μm; IC₅₀ K⁺15, 0.075 μm; Control, n = 9-19; K⁺15, n = 4-12 per data point. Data compared by two-way ANOVA, followed by Sidak's test: ****p < 0.0001. G, Dose–response curves of morphine inhibition of Fsk responses in naive DRG neurons with control or 15 mm [K⁺]_e. Control IC₅₀ = 0.021 μm; K⁺15 IC₅₀ = 0.16 μm; Control, n = 3-5; K⁺15, n = 4 or 5 per data point (two-way ANOVA). Detailed statistical information is provided in Table 1.

Figure 4.

Inhibition of C-Raf partially restores opioid sensitivity after depolarization. A, Inhibition of 15 mm [K⁺]_e effects on DAMGO responses. DRG neurons were preincubated with inhibitors of Src (saracatinib, 10 μm), PKC (sotrastaurin, 1 μm), PKA (H89, 10 μm), C/B-Raf (RAF709, 10 μm), C-Raf (GW5074, 3 μm), or MEK1/2 (UO126, 10 μm) for 30 min and then stimulated with Fsk (3 μm) ± DAMGO (0.1 μm) in control media or 15 mm [K⁺]_e (K⁺15; 5 min). Effects are reported as inhibition of the control Fsk response (1.0). Drug effects were compared against the Control-DAMGO inhibition (*) or K⁺-DAMGO inhibition (#) via one-way ANOVA, followed by Dunnett's test. Significance and n values are reported on each bar. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^##p< 0.01. B, C-Raf inhibition restores DAMGO IC₅₀ in 15 mm [K⁺]_e, but has no effect on DAMGO efficacy (E_max). GW5074 (10 μm) was applied 30 min before simultaneous addition of K⁺(15 mM), Fsk(3 mm), and DAMGO for 5 min. Per data point, Control, n = 10-21; K⁺15, n = 6-22; K⁺15+GW5074, n = 3-10; Control+GW5074, n = 3-11. Treatment effects were compared using two-way ANOVA followed by Tukey's multiple comparisons test. Individual data point comparisons using Sidak's test can be found in Table 1. C, D, Quantification of individual IC₅₀ (C) and E_max (D) values of DAMGO dose–response curves as described in B. C, Control, n = 13; K⁺15(–), n = 10; K⁺15(GW), n = 4. D, Control, n = 14; K⁺15(–), n = 10; K⁺15(GW), n = 4. Data were compared using one-way ANOVA, followed by Tukey's test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. Detailed statistical information is provided in Table 1.

Figure 5.

Depolarization induces C-Raf activation and relief of RKIP inhibition in IB4⁺ neurons. A, C-Raf activity is promoted by phosphorylation (S338). C-Raf inhibition is relieved on phosphorylation of RKIP (S153). B, Phosphorylation of C-Raf (S338) and RKIP (S153) in response to increasing [K⁺]_e (5 min stimulation). pC-Raf_S338, n = 3-7; pRKIP_S153, n = 3. C, Phosphorylation of C-Raf (S338) with 15 mm [K⁺]_e in naive and SCI DRG neurons; Naive, n = 6; SCI, n = 5. Data compared via unpaired t test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. D, E, Quantification of pC-Raf_s338 (D, n = 4) and pRKIP_S153 (E, n = 3) phosphorylation in naive neurons in response to 5 min, 15 mm [K⁺]_e between different neuronal subpopulations (cluster analysis performed as in Fig. 2). Differences in cluster responses determined via one-way ANOVA, followed by Tukey's test. pC-Raf_S338 and pRKIP_S153 responses have been normalized to control baselines for each cluster. p values in the figure correspond to the comparison against IB4. Detailed statistical information is provided in Table 1. F, C-Raf and RKIP phosphorylation in control and 15 mm [K⁺]_e, shown as coordinates of nociceptor Area (X), CGRP (Y), and IB4 (Z) intensity. Dot color saturation is proportional to the intensity of fluorescent signal. Darker colors represent stronger signals. pRKIP: n > 1000 neurons; pC-Raf: n > 3200 neurons. G, Examples of pC-Raf_S338 responses to depolarization (5 min, 15 mm [K⁺]_e). 10× magnification. Scale bar, 25 μm.

Figure 6.

Effects of depolarization and C-Raf activation on HEK-293MOR cells. A, DAMGO dose–response curves in control conditions ([K⁺]_e 5 mm, serum-starved; black empty circles), [K⁺]_e 15 mm (blue circles), C-Raf-CTH overexpression (Raf-CTH; purple triangles, dotted line), serum treatment (not starved, red squares), or serum in presence of 3 μm the C-Raf inhibitor GW5074 (Ser+GW; orange circles, dotted line). Control, n = 7-9; K⁺15, n = 3; Serum, n = 3-11; Raf-CTH, n = 6; Ser+GW, n = 7 or 8 per data point. Treatment effects were compared via two-way ANOVA, followed by Sidak's test. ^vp < 0.05; **p < 0.01; ***p < 0.001; ^{∧∧∧∧,vvvv}p < 0.0001. *Control versus Raf-CTH. ^∧Control versus Serum. ^vSerum versus Ser+GW. B, C, Effects of the different treatments used in A, on DAMGO IC₅₀ (B) and E_max (C); 50 mm K⁺ not shown in A. B, Control, n = 9; K⁺15, n = 3; K⁺50, n = 3; Ser, n = 9; Raf-CTH, n = 6; Ser+GW, n = 7. C, Control, n = 9; K⁺15, n = 3; K⁺50, n = 3; Ser, n = 8; Raf-CTH, n = 6; Ser+GW, n = 7. Treatment effects compared via one-way ANOVA followed by Dunnett's test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. Detailed statistical information is provided in Table 1.

Figure 7.

Inhibition of C-Raf fully restores opioid sensitivity and reverses hyperexcitability after depolarization. A, DAMGO inhibition of Fsk responses in SCI cultured neurons pretreated for 30 min with vehicle (DMSO ≤0.1%), GW5074 (3 μm), or retigabine (10 μm), followed by 5 min stimulation with Fsk (3 μm) +/− indicated concentrations of DAMGO. Vehicle, n = 11; GW5074, n = 7 or 8; retigabine, n = 4. Treatment effects compared via two-way ANOVA, followed by Sidak's test. *p < 0.05. **^,∧∧p < 0.01. Significance symbols: *Vehicle versus retigabine. ^∧Vehicle versus GW5074. B, IC₅₀ values from individual DAMGO dose–response curves for SCI groups treated as in A. Veh, n = 10; GW, n = 8; Ret, n = 4. Treatment effects compared using one-way ANOVA, followed by Dunnett's test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. C, Representative traces of current-clamp recordings (I = 0) of sensory neurons isolated from Naive and SCI groups pretreated (30 min) with vehicle (DMSO 0.03%), C-Raf inhibitor (GW5074, 3 μm) or MEK inhibitor (UO126, 3 μm). APs are clipped at 0 mV so that subthreshold DSFs are more visible. D, A selective C-Raf inhibitor (GW5074) and MEK inhibitor (UO126) decrease the incidence (%) of neurons exhibiting ongoing firing at RMP (SA) or when held at −45 mV (OA). Comparisons of active neuron incidence by Fisher's exact test. n values and p values are indicated over each bar. For multiple comparisons, Bonferroni correction was applied and significance levels are provided on figure. E, Inhibition of C-Raf or MEK by GW5074 or UO126, respectively, hyperpolarizes RMP. The effects of C-Raf and MEK inhibitors on RMP were compared with Brown-Forsythe and Welch ANOVA, followed by Dunnett's test (p = 0.011 for GW5074 and p = 0.035 for UO126). F, Reduction of DSF amplitudes by GW5074 and UO126. Mean DSF amplitudes trended lower when recorded at RMP (p = 0.055 for each inhibitor) and were significantly lower at −45 mV (p = 0.033 for GW5074 and p = 0.013 for UO126). One-way ANOVA followed by Holm–Sidak's test at RMP and Kruskal-Wallis with Dunn's multiple comparison test at −45 mV. MP, Membrane potential. Detailed statistical information is provided in Table 1.

Figure 8.

Model of depolarization-dependent, C-Raf-mediated, self-reinforcing mechanisms driving nociceptor hyperexcitability and reduced opioid responses after SCI. Depolarization of nociceptors (ΔVm) induced by SCI enhances C-Raf activity in IB4⁺ neurons via direct phosphorylation of C-Raf and relief from RKIP inhibition. Active C-Raf promotes hyperexcitability via two different mechanisms acting in parallel. (1) Activation of the MEK-ERK cascade by C-Raf has direct effects on RMP and neuronal excitability. (2) Phosphorylation of AC5/6 by C-Raf reduces the inhibitory effects of Gαi on cAMP generation by AC and downstream PKA/EPAC signaling, which also regulate nociceptor hyperexcitability and RMP. The combined effects of MAPK and cAMP signaling on RMP and hyperexcitability set up positive feedback that maintains OA/SA in nociceptors while also limiting the effectiveness of opioids.