Sustained Suppression of Hyperalgesia during Latent Sensitization by μ-, δ-, and κ-opioid receptors and α2A Adrenergic Receptors: Role of Constitutive Activity

Abstract

Many chronic pain disorders alternate between bouts of pain and periods of remission. The latent sensitization model reproduces this in rodents by showing that the apparent recovery ("remission") from inflammatory or neuropathic pain can be reversed by opioid antagonists. Therefore, this remission represents an opioid receptor-mediated suppression of a sustained hyperalgesic state. To identify the receptors involved, we induced latent sensitization in mice and rats by injecting complete Freund's adjuvant (CFA) in the hindpaw. In WT mice, responses to mechanical stimulation returned to baseline 3 weeks after CFA. In μ-opioid receptor (MOR) knock-out (KO) mice, responses did not return to baseline but partially recovered from peak hyperalgesia. Antagonists of α2A-adrenergic and δ-opioid receptors reinstated hyperalgesia in WT mice and abolished the partial recovery from hyperalgesia in MOR KO mice. In rats, antagonists of α2A adrenergic and μ-, δ-, and κ-opioid receptors reinstated hyperalgesia during remission from CFA-induced hyperalgesia. Therefore, these four receptors suppress hyperalgesia in latent sensitization. We further demonstrated that suppression of hyperalgesia by MORs was due to their constitutive activity because of the following: (1) CFA-induced hyperalgesia was reinstated by the MOR inverse agonist naltrexone (NTX), but not by its neutral antagonist 6β-naltrexol; (2) pro-enkephalin, pro-opiomelanocortin, and pro-dynorphin KO mice showed recovery from hyperalgesia and reinstatement by NTX; (3) there was no MOR internalization during remission; (4) MORs immunoprecipitated from the spinal cord during remission had increased Ser(375) phosphorylation; and (5) electrophysiology recordings from dorsal root ganglion neurons collected during remission showed constitutive MOR inhibition of calcium channels.

Significance statement: Chronic pain causes extreme suffering to millions of people, but its mechanisms remain to be unraveled. Latent sensitization is a phenomenon studied in rodents that has many key features of chronic pain: it is initiated by a variety of noxious stimuli, has indefinite duration, and pain appears in episodes that can be triggered by stress. Here, we show that, during latent sensitization, there is a sustained state of pain hypersensitivity that is continuously suppressed by the activation of μ-, δ-, and κ-opioid receptors and by adrenergic α2A receptors in the spinal cord. Furthermore, we show that the activation of μ-opioid receptors is not due to the release of endogenous opioids, but rather to its ligand-independent constitutive activity.

Keywords: adrenergic receptor; constitutive activity; hyperalgesia; kappa-opioid receptor; latent sensitization; mu-opioid receptor.

Figure 1.

Latent sensitization in MOR KO mice and effect of receptor antagonists. MOR KO mice (MOR KO CFA, n = 8) or their WT littermates were injected with 5 μl CFA (WT CFA, n = 9) or 5 μl of saline (WT saline, n = 3) in the hindpaw. A, Responses to von Frey hairs were tested on the days indicated. Short-term responses to drugs are not shown. Holm-Sidak's post hoc tests: *p < 0.05, ***p < 0.001 compared with WT saline; †p < 0.05, ††p < 0.01, †††p < 0.001 compared with WT CFA. B, On day 34, the MOR inverse agonist NTX (3 mg/kg) was injected subcutaneously and von Frey responses tested every 20 min and at 24 h. C, On day 41, the MOR neutral antagonist 6β-naltrexol (10 mg/kg) was injected subcutaneously. D, On day 48, the α_2AR antagonist BRL44408 (1 mg/kg) was injected subcutaneously. E, On day 55, the DOR antagonist naltrindole (3 mg/kg) was injected subcutaneously. F, New groups of WT mice were injected with 5 μl CFA (n = 14) or saline (n = 6) in the hindpaw and on day 29 with naltrindole (3 mg/kg, s.c.). Holm-Sidak's post hoc tests (for B–F): *p < 0.05, **p < 0.01, ***p < 0.001 compared with 0 min.

Figure 2.

Effect of a MOR inverse agonist and neutral antagonist in rats with CFA-induced latent sensitization. Saline-injected controls. A, PWTs of rats (n = 6) implanted with intrathecal catheters and injected in the hindpaw with 50 μl of CFA subcutaneously. B, On day 30, the rats received intrathecal NTX (2.6 nmol), a MOR inverse agonist. C, On day 38, the MOR neutral antagonist 6β-naltrexol (8.7 nmol) was injected intrathecally. D, On day 45, NTX (2.6 nmol) plus 6β-naltrexol (8.7 nmol) were injected intrathecally. Holm-Sidak's post hoc tests of two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 compared with baseline (0 d or 0 min). E, PWTs of rats (n = 8) with intrathecal catheters that were injected in the hindpaw with 50 μl of saline subcutaneously. F, On day 32, the saline-injected rats (n = 8) received intrathecal NTX (2.6 nmol).

Figure 3.

Effect of the α_2AR antagonist BRL44408 in rats with CFA-induced latent sensitization. A, PWTs of rats (n = 9) implanted with intrathecal catheters and injected in the hindpaw with 50 μl of CFA subcutaneously. B, On day 30, 6 of the rats received 0.3 nmol BRL44408 (α_2AR antagonist) intrathecally. C, On day 40, all rats (n = 9) received 3 nmol BRL44408 intrathecally. Holm-Sidak's post hoc tests of two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 compared with baseline (0 d or 0 min). D, PWTs of rats (n = 8) with intrathecal catheters that were injected in the hindpaw with 50 μl of saline subcutaneously. E, On day 31, the saline-injected rats (n = 8) received intrathecal BRL-44408 (3 nmol).

Figure 4.

Effect of the DOR antagonist naltrindole in rats with CFA-induced latent sensitization. A, PWTs of rats (n = 11) injected in the hindpaw with 50 μl of CFA subcutaneously. B, On day 30, these rats received 2.2 nmol naltrindole (a DOR antagonist) intrathecally. C, On day 48, some of the rats (n = 8) received 1 mg/kg naltrindole subcutaneously. Holm-Sidak's post hoc tests of two-way ANOVA: *p < 0.05, ***p < 0.001 compared with baseline (0 d or 0 min). D, PWTs of rats (n = 8) without intrathecal catheters that were injected in the hindpaw with 50 μl of saline subcutaneously. E, On day 30, the saline-injected rats (n = 8) received naltrindole subcutaneously (1 mg/kg).

Figure 5.

Effect of the KOR antagonists nor-BNI and JDTic in rats and mice with CFA-induced latent sensitization. A, PWTs of rats (n = 5) injected in the hindpaw with 50 μl of CFA subcutaneously. B, On day 30, these rats received 1.3 nmol nor-BNI (a KOR antagonist) intrathecally, which reinstated hyperalgesia 12 h after the injection. C, PWTs of rats (n = 8) with intrathecal catheters that were injected in the hindpaw with 50 μl of saline subcutaneously. D, On day 32, the saline-injected rats (n = 8) received intrathecal nor-BNI (1.3 nmol). E, Mice received 5 μl of CFA (n = 6) or 5 μl of saline (n = 6) subcutaneously in the hindpaw. F, On day 43, the mice received an injection of the KOR antagonist JDTic (10 mg/kg, s.c.), which reinstated hyperalgesia after 12 h. Holm-Sidak's post hoc tests of two-way ANOVA: **p < 0.01, ***p < 0.001 compared with baseline (0 d or 0 min).

Figure 6.

Latent sensitization in pENK, pOMC, and pDYN KO mice. Mice were injected in the hindpaw with 5 μl of CFA and, upon return to baseline, they received NTX (3 mg/kg, s.c.). PWTs to von Frey filaments were obtained on the days indicated. A, pENK KO mice (n = 10) and their WT littermates (n = 5). B, NTX injected on day 28 to pENK KO and WT mice reinstated hyperalgesia. C, pOMC KO mice (n = 5) and their WT littermates (n = 4). D, NTX injected on day 56 to pOMC KO and WT mice reinstated hyperalgesia. E, pDYN KO mice (n = 8) and their WT littermates (n = 8). F, NTX injected on day 35 to pDYN KO and WT mice reinstated hyperalgesia. G, JDTic (10 mg/kg, s.c.) injected on day 58 to pDYN KO and WT mice reinstated hyperalgesia after 1 d. Holm-Sidak's post hoc tests of two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 compared with baseline (time 0 d or 0 min).

Figure 7.

Absence of MOR internalization in the spinal cord during the remission phase of latent sensitization. A, Rats (n = 6) implanted with intrathecal catheters were injected in the hindpaw with 50 μl of CFA subcutaneously and responses to von Frey hairs were tested. B, On day 28, the rats received NTX (1 mg/kg, s.c.) and PWTs were tested. Holm-Sidak's post hoc tests: *p < 0.05, **p < 0.01, ***p < 0.001 compared with baseline (time 0 d or 0 min). C, On day 30, rats injected with CFA (n = 4) or saline-injected controls (n = 4) received intrathecal injections of peptidase inhibitors (amastatin, phosphoramidon, and captopril, 100 nmol) and fixed 15 min later. MOR immunohistochemistry was performed in spinal segments C2, T10, and L4 to measure MOR internalization. D, Images taken from the L4 segment of a rat 30 d after injecting CFA in the hindpaw; MORs are not internalized. E, Images taken from the L4 segment of a rat 30 d after injecting saline in the hindpaw; MORs are not internalized. F, Images taken from the L4 segment of a naive rat; MORs are not internalized. Images are single optical sections taken with a 10× objective (main panels, voxel size 830 × 830 × 5983 nm, scale bar 100 μm) or a 63× objective (insets, voxel size 132 × 132 × 383 nm, scale bar 5 μm). G, Confocal stack of cell d (arrow) in D. H, Confocal stack of cell d (arrow) in E. I, Confocal stack of cell a (arrow) in F.

Figure 8.

Increased phosphorylation of MORs at Ser³⁷⁵ in latent sensitization. Rats were injected in one hindpaw with 50 μl of saline (n = 8) or CFA (n = 8). After 28 d, the rats were killed and the spinal cords collected. MORs were immunoprecipitated using a MOR antibody from Abcam (ab134054). A, Western blots were probed with antibodies to MOR (Neuromics, RA10104), p-Ser³⁷⁵-MOR (Neuromics RA18001, pMOR), and p-Tyr⁴¹⁶-Src (Cell Signaling Technology, pSFK). Two bands at ∼57 and 64 kDa appear in the MOR and pMOR blots and one band at 60 kDa in the pSFK blot. Data are representative from four of eight rats in each group. B, The ratio of the optical density of the pSer³⁷⁵-MOR blot and the MOR blot was obtained and normalized to saline. C, The ratio of the optical density of the pSFK blot and the MOR blot was obtained and normalized to saline. *p = 0.0141, ***p = 0.0009, unpaired t tests with Welch's correction.

Figure 9.

Ligand-independent and ligand-dependent inhibition of Cav channels in DRG neurons from mice with latent sensitization. Mice were injected in one hindpaw with 5 μl of saline or CFA. DRG neurons (L4–L5) were acutely isolated from the saline-injected or the CFA-injected mice after day 21. A, Whole-cell patch-clamp recordings were used to measure Cav currents evoked by a two-pulse protocol (bottom traces). P2 includes a high voltage prepulse from −80 mV to +80 mV to dissociate Gβγ subunits from the Cav channels, whereas P1 consists only of a test pulse from −80 mV to +10 mV. Constitutive inhibition of the Cav channels was indicated by a larger current with the P2 protocol (red traces) resulting in an increase in the P2/P1 ratio. B, P1/P2 ratio obtained from DRG of saline-injected and CFA-injected mice in the absence and presence of NTX (1 μm). Two-way ANOVA: NTX p = 0.023, CFA p = 0.15, interaction p = 0.0497. Holm-Sidak's post hoc tests: **p < 0.01 compared with baseline, †p < 0.05 as shown. C, P1/P2 ratio obtained from DRG of CFA-injected mice untreated (baseline) or in presence of NTX (1 μm), 6β-naltrexol (10 μm), and NTX plus 6β-naltrexol. One-way ANOVA: p < 0.0001. D, DAMGO was used to assess MOR ligand inhibition of Cav currents in saline-injected mice and mice with CFA-induced latent sensitization. A single depolarizing 100 ms pulse from −70 mV to +10 mV was used to evoke Cav currents. An exemplar recording shows the following: (1) the basal Cav current (initial), (2) DAMGO (1 μm) inhibition of Cav current, and (3) the current after DAMGO had been washed off. E, DAMGO inhibition of Cav currents expressed as a percentage of the total current in neurons from the ipsilateral L4–L6 DRG from CFA- and saline-injected mice; there was no effect of CFA. Holm-Sidak's post hoc tests: *p < 0.05 compared with baseline; ††p < 0.01, †††p < 0.001 compared with NTX. Numbers indicate the number of neurons recorded in each group (n).