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. 2010 Mar 31;30(13):4735-45.
doi: 10.1523/JNEUROSCI.5968-09.2010.

Synaptic mechanism for functional synergism between delta- and mu-opioid receptors

Zhi Zhang  1 , Zhizhong Z Pan
Affiliations

Synaptic mechanism for functional synergism between delta- and mu-opioid receptors

Zhi Zhang et al. J Neurosci. .

Abstract

By sustained activation of mu-opioid receptors (MORs), chronic opioids cause analgesic tolerance, physical dependence, and opioid addiction, common clinical problems for which an effective treatment is still lacking. Chronic opioids recruit delta-opioid receptors (DORs) to plasma membrane through exocytotic trafficking, but the role of this new DOR and its interaction with existing MOR in brain functions and in these clinical problems remain largely unknown. In this study, we investigated the mechanisms underlying synaptic and behavioral actions of chronic morphine-induced DORs and their interaction with MORs in nucleus raphe magnus (NRM) neurons important for opioid analgesia. We found that the emerged DOR inhibited GABAergic IPSCs through both the phospholipase A(2) (PLA(2)) and cAMP/protein kinase A (PKA) signaling pathways. MOR inhibition of IPSCs, normally mediated predominantly by the PLA(2) pathway, was additionally mediated by the cAMP/PKA pathway, with MOR potency significantly increased after chronic morphine treatment. Isobologram analysis revealed a synergistic DOR-MOR interaction in their IPSC inhibition, which was dependent on upregulated activities of both the PLA(2) and cAMP/PKA pathways. Furthermore, DOR and MOR agonists microinjected into the NRM in vivo also produced a PLA(2)-dependent synergism in their antinociceptive effects. These findings suggest that the cAMP/PKA pathway, upregulated by chronic opioids, becomes more important in the mechanisms of both MOR and DOR inhibition of GABA synaptic transmission after chronic opioid exposure, and DORs and MORs are synergic both synaptically and behaviorally in producing analgesic effects in a PLA(2)-dependent fashion, supporting the potential therapeutic use of DOR agonists in pain management under chronic opioid conditions.

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Figures

Figure 1.
Figure 1.
Chronic morphine-induced inhibition of GABA synaptic currents by DORs involves both the PLA2 and cAMP/PKA pathways. A, Representative GABA IPSCs (top) before (control) and during application of the DOR agonist deltorphin (1 μm) in a brainstem neuron of the NRM from a morphine-treated rat. Also shown (bottom) is a dose–response curve for the deltorphin inhibition (n = 6–7 cells for each data point). Data points were fitted by a logistic function to determine the EC50 value. B, Representative IPSC pairs (top) and summarized group data (bottom) in neurons from saline-treated rats and from morphine-treated rats without and with addition of the DOR antagonist naltriben (10 μm), showing the effects of deltorphin on the PPR. The numbers in columns denote cell numbers for that group. C, Deltorphin effects on GABA IPSCs in neurons of the morphine group in the presence of the PLA2 inhibitor AACOCF3 (10 μm) or the presynaptic potassium channel blocker 4-AP (100 μm). D, Deltorphin effects on IPSCs in the morphine group in the presence of the PKA inhibitor H89 (1 μm) or in combination of H89 and AACOCF3 or another PKA inhibitor KT5720 (1 μm) and AACOCF3. *p < 0.05, **p < 0.01. Calibration: A, C, D, 100 pA and 50 ms; B, 100 ms.
Figure 2.
Figure 2.
Chronic morphine increases the potency of μ-opioid receptor (MOR) agonist. A, GABA IPSCs in the absence and presence of the MOR agonist DAMGO (1 μm) in neurons of saline and morphine groups. B, C, Summarized data of the DAMGO inhibition expressed as percentage of control (B) or current amplitude (C). D, Dose–response relationship for the IPSC-inhibiting effect of DAMGO in neurons from the saline-treated rats (open circles) and from the morphine-treated rats (filled circles). n = 6–7 cells for each data point. **p < 0.01. Calibration: A, 100 pA and 50 ms.
Figure 3.
Figure 3.
Chronic morphine recruits the cAMP/PKA pathway for MOR inhibition of GABA synaptic currents. A, B, Representative IPSCs (top) and group data (bottom) of the DAMGO inhibition in control and in the presence of AACOCF3 or 4-AP in neurons of the saline group (A) and the morphine group (B). C, DAMGO effects on IPSCs in the presence of H89 from neurons of the saline group and of the morphine group without or with addition of AACOCF3 or 4-AP. D, DAMGO effects in similar experiment groups but in the presence of the AC inhibitor SQ22536 (50 μm). E, Summarized data of the DAMGO effects on IPSCs in the saline group (open columns) and in the morphine group (filled columns). *p < 0.05, **p < 0.01. Calibration: 100 pA and 50 ms. KT, KT5720.
Figure 4.
Figure 4.
Activation of the cAMP/PKA pathway induces additional MOR inhibition of GABA synaptic currents. A, Effect of the AC activator forskolin (10 μm) on IPSCs in a naive neuron (normal) and a neuron in a naive slice treated with forskolin and AACOCF3. B, Summarized data of normalized IPSC amplitude after treatment of naive slices with forskolin, with the cAMP analog 8-bromo-cAMP (100 μm), or with AACOCF3 plus forskolin or cAMP before and after addition of DAMGO. C, D, IPSC pairs and summarized data of effects of forskolin and 8-bromo-cAMP on PPR in naive slices (normal, C), and effects of DAMGO on PPR in naive slices treated with forskolin plus AACOCF3 (D). **p < 0.01. cAMP, 8-bromo-cAMP; Forsk, Forskolin. Calibration: 100 pA and 50 ms.
Figure 5.
Figure 5.
Chronic morphine upregulates PLA2 activity. A, Dose–response relationship for AACOCF3 antagonism of DAMGO-mediated inhibition of IPSCs in neurons of the saline group (open circles) and of the morphine group (filled circles). n = 6–12 cells for each data point. B, Similar experiments of AACOCF3 antagonism but in the presence of KT5720 for both the saline group (n = 8–11) and the morphine group (n = 5 for each AACOCF3 dose), showing AACOCF3 antagonism of PLA2-mediated DAMGO inhibition (normalized). C, D, Representative lanes and group data of Western blots for cPLA2 and phosphorylated cPLA2 as well as for β-actin in NRM tissues from placebo (n = 6) and morphine pellet-implanted (n = 9) rats (C) and from saline (n = 5)- and morphine (n = 5)-injected rats (D). Data of percentage changes were normalized to the expression of β-actin. The molecular mass was 95 kDa for cPLA2 and phosphorylated cPLA2. *p < 0.05, **p < 0.01.
Figure 6.
Figure 6.
Combinations of MOR and DOR agonists produce synaptic synergism. A, Dose-dependent inhibition of IPSCs by a mixture of DAMGO and deltorphin at a fixed ratio of 9:1 in neurons from morphine-treated rats. n = 5–7 cells for each data point. B, Normalized data of dose-dependent IPSC inhibition by DAMGO, deltorphin, and the mixture (experimental), fitted by a regression line, and a calculated theoretical line of additivity in neurons from the morphine group. n = 5–16 cells for each data point. C, Isobolograph of EC50 values and their 95% confidence limits (vertical and horizontal bars) for theoretical additivity and for experimentally derived data (squares) with a DAMGO:deltorphin mixture of 9:1 and 1:1 ratios. For comparison, theoretical EC50 values for simple additivity at both 9:1 and 1:1 mixture ratios are also depicted as cross bars (95% confidence limits) without symbols on the theoretical line. D, Dose–response curves for IPSC inhibition by DAMGO and by the mixture (9:1) plus AACOCF3 (1 μm). n = 5–12 cells for each data point. E, F, Similar dose-dependent data of regression lines and a theoretical line (E) and isobolograph of EC50 values (F) except that the experimental data of the mixture (9:1) were obtained in the presence of AACOCF3 (1 μm). n = 5–16 cells for each data point. G, Effects of lower doses of the AC inhibitor SQ22536 on DAMGO inhibition of IPSCs in neurons of the morphine group. H, Antagonizing effect of 1 μm SQ22536 on the IPSC inhibition induced by the mixture at 10 nm (n = 10) and 30 nm (n = 7). n = 5–16 for other data points. *p < 0.05, **p < 0.01. Calibration: 100 pA and 50 ms.
Figure 7.
Figure 7.
Mixture of MOR and DOR agonists produces behavioral synergism. A, Effects of a single microinjection (arrow) of saline (n = 4 rats), DAMGO (10 ng, n = 5), or deltorphin (1 μg, n = 5) into the brainstem NRM on pain threshold measured by the tail-flick test in morphine-treated rats in vivo. B, Dose-dependent effects of microinjected DAMGO and deltorphin on the pain threshold in morphine-treated rats. n = 4–6 rats. C, Behavioral effects of NRM-microinjected 1:1 mixture composed of subthreshold doses of DAMGO (3 ng) and deltorphin (3 ng) with premicroinjection (1 h before) of saline in placebo-treated (n = 4 rats) and morphine-treated rats (n = 6). D, Behavioral effects of premicroinjections of 0.2 and 2 μg AACOCF3 on the mixture-induced antinociception in morphine-treated rats. n = 6 rats for each group. E, Behavioral effects on pain threshold produced by NRM microinjections of 6 ng DAMGO and by the mixture (6 ng at 1:1 ratio) with premicroinjection of saline or AACOCF3 (0.2 μg and 0.2 μg). F, Behavioral effects of NRM microinjection of saline (n = 4 rats), deltorphin (3 ng, n = 4), DAMGO (6 ng, n = 5), and the mixture (3 ng DAMGO plus 3 ng deltorphin, n = 6) in rats treated with twice daily injections of morphine. * and # p < 0.05, **p < 0.01.
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