cAMP-mediated mechanisms for pain sensitization during opioid withdrawal

Abstract

Chronic opioid-induced drug dependence and withdrawal syndrome after opioid cessation remain a severe obstacle in clinical treatment of chronic pain and opioid drug addiction. One of the key symptoms during opioid withdrawal is a state of sensitized pain. The most significant molecular adaptation induced by chronic opioids in the brain is upregulation of the cAMP-signaling pathway. Although the cAMP system is known to have multiple effects on central neuron functions, how its upregulation mediates behavioral opioid dependence and withdrawal-induced pain in vivo remains unclear. In this study, we demonstrate that withdrawal from chronic morphine significantly upregulates the mRNA level of adenylyl cyclase (AC) VI and VIII isoforms and immunoreactivity of ACV/VI in the nucleus raphe magnus (NRM), a brainstem site critically involved in opioid modulation of pain. In cellular studies of NRM neurons containing mu-opioid receptors, we show that morphine withdrawal significantly increases glutamate synaptic transmission via a presynaptic mechanism mediated by an upregulated cAMP pathway. Morphine withdrawal also enhances the hyperpolarization-activated current in these neurons by increased intracellular cAMP. Both of the withdrawal-induced cAMP actions increase the excitability of these mu-receptor-containing neurons, which are thought to facilitate spinal pain transmission. Furthermore, in morphine-dependent rats in vivo, blocking the cAMP pathway significantly reduces withdrawal-induced pain sensitization. These results illustrate neurobiological mechanisms for the cAMP-mediated withdrawal pain and provide potential therapeutic targets for the treatment of opioid dependence and withdrawal-related problems.

Figure 1.

Morphine withdrawal upregulates AC isoforms in the NRM area. A, B, Representative amplification plots of real-time PCRs for ACVI (A) and ACVIII (B). Con, Control. C, Fold changes in the mRNA of four AC isomers during morphine withdrawal. Error bars indicate SDs (n = 4 rats in placebo control and in withdrawal groups). D, Representative lanes of Western blots for ACI, ACV/VI, ACVIII, and corresponding β-actin from control and withdrawn rats. E, Group data of Western blots showing the percentage of changes in withdrawn rats from controls after normalization to β-actin. ^*p < 0.05; ^**p < 0.01. Error bars indicate SEs (n = 6-8 rats in each group).

Figure 2.

Activation of cAMP pathway presynaptically increases glutamate synaptic transmission in μ-receptor-containing NRM cells from control rats. A-C, Superimposed glutamatergic EPSCs (A) and EPSC pairs normalized to the first EPSC (B, C) in control and in forskolin (10 μm) from the same cell (B) or recorded with an 8-br-cAMP-filled (1 mm) pipette (C). D, Cumulative frequency distribution of glutamate mEPSCs in control, in naloxone (1 μm), and in naloxone plus forskolin. cAMP, 8-Br-cAMP; Con, control; Forsk, forskolin; Nal, naloxone.

Figure 3.

Naloxone-precipitated morphine withdrawal presynaptically increases glutamate synaptic transmission by cAMP upregulation. A, B, Glutamate EPSCs in control, in naloxone, and in naloxone plus forskolin from a μ-receptor-containing cell (A) and from a μ-receptor-lacking cell (B) of a morphine-dependent rat. Abbreviations are as in Figure 2. The following results were obtained from μ-receptor-containing cells. C, Normalized EPSC pairs in control and in naloxone in a morphine-dependent cell. D, Percentage increase by forskolin of the EPSC amplitudes in control (open circles; n = 12) and in withdrawn cells (filled circles; n = 8). ^*p < 0.05; ^**p < 0.01. E, F, Frequency (E) and amplitude (F) distributions of the mEPSCs in control, in naloxone, and in naloxone plus forskolin from the same morphine-dependent cell. G-I, Glutamate EPSCs (G, superimposed) and normalized EPSC pairs (H, I, staggered) in control and in naloxone from morphine-dependent cells treated with MDL12330a (MDL; 100 μm) or H89 (10 μm).

Figure 4.

Morphine abstinence-induced spontaneous withdrawal increases glutamate synaptic transmission by cAMP upregulation in μ-receptor-containing cells. A, Representative EPSCs in neurons from a control slice and from a spontaneously withdrawn slice. B, Group data of EPSC amplitudes from the same two groups as in A. Numbers in the columns indicate the cell numbers. C, Normalized EPSC pairs in neurons from a control slice, a withdrawn slice, and a withdrawn slice treated with MDL12330a (MDL). D, Group data of PPRs from the same three groups as in C. ^*p < 0.05; ^**p < 0.01. Withdr., Withdrawal. Error bars represent SEM.

Figure 5.

Morphine withdrawal enhances the I_h by cAMP upregulation in μ-receptor-containing cells. A, Current traces (aligned on the instantaneous current level) recorded with a control and an 8-br-cAMP-filled (1 mm) pipette during a voltage step (-50 to -80 mV). B, Averaged amplitudes of I_h tails recorded with control (n = 12) and 8-br-cAMP-filled pipettes (n = 15). C-E, I_h activation curves in control (V_1/2 = -76.1 mV; n = 12) and in 8-br-cAMP (V_1/2 = -67.4 mV; n = 15; p < 0.001) (C), in control (same as in C), in morphine-withdrawn cells (V_1/2 = -72.6 mV; n = 14; p < 0.01) (D), in withdrawn cells (same as in D), and in another group of withdrawn cells treated with MDL12330a (MDL) (V_1/2 = -75.8 mV; n = 18; p < 0.01) (E). Con, Control. Error bars represent SEM.

Figure 6.

Inhibition of I_h channels reduces evoked firing of μ-receptor-containing cells in withdrawn slices. Evoked firing of action potentials in cells from a control slice, a withdrawn slice, and a withdrawn slice (Withdr.) treated with ZD7288 (10 μm) is shown.

Figure 7.

Inhibition of the cAMP pathway attenuates morphine withdrawal-induced pain sensitization. A-E, Plots of tail-flick latencies in placebo-treated rats (open circles) and in morphine-treated rats (filled symbols). Naloxone-induced withdrawal hyperalgesia (filled circles) was reduced by NRM microinjection of AP-5 and CNQX (A; filled squares; n = 4 rats), by ZD7288 (B; filled squares; n = 5), by MDL12330a (MDL) (C; filled triangles; n = 6), by H89 (D; filled diamonds; n = 6), and by Rp-cAMP (E; filled diamonds; n = 5). ^*p < 0.05; ^**p < 0.01 (ANOVA for repeated measures and the Tukey-Kramer test of post hoc analysis). F, Same microinjections of Rp-cAMP as in E, with cannula placement dorsal to the NRM (n = 3 rats). Error bars represent SEM.

Figure 8.

Placements of NRM microinjections. Photomicrographs show representative microinjection sites within the NRM area (A) and dorsal to the NRM (B). Scale bar, 1 mm.