Blockade of neuronal dopamine D2 receptor attenuates morphine tolerance in mice spinal cord

Abstract

Tolerance induced by morphine remains a major unresolved problem and significantly limits its clinical use. Recent evidences have indicated that dopamine D2 receptor (D2DR) is likely to be involved in morphine-induced antinociceptive tolerance. However, its exact effect and molecular mechanism remain unknown. In this study we examined the effect of D2DR on morphine antinociceptive tolerance in mice spinal cord. Chronic morphine treatment significantly increased levels of D2DR in mice spinal dorsal horn. And the immunoreactivity of D2DR was newly expressed in neurons rather than astrocytes or microglia both in vivo and in vitro. Blockade of D2DR with its antagonist (sulpiride and L-741,626, i.t.) attenuated morphine antinociceptive tolerance without affecting basal pain perception. Sulpiride (i.t.) also down-regulated the expression of phosphorylation of NR1, PKC, MAPKs and suppressed the activation of astrocytes and microglia induced by chronic morphine administration. Particularly, D2DR was found to interact with μ opioid receptor (MOR) in neurons, and chronic morphine treatment enhanced the MOR/D2DR interactions. Sulpiride (i.t.) could disrupt the MOR/D2DR interactions and attenuate morphine tolerance, indicating that neuronal D2DR in the spinal cord may be involved in morphine tolerance possibly by interacting with MOR. These results may present new opportunities for the treatment and management of morphine-induced antinociceptive tolerance which often observed in clinic.

Figure 1. Chronic morphine treatment increases D2DR expression in mice spinal dorsal horn.

(A,B) Chronic morphine treatment increased the spinal D2DR protein expression after morphine treatment for 3d, 5d and 7d (n = 4, *P < 0.05, **P < 0.01, compared with control group). Representative western blot bands and a data summary were shown. (C,D) Confocal images showed that the mean fluorescence pixels of D2DR were increased after chronic morphine treatment for 7 days. Quantification of D2DR immunofluorescence was represented as mean fluorescence pixels in the superficial dorsal horns. The spinal cord tissues were taken 30 min after morphine injection on day 1, 3, 5, 7. (n = 4, 8 images per animal; *P < 0.05, **P < 0.01 compared with morphine group).

Figure 2. Distribution and co-localization of D2DR with NeuN, but not GFAP and IBA1 following chronic morphine exposure.

(A,D) Confocal images showed that D2DR (green) co-localized with the neurons (red) in the spinal cord (20X magnification and 40X magnification). (B,C) It was difficult to see that D2DR co-localized with GFAP (red) or IBA1 (red) (20X magnification). All spinal cords were collected on day 7, 30 min after chronic morphine exposure (n = 4).

Figure 3. Daily intrathecal injection of D2DR antagonist attenuates morphine-induced antinociceptive tolerance.

Mice were examined daily with tail-flick assay. Data were shown as percentage of maximal possible effect (% MPE). (A,B) D2DR antagonist sulpiride (1, 4 and 8 μg/10 μl, i.t.) attenuated the development of tolerance and exhibited no effects on the pain threshold in naive mice. (C,D) Sulpiride (1, 4 and 8 μg/10 μl, i.t.) partially reversed the established tolerance. (E,F) D2DR specific antagonist L-741,626 (1, 4 and 8 μg/10 μl, i.t.) also significantly attenuated morphine tolerance and exhibited no effects on the pain threshold in naive mice. (G,H) The specific receptor antagonist for dopamine D3 receptor SB 277011A (2.5, 5 and 10 μg/10 μl, i.t.) could not affect morphine tolerance in mice. (n = 12, *P < 0.05, **P < 0.01, compared with morphine group).

Figure 4. D2DR antagonist sulpiride attenuates morphine up-regulated phosphorylation of spinal NR1, PKC, ERK, p38 and JNK.

(A) Sulpiride (1, 4 and 8 μg/10 μl, i.t.) decreased the up-regulated expression of p-ERK, p-p38 and p-JNK in the spinal cord following chronic morphine treatment. (B) Sulpiride (1, 4 and 8 μg/10 μl, i.t.) inhibited morphine induced up-regulated p-NR1, p-PKC expression in the spinal cord. Representative western blot bands and a data summary are shown. All spinal cords were collected on day 7, 30 min after chronic morphine exposure (n = 3, *P < 0.05, **P < 0.01, compared with morphine group).

Figure 5. D2DR antagonist sulpiride suppresses morphine exposure induced activation of astrocytes and microglia in the spinal cord and decreases pro-inflammatory cytokines TNF-α and IL-1β.

(A,B) Confocal images and immunofluorescence showed that chronic morphine treatment activated the astrocytes and microglia in the spinal dorsal horn. Sulpiride (8 μg/10 μl, i.t.) inhibited the activation of the glias in the spinal cord (10X and 20X magnification). Quantification of GFAP and IBA1 immunofluorescence were represented as mean fluorescence pixels in the superficial dorsal horns. (C,D) Western blot showed that sulpiride (1, 4 and 8 μg/10 μl, i.t.) decreased the up-regulated expression of GFAP and IBA1. (E,F) Western blot analysis data showed that the up-regulated expression of the pro-inflammatory cytokines TNF-α and IL-1β were decreased by sulpiride (1, 4 and 8 μg/10 μl, i.t.) (n = 3, *P < 0.05, **P < 0.01, compared with morphine group). Representative western blot bands and a data summary are shown. All spinal cords were collected on day 7, 30 min after chronic morphine exposure (n = 4, *P < 0.05, **P < 0.01, compared with morphine group).

Figure 6. Chronic morphine treatment increases the MOR/D2DR interactions in the spinal cord dorsal horn and spinal D2DR inhibition with D2DR antagonist disrupts these interactions.

(A) Double immunofluorescence staining showed that MOR (green) and D2DR (red) were co-localized in the mice spinal cord (20X magnification). Chronic morphine treatment increased the co-localization of MOR and D2DR in the spinal cord, and D2DR antagonist sulpiride (8 μg/10 μl, i.t.) reduced the increased co-expression of D2DR with MOR (n = 4). (B) Co-IP experiments showed that D2DR could interact with MOR, and the MOR/D2DR interactions were increased in the spinal dorsal horn after chronic morphine treatment for 7 days while D2DR antagonist sulpiride (8 μg/10 μl, i.t.) disrupted the interactions of the MOR/D2DR (n = 3).

Figure 7. Chronic morphine treatment increases the MOR/D2DR interactions in primary spinal cord dorsal horn neurons.

(A) Chronic morphine treatment increased the mean fluorescence density of D2DR in primary spinal cord dorsal horn neurons. Quantification of D2DR immunofluorescence was represented as mean fluorescence pixels in the superficial dorsal horns (n = 4, *P < 0.05, **P < 0.01, compared with morphine group). (B) Confocal images and immunofluorescence analysis data showed that D2DR (green) was co-localized with NeuN (red) and this co-expression was increased after chronic morphine treatment in primary spinal cord dorsal horn neurons (n = 4). (C) Confocal images and immunofluorescence analysis data showed that D2DR (green) was co-localized with MOR (red), and this co-expression was increased after chronic morphine treatment (n = 4).