Cingulate NMDA NR2B receptors contribute to morphine-induced analgesic tolerance

Abstract

Morphine is widely used to treat chronic pain, however its utility is hindered by the development of tolerance to its analgesic effects. While N-methyl-D-aspartate (NMDA) receptors are known to play roles in morphine tolerance and dependence, less is known about the roles of individual NMDA receptor subtypes. In this study, Ro 256981, an antagonist of the NMDA receptor subunit NR2B, was used to reduce the expression of analgesic tolerance to morphine. The mechanisms altered with chronic drug use share similarities with those underlying the establishment of long-tem potentiation (LTP) and behavioral memory. Since NMDA NR2B receptors in the anterior cingulate cortex (ACC) play roles in the establishment of LTP and fear memory, we explored their role in changes that occur in this region after chronic morphine. Both systemic and intra-ACC inhibition of NR2B in morphine-tolerant animals inhibited the expression of analgesic tolerance. Electrophysiological recordings revealed a significant increase in the NR2B component of NMDA receptor mediated excitatory postsynaptic currents (EPSCs), at both synaptic and extra-synaptic sites. However, there was no change in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated EPSCs. This study suggests that selective inhibition of NMDA NR2B receptors may prove useful in combating the development of analgesic tolerance to morphine and proposes a novel role for the ACC in opioid tolerance and morphine induced changes in synaptic plasticity.

Figure 1

The role of NR2B receptors in acute morphine-induced analgesia and analgesic tolerance. (A) Morphine analgesia was greater in mice pretreated with Ro25-6981 (5 mg/kg, i.p., n = 5) compared to mice pretreated with saline (n = 4) beginning 90 min after injection. There was a significant effect of treatment (p < 0.05) with significant differences occurring at 90, 120, 150 and 180 minutes after injection (p < 0.05 for all). * represents a significant difference from saline injected mice. (B) Response latencies were significantly increased by Ro 256981 injection (5 mg/kg, i.p., n = 8). Day 7 vs. Day 8, p < 0.001). * represents a significant difference from responses on Day 6 and 7. (C) Daily co-administration of Ro 256981 (5 mg/kg, i.p.) with morphine (10 mg/kg, s.c.) attenuates opioid analgesic tolerance (p < 0.001) compared to mice receiving morphine (n = 7) or saline (n = 5). There was no difference between morphine treated and Ro256981+morphine treated mice on Day 1 (p = 0.74) but a significant difference arose on Day 2 (p < 0.001) and persisted during Days 3, 4, 6 and 7 (p < 0.001, p < 0.01, p < 0.05, p < 0.05 respectively).

Figure 2

Antagonism of NR2B receptors in the ACC can reverse opioid analgesic tolerance compared to mice receiving saline in the ACC. (A) Analgesic tolerance in ACC cannulated mice developed over the seven-day test period. (B) Bilateral microinjection of Ro 256981 (1 μg in 1 μl, bilaterally, n = 7; filled circles) into the ACC significantly increased response latencies compared to mice receiving saline injections (n = 5; open circles) and to responses on Day 7.

Figure 3

No change in mEPSCs in morphine treated mice. (A) Representative traces showing mEPSCs in saline (upper) or morphine treated mice (lower). (B) Pooled data showed that there is no difference in mEPSC frequency in saline (n = 16) and morphine treated groups (n = 16). (C) No difference in amplitude of mEPSCs in the ACC of saline (n = 16) and morphine treated groups (n = 16).

Figure 4

Enhanced NR2B NMDA receptor function in morphine-treated mice. (A) Representative traces showing that the NR2B component was revealed by application of Ro256981 (3 μM) in saline (upper) or morphine treated mice (lower). (B) Pooled data showed a significant increase in the NR2B component in morphine treated mice (n = 8) compared with that of saline group (n = 6). (C) A short train of stimuli (200 Hz, 7 pulses) induced lager NMDA EPSCs. The NR2B component, including extrasynaptic NR2B, was revealed by application of Ro 256981 (3 μM) in saline (upper) or morphine treated mice (lower). (D) Pooled data showed a significant increase in the NR2B component of short train-induced NMDA current in morphine treated mice (n = 7) compared with that of the saline group (n = 7).

Figure 5

Enhanced cingulate LTP in morphine treated mice. (A) LTP was induced with the spike-timing protocol in ACC neurons in wild-type mice (n = 7). (B) Enhanced potentiation was observed in morphine treated mice (n = 8). The insets show averages of six EPSCs at baseline responses (1) and 30 min (2) after LTP induction (arrow). The dashed line indicates the mean basal synaptic response. (C) There was a significant increase in LTP in morphine treated mice compared to saline treated group.