Altered Chloride homeostasis underlies pain hypersensitivity after chronic morphine treatment

Opioids are widely used to treat acute and chronic pain, and morphine is recommended by the WHO as the analgesic of choice for cancer pain. However prolonged morphine exposure leads to a reduction of the antinociceptive effect (opioid tolerance) and to an increased pain sensitivity. Several lines of evidence suggest that these side effects share similar mechanisms with those underlying neuropathic pain, including microglia activation. We have recently shown that the release of BDNF by activated microglia following peripheral nerve injury, decreases KCC2 activity in the spinal dorsal horn (DH) and weakens Cl−-mediated inhibition through GABAA and glycine receptors (Coull et al. Nature, 2005). We tested the hypothesis that morphine may activate a similar cascade of events leading to a loss of inhibition in DH neurons. Adult rats, receiving either morphine (10 mg/kg s.c. twice a day) or saline, were tested for pain threshold every day before and after (1 h) the injection. This morphine treatment induced tolerance within 2 days and hyperalgesia within 5 days. The hyperalgesia, but not the morphine tolerance, was reversed by intrathecal (i.t.) administration of the anti-mac1 saporin-conjugated antibody (an immunotoxin targeted against microglia) or a TrkB blocking antibody, confirming involvement of both microglia and endogenous BDNF in the morphine-dependent hyperalgesia. After 7 days of treatment with morphine or saline, rats were sacrified and lamina I-II neurons were recorded in whole cell configuration by imposing a Cl- load (29 mM) to better measure any change in Cl- extrusion capacity. A depolarizing shift in EGABA was observed in lamina I neurons from morphine-treated rats (-42±1 mV, n=6) compared to controls (-50±2 mV, n=5, P<0.05) indicating a weaker Cl- extrusion capacity in these cells. An similar effect was also observed following an acute, prolonged incubation of spinal cord slices with morphine (1μM, >3 h; EGABA: -48±1 mV, n=6 in control, -42±1 mV, n=7 after morphine; P<0.05). No change in EGABA was observed either in the presence of opioid receptor antagonists or the TrkB blocking antibody, confirming the involvement of opioid receptors and BDNF in the morphine signalling pathway. Interestingly, morphine did not produce any change in EGABA in lamina II neurons. The impact of altered Cl- homeostasis in morphine-induced hyperalgesia was further investigated in vivo by i.t. administration of the carbonic anhydrase inhibitor acetazolamide (22.5μg). Acetazolamide, by minimizing the depolarizing bicarbonate-mediated component of GABAA/glycine currents, was sufficient to restore inhibition onto spinal DH neurons and reverse the morphine-dependent hyperalgesia. Our data suggest that microglia activation and subsequent BDNF release following morphine treatment may alter Cl- extrusion capacity of spinal lamina I neurons and increase pain hypersensitivity.