Glial TLR4 signaling does not contribute to opioid-induced depression of respiration

Opioids activate glia in the central nervous system in part by activating the toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) complex. TLR4/MD2-mediated activation of glia by opioids compromises their analgesic actions. Glial activation is also hypothesized as pivotal in opioid-mediated reward and tolerance and as a contributor to opioid-mediated respiratory depression. We tested the contribution of TLR4 to opioid-induced respiratory depression using rhythmically active medullary slices that contain the pre-Botzinger Complex (preBotC, an important site of respiratory rhythm generation) and adult rats in vivo. Injection with DAMGO (mu-opioid receptor agonist; 50 mu M) or bath application of DAMGO (500 nM) or fentanyl (1 mu M) slowed frequency recorded from XII nerves to 40%, 40%, or 50% of control, respectively. This DAMGO-mediated frequency inhibition was unaffected by preapplication of lipopolysaccharides from Rhodobacter sphaeroides (a TLR4 antagonist, 2,000 ng/ml) or (+) naloxone (1-10 mu M, a TLR4-antagonist). Bath application of (-) naloxone (500 nM; a TLR4 and mu-opioid antagonist), however, rapidly reversed the opioid-mediated frequency decrease. We also compared the opioid-induced respiratory depression in slices in vitro in the absence and presence of bath-applied minocycline (an inhibitor of microglial activation) and in slices prepared from mice injected (ip) 18 h earlier with minocycline or saline. Minocycline had no effect on respiratory depression in vitro. Finally, the respiratory depression evoked in anesthetized rats by tail vein infusion of fentanyl was unaffected by subsequent injection of (+) naloxone, but completely reversed by (-) naloxone. These data indicate that neither activation of microglia in preBotC nor TLR4/MD2-activation contribute to opioid-induced respiratory depression.