Morphine tolerance in the mouse peripheral nervous system

Emma E. Johnson & Mark Connor. Pain Management Research Institute, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065 Australia.

Analgesic tolerance produced by chronic morphine treatment (CMT) is accompanied by m -opioid receptor (MOP) dependent changes in neuronal signalling in the central nervous system. However, the role of changes in MOP function in analgesic tolerance is obscure. There is little information about MOP signalling in the peripheral nervous system of morphine tolerant animals, although one study reported a down regulation of MOP mRNA following intermittent morphine treatment (Meuser et al., 2003).

We investigated the effects of chronic morphine treatment on the MOP-mediated responses of acutely isolated mouse trigeminal sensory neurons. We mostly examined small diameter (<20 µm) neurons that were shown to express proteins consistent with a nociceptive phenotype, particularly TRPV1 (Borgland et al., 2001). We made standard whole cell voltage clamp recordings of voltage gated calcium channels ( ICa, Borgland et al., 2001) and employed real time RT-PCR to examine changes in MOP mRNA expression.

Male C57 mice (20-30g) littermate pairs received subcutaneous injections of sustained release emulsion containing morphine base (300mg kg-1) or emulsion alone (VT) every second day for 5 days. Injections were made under light halothane anaesthesia (Connor et al., 1999). Mice were sacrificed on day 6 or 7 and trigeminal neurons prepared as outlined in Borgland et al., 2001. All data is mean ± s.e.m. mRNA expression was assessed by quantitative RT-PCR using co-amplification of hypoxanthine phosphoribosyltransferase (HPRT) as an internal control. MOP primers were 5’-TTCTGCATCCCAACTTCCTC-3’ (forward) and 5’-CTGACAGCAACCTGATTCCA-3’ (reverse).

In trigeminal neurons from VT animals, morphine inhibited ICa with a pEC50 of -6.20 ± 0.20M to a maximum of 44 ± 5%. In CMT mice, the pEC50 was -6.25 ± 0.15M and the maximum inhibition was reduced to 24 ± 2% ( P<0.0001, effect of treatment, 2 way ANOVA). The number of cells responding to morphine in CMT (132/163) and VT (125/155) animals was unchanged. The inhibition of ICa in trigeminal neurons by a maximally effective concentration of the MOP agonist DAMGO (10µM) was also significantly reduced (inhibition 49 ± 5% VT vs 28 ± 4% CMT mice, P<0.0001, t -test). The inhibition of ICa by maximally effective concentrations of nociceptin (300nM - 1µM) was unchanged in neurons from CMT animals compared to VT animals (inhibition 43 ± 6% vs 37 ± 3%, respectively, P=0.258, t-test). There was no significant change in expression levels of MOP mRNA relative to HPRT mRNA produced by CMT (CMT 0.44 ± 0.12% of HPRT, n=10 vs VT 0.48 ± 0.15% of HPRT, n=13).

These results indicate that analgesic tolerance to morphine produced by continuous morphine administration is accompanied by a reduction in receptor coupling in trigeminal sensory neurons but that these changes in receptor signalling are not accompanied by changes in MOP mRNA expression.

Borgland et al., 2001, J Physiol 536:35-47.
Connor et al., 1999, Br J Pharmacol 128:1561-9.
Meuser et al., 2003, Anesth Analg 97:1458-63.