Characterization of functional µ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study.

BACKGROUND AND PURPOSE: Regulation of µ receptor dynamics such as its trafficking is a possible mechanism underlying opioid tolerance that contributes to inefficient recycling of opioid responses. We aimed to characterize the functional turnover of µ receptors in the noradrenergic nucleus locus coeruleus (LC). EXPERIMENTAL APPROACH: We measured opioid effect by single-unit extracellular recordings of LC neurons from rat brain slices. Immunocytochemical techniques were used to evaluate µ receptor trafficking. KEY RESULTS: After near-complete, irreversible µ receptor inactivation with ß-funaltrexamine (ß-FNA), opioid effect spontaneously recovered in a rapid and efficacious manner. In contrast, α2 -adrenoceptor-mediated effect hardly recovered after receptor inactivation with the irreversible antagonist EEDQ. When the recovery of opioid effect was tested after various inactivating time schedules, we found that the longer the ß-FNA pre-exposure, the less efficient and slower the functional µ receptor turnover became. Interestingly, µ receptor turnover was slower when ß-FNA challenge was repeated in the same cell, indicating constitutive µ receptor recycling by trafficking from a depletable pool. Double immunocytochemistry confirmed the constitutive nature of µ receptor trafficking from a cytoplasmic compartment. The µ receptor turnover was slowed down when LC neuron calcium- or firing-dependent processes were prevented or vesicular protein trafficking was blocked by a low temperature or transport inhibitor. CONCLUSIONS AND IMPLICATIONS: Constitutive trafficking of µ receptors from a depletable intracellular pool (endosome) may account for its rapid and efficient functional turnover in the LC. A finely-tuned regulation of µ receptor trafficking and endosomes could explain neuroadaptive plasticity to opioids in the LC.

Inhibition of neuronal nitric oxide synthase attenuates the development of morphine tolerance in rats.

Our previous results have shown the involvement of nitric oxide in acute opioid desensitization of mu-opioid receptors in vitro. In the present study, we investigated the effect of repeated administration of 7-nitroindazole (7-NI; 30 mg/kg/12 h, i.p., 3 days), an inhibitor of neuronal nitric oxide synthase in vivo, on mu-opioid receptor tolerance induced by subchronic treatment with morphine in rats. The inhibitory effect of the opioid agonist Met5-enkephalin (ME) on the cell firing rate was evaluated by single-unit extracellular recordings of noradrenergic neurons in the locus coeruleus from brain slices, and the antinociceptive effect of morphine was measured by tail-flick techniques. In morphine-treated animals, concentration-effect curves for ME in the locus coeruleus were shifted by 5-fold to the right as compared to those in sham-treated animals, which confirmed the induction of mu-opioid receptor tolerance. However, tolerance to ME in morphine-treated rats was fully prevented by co-administration of 7-NI when compared to the vehicle-morphine group. Likewise, the antinociceptive effect of morphine was reduced in morphine-treated animals as compared to the sham group, whereas the antinociceptive tolerance was partially prevented by co-administration of 7-NI in morphine-treated rats (when compared to the vehicle-morphine group). Finally, 7-NI administration in sham-treated rats failed to change the effect induced by ME on the locus coeruleus or by morphine in the tail-flick test as compared to vehicle groups. These results demonstrate that subchronic administration of a neuronal inhibitor of nitric oxide synthase attenuates the development of morphine tolerance to the cellular and analgesic effects of mu-opioid receptor agonists.

Morphine withdrawal is modified in pituitary adenylate cyclase-activating polypeptide type I-receptor-deficient mice.

The pituitary adenylate cyclase-activating polypeptide type I-receptor (PAC1) is a G-protein-coupled receptor that is widely expressed in neurons of the central and peripheral nervous system. The strong expression of PAC1 in the second sensory neuron as well as in brainstem regions such as the locus coeruleus prompted us to elucidate the potential in vivo role of PAC1-mediated signalling in pain perception and opioid addiction using a PAC1-deficient mouse line. We observed a selective involvement of PAC1 in the mediation of visceral pain. While there was no impairment in acute somatic pain perception, PAC1-mutants exhibited a dramatically decreased response in the abdominal writhing test. These data in concert with data from the literature implicate PAC1 in the mediation of visceral and chronic pain. In addition, we observed that PAC1 did not influence the motivational aspects of opioid addictive properties, since morphine-induced rewarding effects and sensitization to locomotor responses were completely maintained in PAC1-deficient mice. However, there was a dramatic increase in physical withdrawal signs after naloxone-precipitated morphine withdrawal in PAC1 mutants. At the cellular level, electrophysiological examinations in locus coeruleus neurons from morphine-dependent wild-type and PAC1-deficient mice did not reveal any differences in firing rates. These data therefore suggested that most likely disruption of PAC1-mediated signalling in afferents towards the locus coeruleus but not within the intrinsic locus coeruleus system led to the enhancement of somatic withdrawal signs.