Opioids activate the descending antinociceptive pathway from the ventrolateral periaqueductal gray (vlPAG) by both pre-and postsynaptic inhibition of tonically active GABAergic neurons (i.e., disinhibition). Previous research has shown that short-term desensitization of postsynaptic -opioid receptors (MOPrs) in the vlPAG is increased with the development of opioid tolerance. Given that pre-and postsynaptic MOPrs are coupled to different signaling mechanisms, the present study tested the hypothesis that short-term desensitization of presynaptic MOPrs also contributes to opioid tolerance. Twice-daily injections of morphine (5 mg/kg s.c.) for 2 days caused a rightward shift in the morphine dose-response curve on the hot plate test (D 50 ϭ 9.9 mg/kg) compared with saline-pretreated (5.3 mg/kg) male Sprague-Dawley rats. In vitro whole-cell patch-clamp recordings from vlPAG slices revealed that inhibition of evoked inhibitory postsynaptic currents (eIPSCs) by the MOPrselective agonist [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin was decreased in morphine-tolerant (EC 50 ϭ 708 nM) compared with saline-pretreated rats (EC 50 ϭ 163 nM). However, shortterm desensitization of MOPr inhibition of eIPSCs was not observed in either saline-or morphine-pretreated rats. Reducing the number of available MOPrs with the irreversible opioid receptor antagonist, ␤-chlornaltrexamine decreased maximal MOPr inhibition with no evidence of desensitization, indicating that the lack of observed desensitization is not caused by receptor reserve. These results demonstrate that tolerance to the antinociceptive effect of morphine is associated with a decrease in presynaptic MOPr sensitivity or coupling to effectors, but this change is independent of short-term MOPr desensitization.

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Mu‐opioid receptor (MOP) internalization has been proposed as a cellular mechanism of morphine tolerance. In order to test this hypothesis in vivo, the fluorescent MOP agonist, DERM‐A594, was microinjected into the periaqueductal gray (vlPAG). Rats were pretreated with either saline (0.4 μl) or morphine (5 μg/0.4 μl) 2X/day for 2 days. On day 3, rats were given either a saline or morphine challenge 30 min prior to DERM‐A594 (300 ng/0.4 μl). Intracellular DERM‐A594 intensity was similar in all groups except for a significant decrease in DERM‐A594 label in morphine tolerant rats given a morphine challenge (F (3, 34) = 3.899, p<0.05). These results suggest that repeated morphine administration increases morphine‐induced internalization of MOPs. To further test this idea, a myr‐dynamin inhibitory peptide (Dyn+) and a scrambled peptide (Dyn−) were microinjected into the vlPAG prior to microinjection of DERM‐A594. Dyn+ reduced DERM‐A594 antinociception compared to Dyn− (15 ± 3 s vs. 33 ± 5 s). Dyn+ also reduced morphine antinociception in both saline (D50 = 8.1 μg vs. Dyn− = 3.8 μg) and morphine pretreated rats (D50 = 10.7 μg vs. Dyn− = 5.8 μg). A comparable shift in D50 values in saline and morphine pretreated rats suggests that Dyn+ attenuates antinociception similarly in both groups. Thus, these studies demonstrate that internalization is important for MOP signaling in the vlPAG. Supported by DA023318(TM), DA015498(MM).