Rewarding morphine-induced synaptic function of delta-opioid receptors on central glutamate synapses

Abstract

The rewarding effect of opioids, the driving force for compulsive behaviors of opioid abuse and addiction, is primarily mediated by the mu-opioid receptor. However, the role of the delta-opioid receptor (DOR) in opioid reward and addiction is still poorly understood. The recently discovered adaptive DOR property of exocytotic translocation in sensory neurons after chronic opioid exposure provides a new avenue of conceptual thoughts to exploring the DOR function in this psychoneurological disease. In this study, we investigated potential adaptive function of DOR in neurons of the central nucleus of the amygdala (CeA), a forebrain structure increasingly recognized for mediating stimulus reward learning in drug addiction. Using whole-cell recordings in CeA slices, we found that in rats displaying morphine-induced behavior of conditioned place preference, a behavioral measure of drug reward, the overall synaptic strength of glutamate synapses in CeA neurons was significantly enhanced. The selective DOR agonist [D-Pen(2),D-Pen(5)]-enkephalin, having no apparent effect on glutamatergic excitatory postsynaptic current (EPSC) in neurons from control rats, produced a significant, dose-dependent inhibition of the synaptic current in neurons from those morphine-treated rats. Detailed analyses of EPSC properties revealed that DOR activation inhibited the EPSC by reducing presynaptic release of glutamate, indicating functional DOR emerging on presynaptic glutamate terminals. The morphine treatment also significantly increased DOR proteins in CeA preparations of synaptosomes. These findings provide functional evidence for an adaptive modulation by presynaptic DOR of a key synaptic activity altered by morphine, thus implying likely important involvement of DOR in opioid reward and addiction.

Fig. 1.

Conditioning treatment with repeated morphine induces the reward behavior of CPP through opioid receptors. Data were expressed as percentage of time the rat spent in the morphine-paired chamber versus the sum of the times spent in both morphine- and saline-paired chambers. Data of pretest (before morphine conditioning) and post-test (after morphine conditioning) were compared to determine the CPP behavior. One day after the post-test, a postinjection test was performed after an intraperitoneal injection of saline (n = 7 rats) or naloxone (1.5 mg/kg, n = 8 rats). **, p < 0.01.

Fig. 2.

Morphine increases glutamate synaptic strength in neurons of the CeA. A, representative EPSCs evoked by an electrical stimulus at three intensities (maximum, one third maximum, and two thirds maximum) in a CeA neuron from a saline-treated control rat (control group) and from a morphine-treated rat with CPP behavior (morphine group). B, group data of the evoked EPSCs in neurons of the control group (n = 14) and of the morphine group (n = 12). *, p < 0.05; **, p < 0.01.

Fig. 3.

Morphine increase of glutamate synaptic transmission involves a presynaptic site. A, representative pairs of EPSCs evoked by two consecutive stimuli at an interval of 40 ms in a CeA neuron from a control rat and from a morphine-treated rat. The inlet shows the same two EPSC pairs scaled to the amplitude of the first EPSC, illustrating morphine-induced reduction in the paired-pulse ratio. B, paired-pulse ratios at three between-stimulus intervals as indicated in CeA neurons of the control group (n = 28 at 40 ms and n = 11 at 60 and 80 ms) and of the morphine group (n = 9 at each interval). *, p < 0.05.

Fig. 4.

Morphine recruits new functional DORs on CeA glutamate synapses. A, representative EPSCs in a CeA neuron, from the control and morphine groups, under conditions of control, in the presence of the selective DOR agonist, DPDPE, and DPDPE plus naltrindole, a selective DOR antagonist. B, group data summarizing the effects of DPDPE and naltrindole addition in the two cell groups. C, dose-response curve of the DPDPE inhibition on EPSCs. The estimated EC₅₀ for the DPDPE effect is 50.7 nM (n = 6–11 for each data point). **, p < 0.01.

Fig. 5.

DOR inhibition of EPSCs involves a presynaptic site. A, representative EPSC pairs at the 40-ms interval before and after the addition of DPDPE in a CeA neuron from the morphine group. B, same EPSC pairs as in A, but scaled to the amplitude of first EPSC, illustrating the DPDPE-mediated increase in the paired-pulse ratio. C, group data of the DPDPE effect in the two cell groups. *, p < 0.05.

Fig. 6.

DOR activation reduces presynaptic release of glutamate. A, current traces with spontaneous events of miniature EPSCs in the absence and presence of DPDPE from a CeA neuron of the morphine group. B, graphs showing cumulative probability of the frequency and amplitude of miniature EPSCs in control and in DPDPE for the neuron in A. C, group data of the DPDPE effect on miniature EPSC frequency and amplitude in neurons from the morphine group. *, p < 0.05.

Fig. 7.

Morphine increases DOR protein expression in CeA synaptosomes. A, representative lanes of Western blots of total DOR proteins and total proteins of β-actin in CeA tissues taken from a saline-treated control rat and from a morphine-treated rat. B, Western blot lanes of DOR proteins and synaptophysin, a synaptic terminal marker, in CeA preparations of synaptosomes from a rat of the two indicated groups. C, summary graph showing changes in the expression ratios of total DOR proteins versus β-actin and synaptosomal DOR proteins versus synaptosomal synaptophysin (n = 6 rats for each group). **, p < 0.01. Syn, synaptosome; synpsn, synaptophysin.