Cocaine withdrawal-induced trafficking of delta-opioid receptors in rat nucleus accumbens

Abstract

Interactions between the opioidergic and dopaminergic systems in the nucleus accumbens (NAcb) play a critical role in mediating cocaine withdrawal-induced effects on cell signaling and behavior. In support of this, increased activation of striatal dopamine-D1 receptors (D1R) results in desensitization of delta-opioid receptor (DOR) signaling through adenylyl cyclase during early cocaine withdrawal. A potential cellular substrate underlying receptor desensitization is receptor internalization. The present study examined the effect of cocaine withdrawal on subcellular localization of DOR in dendrites of the NAcb core (NAcbC) and shell (NAcbS) using immunoelectron microscopy. Female and male rats received binge-pattern cocaine or saline for 14 days and subsequently underwent 48 h withdrawal. Animals were transcardially perfused and tissue sections were processed for immunogold-silver localization of DOR. Semi-quantitative analysis revealed that cocaine withdrawal caused an increase in the percentage of DOR localized intracellularly in the NAcbS of male and female rats and the NAcbC of male rats compared to saline controls. In contrast, in the NAcbC of female rats, there was an increase in DOR associated with the plasma membrane following cocaine withdrawal. To determine whether modulation of D1R could directly impact DOR containing neurons, the hypothesis that DOR and D1R co-exist in common neurons of the NAcb was examined in naïve rats. Semi-quantitative analysis revealed a subset of profiles containing both DOR and D1R immunoreactivities. The present findings demonstrate a redistribution of DOR in the NAcb following cocaine withdrawal and provide anatomical evidence supporting D1R regulation of DOR function in a subset of NAcb neurons.

Figure 1. Subcellular distribution of DOR in the NAcbS in both saline and cocaine-treated females

A: Schematic of a coronal hemisection of rat forebrain illustrating the regions of the NAcb sampled for electron microscopic analysis. Trapezoids indicate the NAcb core and shell regions sampled for ultrastructural analysis. ac, anterior commissure; cc, corpus callosum; CPu, caudate putamen; NAcbC, nucleus accumbens core; NAcbS, nucleus accumbens shell. Modified from Paxinos and Watson (Paxinos, 1998). Scale bar = 3 mm. Saline-treated. B: In the NAcbS of saline-treated rats DOR immunolabeling was visualized in association with the plasma membrane (arrows) and the cytoplasm (arrowheads) in dendritic profiles. C: An unlabeled terminal was presynaptically apposed to a dendrite containing DOR along the plasmalemma. Cocaine-treated. D: Withdrawal from chronic cocaine administration resulted in internalization of DOR (arrowheads). Often internalized receptors were associated with endosome-like structures (arrowhead in circle) E: A DOR-labeled dendrite containing cytoplasmic DOR (arrowheads) was postsynaptic to an unlabeled terminal. The large arrow indicates DOR associated with an endosome-like structure. Boxes include enlarged portions of D (arrowhead in circle) and E (large arrow) representing DOR association with endosome-like structures. Abbreviations: ut = unlabeled terminal; ma= myelinated axon; m=mitochondria.

Figure 2. Subcellular distribution of DOR in the NAcbS in both saline and cocaine-treated females Saline-treated

In the NAcbC of saline treated rats, DOR was localized to both membrane (arrow) and intracellular compartments (arrowheads). A: An unlabeled terminal forms a synapse with a spinous process of a DOR-labeled dendrite. B: A dendrite with DOR associated with the outer plasma membrane and intracellular compartments. In the NAcbC, withdrawal from chronic cocaine administration resulted in a slight increase in DOR at the plasma membrane. Cocaine-treated. C: A DOR-labeled dendrite containing plasma membrane and cytoplasmic DOR post-synaptic to an unlabeled terminal. D: Two DOR-labeled dendrites were visualized in the same field. Both contained plasma membrane (arrows) and cytoplasmic (arrowheads) immunogold labeling for DOR. DOR was associated with endosome-like structures (arrowhead in circle) Abbreviations: ut = unlabeled terminal; m=mitochondria.

Figure 3. Quantification of DOR localization to plasma membrane

Bar graphs representing the percentage of DOR on the plasma membrane in saline and cocaine-treated male and female rats. A: In the NAcbS, 35% of DOR was localized to the plasma membrane of saline-treated females compared to 24% in cocaine-treated rats. B: In the NAcbC, 20% of DOR was localized to the plasma membrane of saline-treated females compared to 26% of cocaine-treated females. C: In the NAcbS of male rats, 29% of DOR from saline-treated rats were localized to the plasma membrane compared to 19% in cocaine-treated males. D: In the NAcbC, 22% of DOR was localized to the plasma membrane in saline-treated males compared to 17% in cocaine-treated males. Abbreviations: PM= plasma membrane. Female (n=3 rats/group); Male (n=2 rats/group)

Figure 4. Analysis of Protein Expression

Western blot analysis of NAcb tissue from female cocaine and saline-treated animals. A: Preincubation of DOR antibody with control peptide prior to immunoblot probing resulted in a loss of immunoreactivity (arrow) at 50kDa, the expected molecular weight of the DOR. B: Following two day withdrawal from chronic binge-pattern cocaine, DOR expression was unchanged in NAcb compared to saline controls (C=cocaine; S=saline). An unpaired t-test revealed no significant difference in DOR band density in NAcb tissue extracts between treatment groups following withdrawal from binge-pattern cocaine administration (n=4 rats/group).

Figure 5. Immunofluorescence microscopy showing localization of the D1R and DOR in the NAcbS

Confocal images of a coronal section through the NAcb dually labeled for visualization of A: DOR (TRITC) and B: D1R (FITC). C: Merged image showing neurons expressing both DOR and D1R indicated by arrowheads in A, B, and C. Scale bar = 25 µm.

Figure 6. Co-localization of the D1R and the DOR in the NAcbS and NAcbC

A: A dendrite co-expressing immunoperoxidase labeled D1R (arrows) and immunogold labeled DOR (arrowheads) in the NAcbS. D1R is visualized along the plasma membrane while DOR is located intracellularly. B: Reverse labeling illustrating a dendrite in the NAcbC exhibiting co-localization of immunoperoxidase labeled DOR (arrows) and immunogold labeled D1R (arrowheads). An unlabeled axon terminal containing vesicles is apposed to the co-labeled dendrite. Bar = 500nm. Abbreviations: D1R + DOR-d = D1R and DOR labeled dendrite; ut = unlabeled terminal; D1R-d = D1R labeled dendrite; ma= myelinated axon: m=mitochondria. C: Bar graphs representing the percentage of co-localization between DOR and D1R. In the NAcbS 33% of D1R labeled profiles contained DOR immunolabeling and 34% of DOR labeled profiles contained D1R immunoreactivity. In the NAcbC, 24% of D1R labeled profiles contained DOR immunoreactivity and 38% of DOR labeled profiles contained immunolabeling for D1R.