Morphine-induced trafficking of a mu-opioid receptor interacting protein in rat locus coeruleus neurons

Abstract

Opiate addiction is a devastating health problem, with approximately 2million people currently addicted to heroin or non-medical prescription opiates in the United States alone. In neurons, adaptations in cell signaling cascades develop following opioid actions at the mu opioid receptor (MOR). A novel putative target for intervention involves interacting proteins that may regulate trafficking of MOR. Morphine has been shown to induce a re-distribution of a MOR-interacting protein Wntless (WLS, a transport molecule necessary for secretion of neurotrophic Wnt proteins), from cytoplasmic to membrane compartments in rat striatal neurons. Given its opiate-sensitivity and its well-characterized molecular and cellular adaptations to morphine exposure, we investigated the anatomical distribution of WLS and MOR in the rat locus coeruleus (LC)-norepinephrine (NE) system. Dual immunofluorescence microscopy was used to test the hypothesis that WLS is localized to noradrenergic neurons of the LC and that WLS and MOR co-exist in common LC somatodendritic processes, providing an anatomical substrate for their putative interactions. We also hypothesized that morphine would influence WLS distribution in the LC. Rats received saline, morphine or the opiate agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), and tissue sections through the LC were processed for immunogold-silver detection of WLS and MOR. Statistical analysis showed a significant re-distribution of WLS to the plasma membrane following morphine treatment in addition to an increase in the proximity of gold-silver labels for MOR and WLS. Following DAMGO treatment, MOR and WLS were predominantly localized within the cytoplasmic compartment when compared to morphine and control. In a separate cohort of rats, brains were obtained from saline-treated or heroin self-administering male rats for pulldown co-immunoprecipitation studies. Results showed an increased association of WLS and MOR following heroin exposure. As the LC-NE system is important for cognition as well as decisions underlying substance abuse, adaptations in WLS trafficking and expression may play a role in modulating MOR function in the LC and contribute to the negative sequelae of opiate exposure on executive function.

Keywords: Confocal microscopy; Electron microscopy; G-protein receptor; Norepinephrine; Trafficking; Wntless.

Figure 1

Evidence for localization of WLS in the brainstem nucleus locus coeruleus. A) Representative schematic diagram adapted from the rat brain atlas of Paxinos and Watson at level bregma −10.03mm, plate 58, showing the regional localization of the LC in the dorsal pons (Paxinos, 1997). B) Western blot analysis shows WLS expression in whole cell lysates obtained from the medial prefrontal cortex (mPFC), striatum, hippocampus, locus coeruleus, nucleus (Nucl) accumbens and ventral tegmental area (VTA). A specific band in the 37–50kD range, the predicted molecular weight of WLS, can be observed in many brain regions, as previously reported. C) A tissue section through the dorsal pons that was processed in the absence of the primary antibody reveals a lack of immunoperoxidase staining. D and E) Immunoperoxidase labeling for WLS (black arrows) can be observed in the LC at low and high magnification. superior cerebellar peduncle, scp. 4v: 4^th ventricle, scale bar 50 microns in C-D; 10 microns in E.

Figure 2

High magnification immunofluorescence microscopy depicting colocalization of TH, MOR, and WLS in individual rat locus coeruleus (LC) neurons. A and A’ illustrate MOR labeling (green) in the LC, B and B’ show WLS labeling (red) and C and C’ depict TH labeling (blue) in the same tissue. D and D’ represent merged images for all three labels. Solid single black arrows indicate neurons that contain MOR, WLS and TH while arrowheads depict neurons containing only MOR. Dual headed arrows indicate neurons that only contain WLS. Arrows indicate dorsal (D) and lateral (L) orientation of the sections illustrated. Scale bar 20 microns.

Figure 3

Electron microscopic evidence for localization of WLS in rat noradrenergic dendrites of the locus coeruleus (LC). A) An immunoperoxidase labeled tyrosine hydroxylase dendrite (TH-d) is shown in proximity to an immunogold-silver labeled (arrowheads) WLS dendrite (WLS-d). The singly labeled dendrites receive direct symmetric synaptic contacts (curved arrows) from unlabeled axon terminals (ut). B) A dendrite contains both immunoperoxidase labeling for TH and immunogold-silver labeling (arrowheads) for WLS (WLS + TH-d). ud: unlabeled dendrites Scale bar 0.5 microns.

Figure 4

Electron microscopic evidence for agonist-induced trafficking of WLS in rat locus coeruleus (LC) neurons using single immunogold-silver labeling for WLS. A-B. Sections from vehicle-treated subjects show immunogold-silver grains for WLS within the cytoplasm (arrowheads) and along the plasma membrane (arrow). Note that WLS is distributed both within the cytoplasm of dendrites as well as associated occasionally with the plasma membrane. Unlabeled axon terminals (ut) are shown contacting (zigzag arrows) the WLS-containing dendrite C-D. WLS immunolabeling shows a shift in distribution from the cytoplasm (arrowheads) to the plasma membrane (arrows) following morphine treatment. An unlabeled axon terminal (ut) is contacting (zigzag arrow) the WLS-containing dendrite. E-F. Following DAMGO treatment, immunogold-silver labeling for WLS (arrowheads) is primarily distributed within the cytoplasm. Double arrows point to endosome-like vesicles. An unlabeled axon terminal (ut) is shown contacting (zigzag arrow) the WLS-containing dendrite (Panel E). Scale bars, 0.5µm.

Figure 5

Differential trafficking of WLS is observed in rat locus coeruleus (LC) dendrites from vehicle-treated (A, B), morphine-treated (C–D) and DAMGO-treated rats (E–F) using dual immunogold-silver for WLS (small gold-silver grains) and MOR (large gold-silver grains). A-B. Immunogold-silver labeling for WLS can be seen within the cytoplasm (arrows) while immunogold-silver labeling for MOR is distributed within the cytoplasm (arrowhead) and along the plasma membrane (double arrowheads) in dendrites from vehicle-treated rats. Panel A shows an unlabeled axon terminal (ut) contacting (zigzag arrow) the WLS and MOR-containing dendrite. C-D. Following morphine treatment, WLS labeling shifts from the cytoplasm to the plasma membranes of LC dendrites. Double arrows point to immunogold-silver labeling for WLS along the plasma membranes while double arrowheads point to immunogold-silver for MOR labeling along the plasma membrane in WLS and MOR-containing dendrite. E-F. Immunogold-silver labeling for WLS (arrows) and MOR (arrowheads) can be seen primarily within the cytoplasm of the WLS and MOR-containing dendrites in rats subjected to DAMGO treatment. Two unlabeled axon terminals (ut) can be seen contacting a WLS and MOR-containing dendrite (Panel E). A thick arrow points to endosome-like vesicles. Scale bars, 0.5µm.

Figure 6

Ratio of cytoplasmic to total immunogold–silver labeling for WLS and MOR following opiate agonist treatment. Morphine treatment caused a significant (P < 0.05) shift in WLS from the cytoplasmic compartment to the plasma membrane, while MOR remains on the plasma membrane, similar to control. DAMGO treatment significantly increased (P < 0.05) the ratio of cytoplasmic to total WLS immunogold–silver labeling compared to control and morphine treatment. DAMGO also caused a significant shift in MOR distribution to the cytoplasm compared to morphine and control. Values are mean ± SEM of three rats per group. Values with asterisks are significantly different (*P < 0.05) from each other. Tukey’s multiple comparison tests after ANOVA.

Figure 7

Interaction of MOR and WLS in the locus coeruleus (LC) of saline and heroin self-administering rats. (A) MOR was immunoprecipitated (IP) from the LC of saline (control) and heroin (drug) self-administering rats using a rabbit anti-MOR antibody. Equal amounts of protein from LC tissue were added to each immunoprecipitation reaction. Interaction with WLS was determined by immunoblotting (IB) with a chicken anti-WLS antibody. Lysate (L) lanes contain 1% of the total protein from the LC compared to the mock (M, rabbit IgG) and immunoprecipitation (IP) lanes. Arrow indicates position of WLS. IgG heavy chain migrates as a dimer at ∼100 kDa. (B) Bands were analyzed by densitometry and quantified using Image J software. Data was analyzed using paired Student’s t-test (expressed as a mean ± SEM; n=3, (*P < 0.05).