Chronic morphine treatment-modulated trafficking of AMPA receptors: a potential mechanism for drug addiction

Abstract

Morphine is the benchmark analgesic for treating chronic pain. However, its clinical uses are hindered by its highly addictive nature, as chronic treatment with the drug will produce physical and psychological dependence upon the cessation of use. Drug craving is the main driving force for relapse after prolonged periods of abstinence, and represents an enormous challenge for the treatment of drug addiction. Since addiction is a long-term behavioral alteration, it is believed that addictive drugs produce reorganization of specific neural circuits and adjustment of synaptic strength. The underlying mechanisms of these neural adaptations may represent a promising target for prevention and/or treatment of addiction, but the detailed mechanisms of these processes remain unclear. Therefore, the main goals of this work are to delineate signaling pathways controlling morphine-induced neural adaptations and investigate their functional role in opiate addictive behaviors. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are postsynaptic glutamate receptors, and are responsible for mediating most excitatory synaptic transmission under normal conditions. More importantly, the dynamic localization of AMPA receptors plays a critical role for modifying synaptic strength and synaptic morphology. Here, I hypothesized that regulation of AMPA receptor trafficking by morphine treatment underlies the drug-induced neural modulation implicated in the addiction process. Hence, the first part of this dissertation research examined whether and how chronic exposure to morphine modulates trafficking of surface GluR1 (a subunit of AMPA receptors) in primary hippocampal neurons. Using live-cell imaging techniques together with biochemical studies, I demonstrated that chronic exposure to morphine induced a significant loss of synaptic and extrasynaptic GluR1 by internalization. In mechanistic studies, I found that the GluR1 internalization was attributed to dephosphorylation of the receptor subunit at Ser845 following morphine treatment, but it did not result from altered neural network or NMDA receptor activation. Moreover, dephosphorylation of GluR1 at Ser845 was found to require morphine-evoked calcineurin activation. Therefore, calcineurin-dependent dephosphorylation of AMPA receptor and subsequent AMPA receptor internalization provides a novel mechanism for opioid-induced neural adaptations. The second part of this dissertation research attempted to link morphine's effects on GluR1 phosphorylation and endocytosis to addictive behavior, especially formation of memory for the environmental context of the drug experience, because recall of this memory by encountering the drug-paired cues triggers relapse to drug seeking. In this approach, a mutant mouse line was used, in which GluR1 at Ser845 was mutated to Ala (S845A) leading to an absence of morphine-induced GluR1 endocytosis. A behavioral test, conditioned place preference (CPP), was carried out to assess the ability of morphine to produce a positive association with environmental cues. I found that S845A mice were significantly slower to acquire morphine-induced CPP when compared to wild types (WT). This decreased sensitivity to morphine CPP in mutants was neither related to contextual memory deficits or abnormal locomotor activity, as there was no difference between WT and S845A mice in the contextual memory acquisition in the Morris water maze test or locomotion with or without morphine injection. To examine the persistence of morphine-associated contextual memory in the mutant mice, I also performed extinction tests on mice conditioned with 10 mg/kg morphine for four sessions, by which both WT and S845A mice exhibited similar CPP responses. Interestingly, a prolonged extinction was observed in S845A mutant mice, suggesting the S845A mutation either impaired the learning of the new conditioning or prolonged the retention of the old conditioning. Nevertheless, these results suggest that an alteration in GluR1 phosphorylation at Ser845 and subsequent receptor endocytosis/insertion are involved in acquisition and extinction of morphine CPP. Altogether, the present findings indicate that calcineurin-mediated GluR1-S845 dephosphorylation is required for morphine-induced internalization of GluR1-containing AMPA receptors, providing a molecular basis for the drug-induced neural modulation. This work also suggests that this regulation of GluR1 phosphorylation and trafficking by morphine is involved in the modulation of the drug-associated contextual memory, which reflects the involvement of AMPA receptor trafficking in the mechanisms underlying opiate-seeking behaviors.