Abstract
Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of μ‐opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro. Here, we report that the Src‐mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone‐precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn−/− mice. The stereotaxic injection of wild‐type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR−/− mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.
Highlights
Cravings for opioids have been linked to the initial rewards associated with the drugs as well as to withdrawal and the adverse motivational state (Koob, 2009)
To demonstrate that MOR is phosphorylated at Tyr336, we developed an affinity-purified polyclonal antibody against phosphorylated Tyr336
Anti-pMORY336 is specific because of the disappearance of phosphorylated Tyr336 (pY336) immunoreactivity when the pMORY336 antibody was pre-incubated with the immunoprecipitated MOR complex that had been extracted from the locus coeruleus (LC) of morphine-dependent WT mice undergoing naloxone-precipitated withdrawal (Fig 1B) and with the corresponding phospho-peptide (Fig 2A)
Summary
Cravings for opioids have been linked to the initial rewards associated with the drugs as well as to withdrawal and the adverse motivational state (Koob, 2009). The neurocircuitry involved in the three stages of the addiction cycle has been mapped, with the extended amygdala as the focal point in the withdrawal/negative affect stage (Stinus et al, 1990; Nestler, 2004; Koob & Volkow, 2010). The extended amygdala is composed of the central nucleus of the amygdala, the bed nucleus of the stria terminalis (BNST), and the medial (shell) subregion of the nucleus accumbens (NAc) It receives projections from limbic structures such as the basolateral amygdala and hippocampus, and sends fibers to brain areas that interface with limbic structures with output to the extrapyramidal motor system (Alheid et al, 1995). To develop a successful treatment for opioid addiction, the cellular events involved in the drug withdrawal episodes that enhance an opioid’s incentive value to the extent that compulsive drug-taking and drugseeking take over one’s behavior need to be elucidated
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