Applying a candidate gene approach to investigate the molecular changes that accompany the development of addiction-like behaviors to opioids in rats
Open Access
- Author:
- Tacelosky, Diana Marie
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 16, 2013
- Committee Members:
- Robert G Levenson, Dissertation Advisor/Co-Advisor
Robert G Levenson, Committee Chair/Co-Chair
Richard Bernard Mailman, Committee Member
Arthur Steven Berg, Special Member
Patricia Sue Grigson Kennedy, Special Member
Willard Freeman, Committee Member - Keywords:
- opioids
heroin
addiction
animal model
mu-opioid receptor
addiction-like behavior - Abstract:
- Opioid addiction is a devastating chronic disorder characterized by compulsive drug seeking and a loss of control over drug use. Activation of the mu-opioid receptor (MOR) is critical for mediating the rewarding and addictive properties of opioids. Recent research suggests that MOR signaling is modulated by proteins that interact with MOR to form multiprotein signalplexes. The goal of my thesis research is to understand how MOR interacting proteins contribute to the molecular adaptations induced by opioid exposure and addiction. To achieve this goal, I developed a rodent model for "addiction-like" behaviors for heroin that serves as a platform for investigating potential underlying neural substrates involved in the development of opioid addiction. My central hypothesis is that the transition from opioid use to opioid addiction is accompanied by differential regulation of MOR interacting proteins. Criteria for "addiction-like" behaviors in rats have been established for cocaine (Deroche-Gamonet et. al., 2004), and I evaluated whether similar behaviors can be observed in rats following the intake of an opioid drug. I used a rat model of heroin self-administration that included key features of human addiction. Thus, responding for heroin was examined in 43 male Sprague-Dawley rats given the opportunity to intravenously self-administer heroin for 27 sessions. Criteria for addiction-like behaviors included greater drug seeking during periods of signaled non-availability, an increased willingness to work for drug when tested with a progressive ratio schedule of reinforcement, and greater lack of satisfaction and increased drug-seeking during timeout periods. My study revealed that although all rats self-administered approximately the same amount of heroin, only 9.3% of rats met all criteria for "addiction-like" behaviors. Additionally, behavior during the initial timeout and signaled non-availability periods (onset activity) was found to be an early predictor of the severity of "addiction-like" behavior that a rat would develop by the end of the study. To gain an understanding of the molecular changes that accompany the development of behavioral criteria for "addiction-like" behavior, I analyzed the protein expression of known MOR interacting proteins and the D2 dopamine receptor. Western blotting was used to assess protein expression in the prefrontal cortex, hippocampus, and nucleus accumbens from 14 rats from the heroin self-administration study. I found a differential expression of the D2 dopamine receptor, spinophilin, and wntless that correlated with behavioral evidence of "addiction-like behaviors." These changes in protein expression may reflect molecular correlates of the development of opioid addiction. Using a modified membrane yeast two-hybrid screen, our laboratory recently identified VAMP-associated protein A (VAPA) as a candidate MOR interacting protein. VAPA is highly expressed in the endoplasmic reticulum and Golgi apparatus and functions in vesicular docking and exocytosis. In this dissertation, I confirmed that VAPA is a bona fide MOR interacting protein in vivo using co-immunoprecipitation and immunoelectron microscopy. To determine if VAPA plays a role in the transition to opioid dependence, I examined VAPA expression in morphine-treated mice harboring a common genetic variant of MOR (A112G allele). In both the cerebral cortex and hippocampus of mice expressing the variant allele, an increase in VAPA protein expression was found. These results suggest that changes in VAPA expression may potentially contribute to the development of opioid addiction. The research in this thesis is important because it uses a novel model of "addiction-like" behavior in rats self-administering heroin to analyze the expression of MOR interacting proteins in key brain regions that mediate reward and addiction. My data suggest that spinophilin and wntless are potential molecular substrates that accompany the transition from opioid exposure to opioid addiction. Future research is needed to demonstrate a cause and effect relationship between the expression of MOR interacting proteins and "addiction-like" behaviors. To test whether wntless or spinophilin are important neural substrates for opioid addiction, experiments can be designed to determine if the development of "addiction-like" behaviors can be prevented by blocking the interaction of the MOR with wntless or spinophilin. Further studies are also required to test my working hypothesis that VAPA is involved in the intracellular trafficking of MOR along the vesicular pathway and that this process is altered in the presence of a chronic opioid agonist. Understanding the role of VAPA in MOR translocation to the cell surface will provide insight into additional mechanisms of receptor regulation.