The Addicted Phenotype: Protein Phosphorylation Status and Dopamine Receptor Responsiveness

Up-regulation of cAMP/PKA signaling by drugs of abuse may contribute to escalation and relapse, possibly by differentially altering dopamine receptor-responsiveness in the mesolimbic dopamine system. To investigate this hypothesis, our initial studies measured alterations in cAMP-dependent and -independent protein phosphorylation in vivo produced by chronic cocaine and heroin self-administration, changes in mesolimbic protein phosphorylation compared to individual differences in the propensity for escalating cocaine self-adminstration, and, ultimately, dopamine receptor-mediated regulation of relapse to cocaine seeking in withdrawal. Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviors, but whether differential alterations develop in the biochemical response to cocaine is less clear. In Chapter 2, we studied cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify regulation related to the context of behavioral reinforcement, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 hrs of intravenous cocaine administration, and subjected to western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including phosphorylation of the AMPA GluR1 receptor subunit at serine 845 in striatal and amygdala subregions, and the NMDA NR1 receptor subunit at serine 897 in the CA1 subregion of hippocampus. Tolerance also developed to cAMPindependent GluR1 S831 phosphorylation in the prefrontal cortex. In contrast, sensitization to cocaine-induced phosphorylation of the pre-synaptic vesicle protein synapsin I at serine 9 developed in amygdala and hippocampal subregions, while cAMP-dependent phosphorylation of the dopamine-synthesizing enzyme tyrosine hydroxylase at serine 40 decreased in pre-synaptic striatal dopamine terminals in striatal subregions. Cocaineinduced phosphorylation of extracellular signal-regulated kinase (ERK) was dissociated from downstream phosphorylation of the transcription factor cAMP-response element binding protein (CREB) in many brain regions, and failed to develop either tolerance or sensitization with chronic administration, and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in selfadministering animals, while negative dose-related correlations were found primarily with passive yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in lieu of their potential impact on the development of cocaine addiction. In Chapter 3, we studied alterations in protein kinase A (PKA)-dependent and PKA-independent phosphorylation in multiple brain regions in rats undergoing either spontaneous or naltrexone-precipitated withdrawal (WD) from chronic intravenous heroin self-administration. Spontaneous WD from heroin self-administration produced region-specific increases in PKA-dependent GluR1S845 phosphorylation in the nucleus accumbens shell, basolateral amygdala, hippocampal CA1 and CA3 regions, and premotor cortex after 24 but not 12 hrs, and there were no changes in prefrontal cortex, nucleus accumbens core or caudate-putamen. Increased GluR1</sub