The Addicted Phenotype: Protein Phosphorylation Status and Dopamine Receptor Responsiveness
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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 cAMP-independent GluR1S831 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. Cocaine-induced 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 self-administering 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 GluR1S845 phosphorylation occurred earlier (12 hrs) in the central amygdala, ventral tegmental area, and substantia nigra. In contrast, prominent ERK phosphorylation was found in both prefrontal and premotor cortex, CA1 and CA3, caudate-putamen, and basolateral amygdala, but not in nucleus accumbens, or central amygdala in spontaneous WD. Phosphorylation of striatal CREB increased in caudate-putamen but not in nucleus accumbens, paralleling ERK rather than PKA activity in heroin WD. Naltrexone administration potentiated GluR1S845 and ERK phosphorylation in the central amygdala, and ERK phosphorylation in nucleus accumbens core and shell. Thus, spontaneous WD from heroin self-administration produces region- and time-dependent changes in PKA and ERK activity that could contribute to the behavioral manifestation of opiate dependence. In Chapter 4 we studied PKA-dependent GluR1S845 phosphorylation and ERK phosphorylation mediated by ERK kinase in striatal subregions in an animal model of cocaine craving. Here, animals with chronic cocaine self-administration experience were re-exposed to the self-administration test chambers for 1 hr in the absence of cocaine to measure phosphorylation induced by the environmental context paired with cocaine reinforcement. After 1 day WD, GluR1S845 levels were elevated in both self-administering and yoked groups in the nucleus accumbens shell, but this effect persisted only in self-administering animals after 3 weeks WD. In the nucleus accumbens core, context-induced phosphorylation of both GluR1S845 and ERK increased from early to late WD from chronic cocaine self-administration, implicating this region in mediating the intensification of cocaine craving with longer periods of abstinence. These differential region- and substrate-specific adaptations to withdrawal- and context-induced protein phosphorylation could underlie the maintenance of cocaine addiction by exacerbating the potential for drug relapse in withdrawal. Finally, as a behavioral correlate, studies in Chapter 5 sought to compare these changes in protein phosphorylation status with alterations in dopamine-receptor mediated regulation of relapse to cocaine seeking in withdrawal. Here, the cocaine-addicted phenotype was modeled in rats based on individual differences in preferred levels of cocaine intake and a propensity for relapse in withdrawal. Since these cocaine-taking and -seeking behaviors are strongly but differentially regulated by postsynaptic dopamine D1 and D2 receptors in the mesolimbic system, we determined whether the development of cocaine addiction would be related to differential sensitivity in functional D1 and D2 receptor responses. Using a population of 40 outbred Sprague-Dawley rats trained to self-administer cocaine for 3 weeks, we found that animals with higher preferred levels of cocaine intake exhibited a vertical and rightward shift in the self-administration dose-response function, and were more resistant to extinction from cocaine self-administration, similar to phenotypic changes reported in other models of cocaine addiction. After 3 weeks of withdrawal from cocaine self-administration, high intake rats were subsensitive to the ability of the D1 agonist SKF 81297 to inhibit cocaine-seeking behavior, but supersensitive to cocaine seeking triggered by the D2 agonist quinpirole, when compared to low intake rats. Additionally, high intake rats developed profound increases in locomotor responses to D2 receptor challenge from early to late withdrawal times, whereas low intake rats developed increased responsiveness to D1 receptor challenge. In a second experiment, responses to the mixed D1/D2 agonist apomorphine and the NMDA glutamate receptor antagonist MK-801 failed to differ between low and high intake rats. These findings suggest that cocaine addiction is specifically related to differential alterations in functional D1 and D2 receptors that mediate opposing influences on cocaine-seeking behavior. Alterations in dopamine receptor signaling may issue from an enduring up-regulation of cAMP/PKA signaling in drug withdrawal. The potential for reduced D1 receptor (Gs/Golf-coupled) sensitivity to account for tolerance to cocaine-regulated PKA-dependent protein phosphorylation during self-administration (Chapter 2) is discussed in Chapter 6. Conversely, it is possible that PKA up-regulation in cocaine WD (Chapter 4) could account for the paradoxical sensitization of subsequent Gi/Go-coupled D2 receptor responses. Thus, addiction-related alterations in D1 and D2 dopamine receptor responses may ultimately involve complex reciprocal interactions between adaptations in PKA signaling pathways that differentially influence D1 and D2 receptor signaling.