AMPA receptor subunit composition and its regulation by cocaine.

摘要

In order to combat the drug addiction problem, it is necessary to learn about the underpinnings of the neural systems it affects. Plasticity of the glutamate system, specifically alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), plays a key role in the neuroadaptations associated with cocaine addiction. The subunit composition of AMPARs is an important determinant of their biological properties and trafficking. However, AMPAR subunit composition has only been determined in the hippocampus. One goal of this thesis was to characterize AMPAR subunit composition in addiction-related brain regions such as the nucleus accumbens (NAc), dorsal striatum (DS), and prefrontal cortex (PFC), focusing on the GluA1-3 subunits important in these regions. To this end, I used a quantitative co-immunoprecipitation (co-IP) protocol along with blue native electrophoresis (BNE) to study the assembly state of the receptors. I found that, in all brain regions studied, the majority of the GluA1 was associated with GluA2, and a small amount with GluA3. Although about half of the GluA2 was not associated with GluA1, this represents an over-estimate of the percent of GluA2 present in GluA2A3 receptors, based on BNE results demonstrating that most GluA2 exists as dimers, rather than functional tetrameric receptors, whereas relatively more GluA1 was present in tetramers. These results along with others suggest a dominant role for the GluA1A2 receptor in all brain regions. Each region also contained a small number of GluA2-lacking AMPAR, which have higher conductance due to calcium permeability.;A second goal was to use the same quantitative co-IP technique to determine if repeated cocaine treatment and withdrawal alters AMPAR subunit composition in the NAc. To do this, two different models of cocaine addiction were used: behavioral sensitization produced by non-contingent cocaine exposure and incubation (time-dependent intensification) of cue-induced cocaine craving after withdrawal from extended access cocaine self-administration. AMPAR subunit composition in the NAc was determined during withdrawal in each model. In the behavioral sensitization model, after using a protein crosslinking assay to determine that there was a withdrawal-dependent increase in surface GluA1A2 receptors, co-IP studies revealed a slight increase in the amount of GluA1 not associated with GluA2, suggesting an increase in total cellular GluA2-lacking AMPARs. However, in contrast to results in the incubation model, described next, these receptors were not found to contribute significantly to synaptic transmission during withdrawal from non-contingent cocaine exposure. In the incubation of cocaine craving model, after 45 days of withdrawal from cocaine self-administration, I used the same quantitative co-IP techniques as previously mentioned to demonstrate a more robust increase in the amount of GluA2 that was not associated with GluA1, as well as an increase in GluA1 not associated with GluA2 or any other subunit. These data complemented other biochemical data showing an increase in surface GluA1 after 45 days of withdrawal, without an increase in surface GluA2 or GluA3, as well as electrophysiological data showing the presence of GluA2-lacking AMPARs in the NAc. Finally, these GluA2-lacking AMPARs were demonstrated to mediate the expression of incubated cue-induced cocaine seeking after prolonged withdrawal. Together, results in both models suggest that AMPAR upregulation in the NAc increases cocaine-related behaviors by increasing the reactivity of MSN to glutamate inputs from cortical and limbic regions that trigger cocaine-related behaviors, although this increase in reactivity is greater in the incubation model due to synaptic incorporation of high conductance GluA2-lacking AMPARs.;Brain-derived neurotrophic factor (BDNF) plays a critical role in plasticity at glutamate synapses and the effects of repeated cocaine exposure. BDNF protein levels increase in nucleus accumbens (NAc) and other addiction-related regions in association with the incubation of cocaine craving. As BDNF promotes synaptic delivery of GluA2-lacking AMPARs in other systems, I hypothesized that increased BDNF levels might underlie synaptic incorporation of GluR2-lacking AMPARs in the incubation model. As a first step towards testing this hypothesis, I investigated the effects of BDNF on AMPAR expression and distribution in cultured NAc neurons obtained from postnatal rats. Our results indicate an important role for BDNF in regulating AMPAR expression on NAc medium spiny neurons. Although BDNF is not specifically linked to regulation of GluA2-lacking AMPARs in cultured NAc neurons, our results warrant in vivo experiments to examine the role of BDNF in AMPAR adaptations occurring in the intact NAc during cocaine withdrawal. (Abstract shortened by UMI.)