While dopamine receptors (DA) mediate acute effects of amphetamine and cocaine, neuroadaptations produced by their chronic administration involve glutamate-dependent synaptic plasticity. Many such neuroadaptafions occur in the nucleus accumbens (NAc), a brain region that is critical to addiction and receives convergent DA and glutamate inputs. Recent work suggests that critical mechanisms for regulating excitatory transmission involve alterations in the phosphorylation and surface expression of AMPA receptors. We developed cultures of postnatal NAc neurons as a model system in which to investigate whether DA receptors, activated during psychostimulant administration, can influence these fundamental mechanisms for altering synaptic strength. We found that D1 DA receptor stimulation enhances phosphorylation of the AMPA receptor subunit GIuR1. Then, using antibody to a cell surface epitope of GluR1 and fluorescence microscopy, we demonstrated rapid down-regulation of surface GluR1 by glutamate and rapid up-regulation by a D1 agonist. We propose to further characterize interactions between DA and AMPA receptors in this model system.
Aim 1 will use fluorescence microscopy to examine colocalization of surface GIuR1 puncta with the synoptic marker synaptophysin, other AMPA receptor subunits, and the NMDA receptor subunit NR1.
Aim 2 will further characterize regulation of GluR1 surface expression by glutamate and DA agonists. Surface biotinylation experiments will be used to verify prior results obtained with quantitative immunofluorescence assays. Then, the latter approach will be used to study effects of D1 and D2 agonists, glutamate agonists (glutamate, NMDA and AMPA), and interactions between these agents.
Aim 3 will test the hypothesis that D1 agonists increase GluR1 surface expression through a protein kinase A (PKA)-dependent mechanism. We will determine if D1 agonist effects are reproduced by activators of the cAMP-PKA pathway and prevented by inhibitors.
Aim 4 will establish methods for co-culturing NAc and cortical neurons that preserve the ability to selectively analyze GluR1 on the processes of NAc neurons. Addingcortical neurons will restore synoptic glutamatcrgic input. In these co-cultures, we will determine the extent to which GluR1 on NAc neurons is localized to synapses and whether this is altered by D1 receptor stimulation. We will also examhle the effect of repeated D1 receptor stimulation, as all initial attempts to model the effects of long-term psychostimulant administration in a culture system. These studies will provide insight into regulatory mechanisms that, when disrupted during chronic drug exposure, may contribute to inappropriate neuroplasticity underlying addiction.
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