Quantitative analysis of AMPA receptor subunit composition in addiction-related brain regions.

The subunit composition of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) is an important determinant of AMPAR biophysical properties and trafficking. To date, AMPAR subunit composition has been quantitatively evaluated only for the hippocampus, where different experimental approaches have yielded different results. Here, we used quantitative co-immunoprecipitation to characterize GluA1-3 associations in the adult rat nucleus accumbens, dorsal striatum, prefrontal cortex, and hippocampus, and blue native electrophoresis (BNE) to study GluA1-3 assembly state. In all brain regions, co-immunoprecipitation experiments showed that ~90% of GluA1 was associated with GluA2 or GluA3 (most was GluA1A2). All regions contained a small number of GluA1A3 receptors. Homomeric GluA1 receptors may also exist. More than half of the GluA2 (53%-65% depending on the region) was not associated with GluA1. However, this represents an over-estimate of the percent of GluA2 present in GluA2A3 receptors, based on BNE results demonstrating that the majority of GluA2 exists as dimers, rather than functional tetrameric receptors. Relatively more GluA1 was present in tetramers. Together with other findings, our results suggest a dominant role for GluA1A2 receptors in all brain regions examined. They also help explain why different results for hippocampal AMPAR subunit composition were obtained using co-immunoprecipitation, which assesses the total cellular pool of AMPARs including partially assembled AMPARs in intracellular compartments, and electrophysiological approaches, which can selectively assess tetrameric (functional) AMPARs on the cell surface.

Behavioral sensitization to amphetamine is not accompanied by changes in glutamate receptor surface expression in the rat nucleus accumbens.

We examined whether behavioral sensitization to amphetamine is associated with redistribution of glutamate receptors (GluR) in the rat nucleus accumbens (NAc) or dorsolateral striatum (DLSTR). Following repeated amphetamine treatment and 21 days of withdrawal, surface and intracellular levels of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or NMDA receptor subunits were determined using a protein cross-linking assay. In contrast to our previous results in cocaine-sensitized rats, we did not observe redistribution of GluR1 or GluR2 to the cell surface in the NAc after amphetamine withdrawal, although a small increase in total GluR1 was found in the shell subregion. Nor did we observe activation of signaling pathways associated with cocaine-induced AMPA receptor trafficking or changes in NMDA receptor subunits. No significant changes were observed in the DLSTR. We also investigated the effect of administering a challenge injection of amphetamine to amphetamine-sensitized rats 24 h prior to biochemical analysis based on prior studies showing that cocaine challenge decreases AMPA receptor surface expression in the NAc of cocaine-sensitized rats. GluR1 and GluR2 were not significantly altered in either NAc or DLSTR, although a modest effect on GluR3 cannot be ruled out. Our results suggest that glutamate transmission in the NAc is dramatically different in rats sensitized to amphetamine versus cocaine.

Acute and chronic dopamine receptor stimulation modulates AMPA receptor trafficking in nucleus accumbens neurons cocultured with prefrontal cortex neurons.

Postsynaptic interactions between dopamine (DA) and glutamate receptors in the nucleus accumbens (NAc) are critical for addiction. To determine the effect of acute and repeated DA receptor stimulation on AMPA receptor (AMPAR) synaptic targeting in medium spiny NAc neurons, we developed a model system consisting of rat NAc neurons cocultured with prefrontal cortex neurons from enhanced green fluorescent protein-expressing mice. Cortical neurons restore excitatory input onto NAc neurons but are distinguishable based on fluorescence. First, we showed that brief D1-like agonist exposure increased AMPAR insertion onto extrasynaptic regions of NAc neuronal processes through a mechanism requiring protein kinase A. This facilitated the Ca2+/calmodulin dependent protein kinase II (CaMKII)-dependent synaptic incorporation of AMPARs in response to subsequent NMDA receptor (NMDAR) stimulation. Through this mechanism, DA may promote reward- and drug-related plasticity in the NAc. Then, to model effects of repeated in vivo cocaine exposure, we treated cocultures with DA (1 microm, 30 min) on days 7, 9, and 11 in culture. On day 15, NAc neurons exhibited increased synaptic AMPAR levels. This was associated with CaMKII activation and was blocked by the CaMKII inhibitor KN-93 (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide phosphate salt). Furthermore, D1-like agonist exposure on day 15 no longer increased AMPAR surface expression. This refractoriness was associated with decreased D1 receptor surface expression. NMDAR surface expression was not altered by acute or repeated DA receptor stimulation. These results suggest that (1) after repeated DA treatment, NAc neurons are more responsive to glutamate inputs but D(1)-like receptor regulation of plasticity is impaired, and (2) NAc/prefrontal cortex cocultures are useful for studying dopamine-induced neuroadaptations.

Cell surface AMPA receptors in the rat nucleus accumbens increase during cocaine withdrawal but internalize after cocaine challenge in association with altered activation of mitogen-activated protein kinases.

Although some studies report increased responsiveness of nucleus accumbens (NAc) AMPA receptors (AMPARs) after withdrawal from repeated cocaine treatment, others report decreased responsiveness after withdrawal plus cocaine challenge. Here we examine this apparent contradiction by quantifying cell surface and intracellular AMPAR subunits in the NAc before and after a challenge injection in behaviorally sensitized rats. Because MAPKs (mitogen-activated protein kinases) regulate AMPAR trafficking and are implicated in addiction, we also evaluated phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Glutamate receptor 1 (GluR1) and GluR2 surface/intracellular (S/I) ratios were increased after 14 d of withdrawal in sensitized rats but were decreased 24 h after challenge with cocaine (which elicited a sensitized locomotor response) or saline (which elicited conditioned locomotion). These findings suggested redistribution of GluR1/2-containing receptors, a possibility supported by immunoprecipitation experiments indicating that most AMPARs in the NAc are GluR1/2 or GluR2/3, with few homomeric GluR1 or GluR1/3 receptors. In sensitized rats, ERK phosphorylation in the NAc increased during withdrawal and normalized after cocaine challenge. JNK phosphorylation also increased after withdrawal, but after cocaine challenge, it was inversely related to GluR1 and GluR2 S/I ratios. After saline challenge, p38 phosphorylation was increased. In summary, surface expression of GluR1/2-containing AMPARs increased in the NAc of sensitized rats, but AMPARs internalized after a single reexposure to cocaine or cocaine-related cues. ERK phosphorylation paralleled AMPAR surface expression. Although JNK results were complex, JNK and p38 may be involved in AMPAR internalization after cocaine or saline challenge, respectively.

Crystallization and X-ray diffraction analysis of an all-RNA U39C mutant of the minimal hairpin ribozyme.

The hairpin ribozyme is a naturally occurring catalytic RNA composed of two helix-loop-helix domains, A and B, that dock to form the biologically active enzyme. Previously, the crystal structure of the hairpin has been solved as a four-way helical junction that incorporated the U1A protein as an artificial crystal-packing motif [Rupert & Ferré-D'Amaré (2001), Nature (London), 410, 780-786]. Here, the crystallization of a minimal junctionless hairpin ribozyme 64-mer is reported in the absence of protein. Crystals grow in space group P6(1)22, with unit-cell parameters a = 93.1, c = 123.2 A. Complete diffraction data have been collected to 3.35 A resolution. Structural analysis should provide details of intermolecular RNA docking, including the ground-state conformations of the U(39)C mutation relevant to hairpin catalysis.