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

Abstract

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.

Fig. 1

Schematic diagram to illustrate the time-line of repeated amphetamine experiments. In all experiments, rats received repeated injections (REP INJ) of amphetamine (AMPH) or saline (SAL) on six consecutive treatment days (TD). For Withdrawal Experiments 1–3, rats were challenged (CHAL) on withdrawal day 7 (WD7) and killed (KILL) on WD21 (indicated by labeling above horizontal line). For the Challenge Experiment, rats were challenged on WD20 and killed on WD21 (indicated by labeling below horizontal line). See Materials and Methods for details of each regimen.

Fig. 2

Representative immunoblots illustrating analysis of glutamate receptor subunit surface expression in nucleus accumbens tissue. Tissue was incubated for 15 min with the membrane-impermeant protein cross-linking reagent BS³, and then processed as described in the Materials and Methods section. BS³ selectively cross-links cell surface receptors, forming high molecular weight aggregates, while intracellular receptors are not modified. Thus, surface (S, arrowheads) and intracellular (I, arrows) pools can be distinguished based on molecular weight using SDS–PAGE and western blotting. Intracellular bands ran at their predicted molecular weights (in kD; GluR1, ∼101; GluR2, ∼99; GluR3, ∼99; NR2A, ∼166; NR2B, ∼166). Note that apparent differences in surface/intracellular ratios between particular subunits do not necessarily reflect differences in the relative portion of each subunit expressed on the surface because cross-linking conditions and antibody differences can affect the S/I ratio. The value of the assay lies in providing a measure of relative differences in S/I ratios between samples that are processed identically and probed with the same antibody.

Fig. 3

Effect of acute amphetamine (AMPH) injection on GluR1 distribution in the nucleus accumbens. Rats were killed 30 min [top: n = 16, saline (SAL); n = 17, AMPH], 2 h (middle; n = 8, SAL; n = 9, AMPH), or 24 h (bottom; n = 8, SAL; n = 8, AMPH) after injection of saline or 2.5 mg/kg amphetamine. A protein cross-linking assay was used to measure surface (S), intracellular (I), and total (S + I) GluR1 levels, and determine the GluR1 S/I ratio. S, I, and S + I values are normalized to total protein in the lane determined with Ponceau S staining. All data are mean ± SEM expressed relative to SAL control values. *p < 0.05 compared with saline group (anova and post hoc t-test).

Fig. 4

Ambulation counts (mean ± SEM) in response to amphetamine (AMPH) injection (2.5 mg/kg) on treatment day 1 and on the test (challenge) day. Panels a–d show results from Withdrawal Experiment 1 (n = 12), 2 (n = 15), 3 (n = 12) and the Challenge Experiment (n = 13). For Withdrawal Experiments 1–3, the test day was on withdrawal day 7 after discontinuing repeated amphetamine injections. For the Challenge Experiment, the test day was withdrawal day 20. The more aggressive regimen used in Withdrawal Experiments 1 and 2 (see Materials and Methods) produced a biphasic response pattern on the test day characterized by stereotypy during the first hour (reflected in decreased ambulation counts) followed by locomotor hyperactivity over the next 2 h. The more moderate regimen used for Withdrawal Experiment 3 and the Challenge Experiment did not result in consistent sensitization of stereotyped behaviors but did lead to robust post-stereotypy locomotor hyperactivity. See Results for statistical analyses.

Fig. 5

Effects of amphetamine challenge on GluR1 distribution in the nucleus accumbens (NAc; top) and dorsolateral striatum (DLSTR: bottom). Rats were treated repeatedly with saline or amphetamine for 6 days (see Materials and Methods), administered a challenge injection of saline or 2.5 mg/kg amphetamine on withdrawal day 20 and killed 24 h later. This resulted in four experimental groups: repeated saline/saline challenge, S/S (NAc, n = 14; DLSTR, n = 13); repeated saline/amphetamine challenge, S/A (NAc, n = 14; DLSTR, n = 14); repeated amphetamine/saline challenge, A/S (NAc, n = 14; DLSTR n = 13); and repeated amphetamine/amphetamine challenge, A/A (NAc, n = 13; DLSTR n = 13). A protein cross-linking assay was used to determine surface (S), intracellular (I), and total (S + I) GluR1 levels and the S/I ratio. S, I, and S + I values are normalized to total protein in the lane determined with Ponceau S staining. All data are mean ± SEM expressed relative to S control values. anova did not indicate significant group differences.