Contrasting the Role of xCT and GLT-1 Upregulation in the Ability of Ceftriaxone to Attenuate the Cue-Induced Reinstatement of Cocaine Seeking and Normalize AMPA Receptor Subunit Expression

Abstract

Long-term treatment with ceftriaxone attenuates the reinstatement of cocaine seeking while increasing the function of the glutamate transporter 1 (GLT-1) and system xC- (Sxc) in the nucleus accumbens core (NAc). Sxc contributes the majority of nonsynaptic extracellular glutamate in the NAc, while GLT-1 is responsible for the majority of glutamate uptake. Here we used antisense to decrease the expression of GLT-1 and xCT (a catalytic subunit of Sxc) to determine the relative importance of both proteins in mediating the ability of ceftriaxone to prevent cue-induced reinstatement of cocaine seeking and normalize glutamatergic proteins in the NAc of rats. Intra-NAc xCT knockdown prevented ceftriaxone from attenuating reinstatement and from upregulating GLT-1 and resulted in increased surface expression of AMPA receptor subunits GluA1 and GluA2. Intra-NAc GLT-1 knockdown also prevented ceftriaxone from attenuating reinstatement and from upregulating xCT expression, without affecting GluA1 and GluA2 expression. In the absence of cocaine or ceftriaxone treatment, xCT knockdown in the NAc increased the expression of both GluA1 and GluA2 without affecting GLT-1 expression while GLT-1 knockdown had no effect. PCR and immunoprecipitation of GLT-1 revealed that ceftriaxone does not upregulate GLT-1 and xCT through a transcriptional mechanism, and their coregulation by ceftriaxone is not mediated by physical interaction. These data support important and distinct roles for xCT and GLT-1 in the actions of ceftriaxone and add to a body of literature finding evidence for coregulation of these transporters. Our results also point to xCT expression and subsequent basal glutamate levels as being a key mediator of AMPA receptor expression in the NAc.SIGNIFICANCE STATEMENT Ceftriaxone attenuates the reinstatement of cocaine, alcohol, and heroin seeking. The mechanism of action of this behavioral effect has been attributed to glutamate transporter 1 (GLT-1) and xCT (a catalytic subunit of Sxc)/Sxc upregulation in the nucleus accumbens core. Here we used an antisense strategy to knock down GLT-1 or xCT in the nucleus accumbens core and examined the behavioral and molecular consequences. While upregulation of both xCT and GLT-1 are essential to the ability of ceftriaxone to attenuate cue-induced reinstatement of cocaine seeking, each protein uniquely affects the expression of other glutamate receptor and transporter proteins. We also report that reducing basal glutamate levels through the manipulation of xCT expression increases the surface expression of AMPA receptor subunits, providing insight to the mechanism by which cocaine alters AMPA surface expression.

Keywords: glutamate; glutamate transporters; microdialysis; nucleus accumbens; relapse.

Figure 1.

Self-administration, extinction, and reinstatement lever pressing in rats receiving intranucleus accumbens core AS directed at xCT or Ctrl oligo and Cef or Veh. A, Timeline for the experiments yielding the data graphed in Figures 1, 2, and 3. B, Active lever presses during self-administration and extinction. C, Inactive lever pressing during self-administration and extinction. D, Active lever pressing during the last 2 d of extinction (mean) and during a cue-primed reinstatement test. Reinstatement of cocaine seeking is defined as a significant increase in active lever pressing from extinction to test. The knockdown of xCT blocked the ability of Cef to prevent reinstatement. *p < 0.05 comparing extinction to cue test (n = 7–12/group, indicated in D). E, Inactive lever presses did not differ by group or test. F, A subset of the correct placements are noted here as examples of the total of 40 bilateral cannulae locations.

Figure 2.

Self-administration, extinction, and reinstatement lever pressing in rats receiving intra-nucleus accumbens core AS directed at GLT-1 or Ctrl oligo. A, Active lever presses during self-administration and extinction. B, Inactive lever pressing during self-administration and extinction. C, Active lever pressing during the last 2 d of extinction (mean) and during a cue-primed reinstatement test. The knockdown of GLT-1 blocked the ability of Cef to prevent reinstatement. *p < 0.05 comparing extinction to cue test. #p < 0.05 compared to Cef-Ctrl (n = 7/group). D, Inactive lever pressing. E, A subset of the 28 correct placements are noted here as example cannulae placements.

Figure 3.

Surface expression of GluA1 and GluA2 and total protein expression of xCT and GLT-1. Rats self-administered Coc or received yoked-saline infusions (Sal) and were treated with either Cef or Veh during extinction training. Rats received intra-NAc AS directed at either GLT-1 or xCT or received Ctrl oligo according to the timeline in Figure 1A. Protein expression is expressed as a percentage of the Sal-Veh-Ctrl group. A, Active lever pressing during self-administration and extinction. B, Inactive lever pressing during self-administration. C, Total protein expression of GLT-1 is decreased by Coc administration and restored by Cef administration. The knockdown of both xCT and GLT-1 itself decreases the total expression of GLT-1 in Coc-Cef-treated rats. D, Total protein expression of xCT is upregulated by Cef relative to Veh-treated Coc and Sal-Veh-Ctrl rats. The knockdown of xCT and GLT-1 prevents this upregulation by Cef. E, Surface GluA1 expression was increased by cocaine and normalized by Cef. The knockdown of xCT negated the ability of Cef to normalize GluA1 expression. F, Surface GluA2 was unaltered by Coc, but the knockdown of xCT increased GluA2 expression. *p < 0.05 compared to the Sal-Veh-Ctrl group. #p < 0.05 compared to the Coc-Cef-Ctrl group. SVC, Sal-Veh-Ctrl; CVC, Coc-Veh-Ctrl; CCC, Coc-Cef-Ctrl; CCG, Coc-Cef-GLT-1 AS; CCx, Coc-Cef-xCT AS.

Figure 4.

Surface expression of GluA1 and GluA2 and total protein expression of xCT and GLT-1. Cocaine-naive rats received intra-NAc AS directed at xCT (n = 12) or Ctrl oligo (n = 10). Protein expression was expressed as a percentage of the Ctrl group. A, Overall, there was no effect of xCT knockdown on GLT-1 expression in the total protein fraction. B, The infusion of xCT AS successfully reduced xCT expression. C, Surface GluA1 was increased by xCT knockdown. D, Surface GluA2 was increased by xCT knockdown. *p < 0.05 compared to Ctrl. C, Ctrl oligo, x, xCT AS.

Figure 5.

Surface expression of GluA1 and GluA2 and total protein expression of xCT and GLT-1. Cocaine-naive rats received intra-NAc AS directed at GLT-1 (n = 5) or Ctrl oligo (n = 5). Protein expression was expressed as a percentage of the Ctrl group. A, GLT-1 antisense decreased total protein expression of GLT-1. B, The infusion of GLT-1 AS had no effect on total xCT expression. C, Surface GluA1 was not affected by GLT-1 knockdown. D, Surface GluA2 was not affected by GLT-1 knockdown. *p < 0.05 compared to Ctrl. C, Ctrl oligo; G, GLT-1 AS.

Figure 6.

No-net-flux microdialysis revealed that basal extracellular glutamate is reduced by intra-NAc infusion of xCT antisense. A, The concentration of glutamate perfused was plotted against the net flux of glutamate. B, Computing the point of no net-flux across the dialysis membrane in A and making a comparison between groups revealed that xCT antisense significantly reduced the basal glutamate level relative to the Ctrl group. C, Microdialysis probe tracks through the NAc; representative examples of dialysis probes are depicted along the rostrocaudal axis of the NAc. *Indicates significant difference between groups (p < 0.05).

Figure 7.

Nucleus accumbens core total protein and mRNA expression of GLT-1 isoforms following cocaine self-administration and 2–3 weeks of extinction (Coc) or yoked-saline infusions (Sal). Cef or Veh treatment occurred during the last 6 d of extinction training. A, Rats later assigned to receive Cef or Veh did not differ in active lever pressing during self-administration and extinction. B, Inactive lever presses during self-administration and extinction. C–E, GLT-1 (C), GLT-1b (D), and xCT (E) mRNA expression were not altered by either Coc or Cef. F–H, However, total protein expression of GLT-1a (F), GLT-1b (G), and xCT (H) in the same animals was altered by Coc and Cef. *p < 0.05 compared to Sal-Veh group; line with * indicates significant (p < 0.05) effect of group with one-way ANOVA.

Figure 8.

GLT-1 and xCT are not physically associated with one another in the nucleus acccumbens core after cocaine or ceftriaxone administration. Rats self-administered cocaine or received yoked saline infusions, underwent extinction training, and were treated with Veh or Cef. A GLT-1 pulldown was conducted on freshly dissected NAc tissue. A, B, Western blots against nGLT-1 confirmed that we successfully immunoprecipitated GLT-1 (A) and Na⁺/K⁺ ATPase α3 (B), a protein known to be physically associated with GLT-1. C, xCT protein did not immunoprecipitate with GLT-1 under any condition. Negative control, Immunoprecipitation with an irrelevant antibody; α3 (m), α3 monomer; α3 (d), α3 dimer; S-V, yoked-saline plus vehicle; C-V, cocaine plus vehicle; S-C, cocaine plus ceftriaxone; C-C, cocaine plus ceftriaxone.