Elevation of Extracellular Glutamate by Blockade of Astrocyte Glutamate Transporters Inhibits Cocaine Reinforcement in Rats via a NMDA-GluN2B Receptor Mechanism

Abstract

It is well established that glutamate plays an important role in drug-induced and cue-induced reinstatement of drug seeking. However, the role of glutamate in drug reward is unclear. In this study, we systemically evaluated the effects of multiple glutamate transporter (GLT) inhibitors on extracellular glutamate and dopamine (DA) in the nucleus accumbens (NAc), intravenous cocaine self-administration, intracranial brain-stimulation reward (BSR), and reinstatement of cocaine seeking in male and female rats. Among the five GLT inhibitors we tested, TFB-TBOA was the most potent. Microinjections of TFB-TBOA into the NAc, but not the ventral tegmental area (VTA), or dorsal striatum (DS), dose-dependently inhibited cocaine self-administration under fixed-ratio and progressive-ratio (PR) reinforcement schedules, shifted the cocaine dose-response curve downward, and inhibited intracranial BSR. Selective downregulation of astrocytic GLT-1 expression in the NAc by GLT-1 antisense oligonucleotides also inhibited cocaine self-administration. The reduction in cocaine self-administration following TFB-TBOA administration was NMDA GluN2B receptor dependent, and rats self-administering cocaine showed upregulation of GluN2B expression in NAc DA- and cAMP-regulated phosphoprotein 32 (DARPP-32)-positive medium-spiny neurons (MSNs). In contrast, TFB-TBOA, when locally administered into the NAc, VTA, or ventral pallidum (VP), dose-dependently reinstated cocaine-seeking behavior. Intra-NAc TFB-TBOA-evoked drug-seeking was long-lasting and NMDA/AMPA receptor dependent. These findings, for the first time, indicate that glutamate in the NAc negatively regulates cocaine's rewarding effects, while an excess of glutamate in multiple brain regions can trigger reinstatement of drug-seeking behavior.SIGNIFICANCE STATEMENT It is well known that glutamate plays an important role in relapse to drug seeking. However, the role of glutamate in drug reward is less clear. Here, we report that TFB-TBOA, a highly potent glutamate transporter (GLT) inhibitor, dose-dependently elevates extracellular glutamate and inhibits cocaine self-administration and brain-stimulation reward (BSR), when administered locally into the nucleus accumbens (NAc), but not other brain regions. Mechanistic assays indicate that cocaine self-administration upregulates NMDA-GluN2B receptor subtype expression in striatal dopaminoceptive neurons and activation of GluN2B by TFB-TBOA-enhanced glutamate inhibits cocaine self-administration. TFB-TBOA also reinstates cocaine-seeking behavior when administered into the NAc, ventral tegmental area (VTA), and ventral pallidum (VP). These findings demonstrate that glutamate differentially regulates cocaine reward versus relapse, reducing cocaine reward, while potentiating relapse to cocaine seeking.

Keywords: TFB-TBOA; astrocyte; cocaine; glutamate; glutamate transporter; reward.

Figure 1.

Effects of five EAAT inhibitors on extracellular dopamine (DA) and glutamate in the NAc. A, Diagram showing the cellular distributions of EAAT1, EAAT2, and EAAT3 in the brain and the targets each EAAT inhibitor acts on; B–D, Effects of intra-NAc local infusion of the EAAT1 (UCPH-101), EAAT2 (DHK), or EAAT3 (L-β-BA) inhibitors on extracellular DA and glutamate, respectively. E, F, Effects of the nonselective EAAT inhibitor (DL-TBOA) or the astrocytic EAAT1 and EAAT2 inhibitor (TFB-TBOA) on extracellular DA and glutamate. All EAAT inhibitors increased extracellular glutamate, but not DA, in the NAc; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the baseline before the drug infusion.

Figure 2.

Effects of microinjections of EAAT inhibitors on cocaine self-administration under an FR2 reinforcement schedule. A, Representative histologic images showing the tips of guide cannulae and the predicted microinjection areas in the NAc, DS, and VTA. B, Intra-NAc microinjections of UCPH-101, DHK, or L-β-BA did not alter cocaine self-administration. C, Intra-NAc DL-TBOA or TFB-TBOA dose-dependently attenuated lever presses for cocaine. D, TFB-TBOA microinjections in the VTA or DS had no effect on cocaine self-administration. E, F, G, Representative cocaine self-administration (infusions) records showing the extinction-like pattern of cocaine self-administration (infusions) observed 30 min after TFB-TBOA microinjection into the NAc (D), but not into the VTA (E) or DS (F); *p < 0.05, ***p < 0.001, compared with the vehicle control group.

Figure 3.

Effects of intra-NAc TFB-TBOA on cocaine or sucrose reward and locomotor activity. A, Intra-NAc TFB-TBOA microinjections shifted the cocaine self-administration dose–response curve downward. B, Intra-NAc TFB-TBOA lowered break-points for cocaine self-administration under a PR reinforcement schedule. C, Intra-NAc TFB-TBOA inhibited oral sucrose self-administration under a FR2 reinforcement schedule. D, Microinjections of TFB-TBOA into the DS had no effect on sucrose self-administration. E, Microinjections of TFB-TBOA into the NAc had no effect on cocaine-induced hyperactivity. F, Intra-NAc TFB-TBOA failed to alter basal locomotor activity; *p < 0.05, **p < 0.01, ***p < 0.001, compared with vehicle control group.

Figure 4.

Effects of intra-NAc administration of TFB-TBOA on electrical BSR in rats. A, A diagram illustrating the location of a stimulation electrode in the MFB at the anterior–posterior level of the lateral hypothalamus. B, A schematic diagram, showing that cocaine shifted the curve to the left and decreased the BSR stimulation threshold (θ₀), while TFB-TBOA shifted the curve to the right and increased θ₀. C, D, Cocaine dose-dependently decreased, while TFB-TBOA dose-dependently increased BSR thresholds (θ₀). E, TFB-TBOA failed to alter Y_max; *p < 0.05, ***p < 0.001, compared with the vehicle control group.

Figure 5.

Downregulation of GLT-1 expression via antisense oligo infusions into the NAc inhibits cocaine self-administration. A, Experimental timeline of events for GLT-1 antisense infusions and cocaine self-administration. B, C, Representative images of GLT-1-immunostaining in NAc sections taken at three magnifications (4×, 20×, 40×) from rats exposed to non-sense (control) or antisense oligos. D, GLT-1 density was significantly lower in the NAc of rats receiving GLT-1 antisense oligos than those receiving non-sense oligos. E, Intra-NAc antisense oligo administration inhibited cocaine self-administration in a dose-dependent manner; *p < 0.05, **p < 0.01, ***p< 0.001, compared with vehicle control group.

Figure 6.

Receptor mechanisms underlying TFB-TBOA-induced reduction in cocaine self-administration. A, Intra-NAc pretreatment with DNQX had no effect on TFB-TBOA-induced reduction in cocaine self-administration. DNQX alone (10 µg) inhibited cocaine self-administration. B, Pretreatment with accumbal AP5 dose-dependently reversed TFB-TBOA-mediated decrease in cocaine self-administration. C, NVP-AAM077 pretreatment (selective GluN2A blocker) failed to alter TFB-TBOA-induced suppression of cocaine self-administration. D, Intra-NAc ifenprodil (a GluN2B blocker) blocked the decrease in cocaine self-administration caused by TFB-TBOA in a dose-dependent manner. E, Microinjection of Ro-25-6981 (a selective GluN2B blocker) into the NAc prevented the TFB-TBOA-induced reduction in cocaine self-administration; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the vehicle group without TFB-TBOA; #p < 0.05, ##p < 0.01, ###p < 0.01, compared with each glutamate receptor antagonist vehicle group in the presence of TFB-TBOA.

Figure 7.

Cocaine self-administration increases NMDA-GluN2B receptor expression in NAc DARPP32 neurons. A–D, Representative fluorescent images illustrating GluN2B cells (green), DARPP32 cells (red), and their co-expression (yellow) in the NAc shell (A, B) or core (C, D) in rats with a history of cocaine self-administration or yoked saline treatment. E, No difference in the density of DARPP32 was observed between cocaine-treated and saline-treated rats in either the NAc core or shell. F, No difference in the density of GluN2B was observed between cocaine-treated and saline-treated rats. G, Cocaine self-administration increased GluN2B expression in DARPP32 neurons in the NAc core and shell relative to controls; *p < 0.05, ***p< 0.001, compared with saline control rats.

Figure 8.

Reinstatement responses caused by intracranial microinjections of TFB-TBOA into the NAc, VP, VTA, or DS in rats after extinction from cocaine self-administration. A–D, Representative histologic images showing the traces of guide cannulae in the brain and the predicted microinjection areas in the NAc (A), VP (B), VTA (C), or DS (D). E–H, Microinjections of TFB-TBOA into the NAc (E), VP (F), and VTA (G), but not the DS (H), induced reinstatement of cocaine seeking in a dose-dependent manner following extinction from cocaine self-administration. I, J, The time course of active lever responding (drug seeking) before and after TFB-TBOA microinjections into the NAc (I) or VTA (J), illustrating that intra-NAc TFB-TBOA caused a long-term increase in cocaine-seeing behavior. K, Pretreatment with AP5 (2 µg/side, NAc) or DNQX (2 µg/side, NAc) blocked TFB-TBOA-induced reinstatement of cocaine-seeking behavior; *p < 0.05, **p < 0.01, ***p< 0.001, compared with vehicle (E–G, K) or baseline (I, J); ##p < 0.01 compared with the group administered (Veh + 3 µg/side TFB-TBOA) (K).

Figure 9.

Diagram showing the astrocyte EAAT-glutamate-extrasynaptic NMDA-GluN2B (NR_2B) receptor mechanism underlying TFB-TBOA modulation of cocaine reward and relapse. TFB-TBOA is a highly potent and selective astrocytic EAAT1/2 inhibitor. Chronic cocaine administration causes a reduction in basal glutamate transmission and NMDA-GluN2B up-regulation in NAc DARPP-32⁺ dopaminoceptive neurons. Intra-NAc local administration of TFB-TBOA blocks glutamate reuptake causing an increase in extracellular glutamate levels. Glutamate then activates extrasynaptic NMDA-GluN2B receptors, which subsequently antagonizes cocaine-induced or DA-induced reduction in DARPP-32⁺ dopaminoceptive neurons (D2-MSNs). This causes a reduction in cocaine reward in the presence of cocaine and an increase in relapse to cocaine seeking in the absence of cocaine. NR_2A (GluN2A); NR_2B (GluN2B).