Increased extracellular glutamate in the nucleus accumbens promotes excessive ethanol drinking in ethanol dependent mice

Abstract

Using a well-established model of ethanol dependence and relapse, this study examined adaptations in glutamatergic transmission in the nucleus accumbens (NAc) and their role in regulating voluntary ethanol drinking. Mice were first trained to drink ethanol in a free-choice, limited access (2 h/day) paradigm. One group (EtOH mice) received repeated weekly cycles of chronic intermittent ethanol (CIE) exposure with intervening weeks of test drinking sessions, whereas the remaining mice (CTL mice) were similarly treated but did not receive CIE treatment. Over repeated cycles of CIE exposure, EtOH mice exhibited significant escalation in drinking (up to ∼3.5 g/kg), whereas drinking remained relatively stable at baseline levels (2-2.5 g/kg) in CTL mice. Using in vivo microdialysis procedures, extracellular glutamate (GLUEX) levels in the NAc were increased approximately twofold in EtOH mice compared with CTL mice, and this difference was observed 7 days after final CIE exposure, indicating that this hyperglutamatergic state persisted beyond acute withdrawal. This finding prompted additional studies examining the effects of pharmacologically manipulating GLUEX in the NAc on ethanol drinking in the CIE model. The non-selective glutamate reuptake antagonist, threo-β-benzyloxyaspartate (TBOA), was bilaterally microinjected into the NAc and found to dose-dependently increase drinking in nondependent (CTL) mice to levels attained by dependent (EtOH) mice. TBOA also further increased drinking in EtOH mice. In contrast, reducing glutamatergic transmission in the NAc via bilateral injections of the metabotropic glutamate receptor-2/3 agonist LY379268 reduced drinking in dependent (EtOH) mice to nondependent (CTL) levels, whereas having a more modest effect in decreasing ethanol consumption in CTL mice. Taken together, these data support an important role of glutamatergic transmission in the NAc in regulating ethanol drinking. Additionally, these results indicate that ethanol dependence produces adaptations that favor elevated glutamate activity in the NAc which, in turn, promote excessive levels of ethanol consumption associated with dependence.

Figure 1

Escalation of ethanol drinking in dependent (chronic intermittent ethanol, CIE-exposed) mice compared with relatively stable ethanol intake in nondependent controls. The data shown are weekly averages (means±SEM) for ethanol consumption (g/kg) during Baseline and Test cycles for EtOH and CTL groups. *P<0.05 significantly different from CTL group; ^P<0.05 significantly different from respective baseline level.

Figure 2

Extracellular glutamate concentrations in dialysate samples from NAc of ethanol dependent (EtOH) and nondependent (CTL) mice collected at baseline and after administration of 1 or 2 g/kg ethanol gavage. (a) Extracellular glutamate (GLU_EX; μM) levels were approximately twofold higher in EtOH (n=19) compared with CTL (n=16) mice. Analysis indicated that EtOH mice displayed significantly higher baseline of GLU_EX levels than CTL mice, but there was no effect of ethanol gavage in either group or either dose. *P<0.05 significantly differs from CTL values. (b) Significantly higher baseline GLU_EX levels in EtOH mice compared with CTL mice was confirmed by expressing data collapsed across Dose and Time factors. *P<0.05 significantly differs from CTL group. (c) Administering 1 g/kg ethanol by gavage did not alter GLU_EX levels in either EtOH or CTL groups of mice; data are expressed as percent of average baseline values for EtOH and CTL groups. (d) Administration of 2 g/kg ethanol by gavage did not significantly alter GLU_EX levels in either EtOH or CTL groups; data are expressed as percent of average baseline values for EtOH and CTL groups.

Figure 3

Effects of pharmacologically increasing extracellular glutamate (GLU_EX) levels in NAc via microinjection of the non-selective glutamate transporter blocker threo-β-benzyloxyaspartate (TBOA) on voluntary ethanol drinking in dependent and nondependent mice. (a) Ethanol consumption (g/kg) significantly increased over successive chronic intermittent ethanol (CIE) exposure cycles in EtOH mice (n=30) compared with CTL mice (n=39) before intra-NAc TBOA treatment. *P<0.05 significantly different from CTL group; ^P<0.05 significantly different from respective baseline level. (b) Bilateral microinjection of TBOA 30 min before limited access drinking sessions increased voluntary ethanol consumption in a dose-related manner in both CTL and EtOH groups. *P<0.05 significantly differs from CTL group; ^P<0.05 significantly differs from vehicle condition. Values are means±SEM.

Figure 4

Effects of pharmacologically reducing extracellular glutamate (GLU_EX) levels in NAc via microinjection of the metabotropic glutamate receptor-2/3 (mGluR2/3) agonist LY379268 on voluntary ethanol drinking in dependent and nondependent mice. (a) Ethanol consumption (g/kg) significantly increased over successive chronic intermittent ethanol (CIE) exposure cycles in EtOH mice (n=23) compared to CTL mice (n=37) prior to intra-NAc LY379268 treatment. *P<0.05 significantly different from CTL group; ^P<0.05 significantly different from respective baseline level. (b) Bilateral microinjection of LY379268 given 30 min before limited access drinking sessions decreased voluntary ethanol consumption in a dose-related manner in EtOH mice while producing a modest (non-significant) reduction in ethanol intake in CTL mice. *P<0.05 significantly differs from CTL group; ^P<0.05 significantly differs from respective vehicle condition. Values are means±SEM.

Figure 5

Microdialysis probe and microinjector tip placements in the NAc. Cresyl violet stained tissue sections showing typical placements for microdialysis probes (a) and the bilateral microinjector guides and tips (b). (c) Shaded areas depicted on plates from the atlas of Paxinos and Franklin (2001) indicating the areas where microdialysis probes and microinjection tips were located. Data were not included in analyses if more than 70% of the dialysis probe or one or both microinjector tips were outside the boundaries of the shaded area.