Alcohol drinking and deprivation alter basal extracellular glutamate concentrations and clearance in the mesolimbic system of alcohol-preferring (P) rats

Abstract

The present study determined the effects of voluntary ethanol drinking and deprivation on basal extracellular glutamate concentrations and clearance in the mesolimbic system and tested the hypothesis that chronic ethanol drinking would persistently increase basal glutamate neurotransmission. Three groups of alcohol-preferring (P) rats were used: 'water group (WG),' 'ethanol maintenance group (MG; 24-hour free choice water versus 15% ethanol)' and 'ethanol deprivation group (DG; 2 weeks of deprivation).' Quantitative microdialysis and Western blots were conducted to measure basal extracellular glutamate concentrations, clearance and proteins associated with glutamate clearance. Chronic alcohol drinking produced a 70-100% increase of basal extracellular glutamate concentrations in the posterior ventral tegmental area (4.0 versus 7.0 μM) and nucleus accumbens shell (3.0 versus 6.0 μM). Glutamate clearances were reduced by 30-40% in both regions of MG rats compared with WG rats. In addition, Western blots revealed a 40-45% decrease of excitatory amino transporter 1 (EAAT1) protein, but no significant changes in the levels of EAAT2 or cystine-glutamate antiporter in these regions of MG versus WG rats. The enhanced glutamate concentrations returned to control levels, accompanied by a recovery of glutamate clearance following deprivation. These results indicated that chronic alcohol drinking enhanced extracellular glutamate concentrations in the mesolimbic system, as a result, in part, of reduced clearance, suggesting that enhanced glutamate neurotransmission may contribute to the maintenance of alcohol drinking. However, because the increased glutamate levels returned to normal after deprivation, elevated glutamate neurotransmission may not contribute to the initiation of relapse drinking.

Figure 1

Representative placements of microdialysis probes and micro-punch samples in the nucleus accumbens shell (NACsh, A) and posterior ventral tegmental area (VTA, B). The posterior VTA was determined based on previous findings (Ding et al 2009, Rodd et al 2004). The lines represent the microdialysis membrane and circles represent micro-punch samples. Micro-punch samples were taken from both sides of the NACsh and posterior VTA, but are only indicated in the left side of the brain. Overlapping probes and micro-punch samples are not shown for clarity purposes.

Figure 2

Effects of ethanol maintenance (MG) and deprivation (DG) on extracellular glutamate concentrations and extraction fractions (Eds) in the nucleus accumbens shell (NACsh, n = 8-10 /group). A) Linear regression plots of glutamate for the water (WG), MG and DG groups; B) extracellular glutamate concentrations determined from points of no-net-flux in each group; C) Ed values of glutamate determined from the slope of the plots for the linear regression analysis in each group. * p < 0.05, significantly different from the WG group.

Figure 3

Effects of ethanol maintenance (MG) and deprivation (DG) on extracellular glutamate concentrations and extraction fractions (Eds) in the posterior ventral tegmental area (VTA, n= 7-8 / group). A) Linear regression plots of glutamate for the water (WG), MG and DG groups; B) extracellular glutamate concentrations determined from points of no-net-flux in each group; C) Ed values of glutamate determined from the slope of the plots for the linear regression analysis in each group. * p < 0.05, significantly different from the WG group.

Figure 4

Western blot analysis indicating effects of ethanol maintenance (MG) and deprivation (DG) on the protein level of excitatory amino acid transporter 1 & 2 (EAAT1 & 2), and the cystine-glutamate antiporter (xCT) in the nucleus accumbens shell (NACsh, n = 6-9 / group). * p < 0.05, significantly different from the WG group.

Figure 5

Western blot analysis indicating effects of ethanol maintenance (MG) and deprivation (DG) on the protein level of excitatory amino acid transporter 1 & 2 (EAAT1 & 2), and the cysteine-glutamate antiporter (xCT) in the posterior ventral tegmental area (n = 6-9 / group). * p < 0.05, significantly different from the WG group.