Changes in gene expression in regions of the extended amygdala of alcohol-preferring rats after binge-like alcohol drinking.

The objective of this study was to determine time-course changes in gene expression within two regions of the extended amygdala after binge-like alcohol drinking by alcohol-preferring (P) rats. Adult male P rats were given 1-h access to 15 and 30% ethanol three times daily for 8 weeks. Rats (n = 10/time point for ethanol and n = 6/time point for water) were killed by decapitation 1, 6, and 24 h after the last drinking episode. RNA was prepared from individual micropunch samples of the nucleus accumbens shell (ACB-shell) and central nucleus of the amygdala (CeA); analyses were conducted with Affymetrix Rat Genome 230.2 GeneChips. Ethanol intakes were 1.5-2 g/kg for each of the three sessions. There were no genes that were statistically different between the ethanol and water control groups at any individual time point. Therefore, an overall effect, comparing the water control and ethanol groups, was determined. In the ACB-shell and CeA, there were 276 and 402 probe sets for named genes, respectively, that differed between the two groups. There were 1.5-3.6-fold more genes with increased expression than with decreased expression in the ethanol-drinking group, with most differences between 1.1- and 1.2-fold. Among the differences between the ethanol and water control groups were several significant biological processes categories that were in common between the two regions (e.g., synaptic transmission, neurite development); however, within these categories, there were few genes in common between the two regions. Overall, the results indicate that binge-like alcohol drinking by P rats produces region-dependent changes in the expression of genes that could alter transcription, synaptic function, and neuronal plasticity in the ACB-shell and CeA; within each region, different mechanisms may underlie these alterations because there were few common ethanol-responsive genes between the ACB-shell and CeA.

Differential effects of ethanol in the nucleus accumbens shell of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar rats: a proteomics study.

The objective of this study was to determine the effects of ethanol injections on protein expression in the nucleus accumbens shell (ACB-sh) of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar (W) rats. Rats were injected for 5 consecutive days with either saline or 1 g/kg ethanol; 24 h after the last injection, rats were killed and brains obtained. Micro-punch samples of the ACB-sh were homogenized; extracted proteins were subjected to trypsin digestion and analyzed with a liquid chromatography-mass spectrometer procedure. Ethanol changed expression levels (1.15-fold or higher) of 128 proteins in NP rats, 22 proteins in P, and 28 proteins in W rats. Few of the changes observed with ethanol treatment for NP rats were observed for P and W rats. Many of the changes occurred in calcium-calmodulin signaling systems, G-protein signaling systems, synaptic structure and histones. Approximately half the changes observed in the ACB-sh of P rats were also observed for W rats. Overall, the results indicate a unique response to ethanol of the ACB-sh of NP rats compared to P and W rats; this unique response may reflect changes in neuronal function in the ACB-sh that could contribute to the low alcohol drinking behavior of the NP line.

In vivo time-course changes in ethanol levels sampled with subcutaneous microdialysis.

BACKGROUND: The objective of this study was to determine time-course changes in in vivo ethanol (EtOH) concentrations using a novel subcutaneous (s.c.) microdialysis sampling technique. The hypothesis to be tested was that EtOH concentrations in the s.c. fluid would reflect blood EtOH concentrations. If this is the case, then s.c. microdialysis could allow a more detailed analysis of changes in in vivo levels of EtOH under different drinking paradigms. METHODS: Adult male and female Wistar rats and male alcohol-preferring (P) rats were used in this study. A loop-style microdialysis probe was designed for s.c. applications. After initial in vitro characterization, probes were implanted under the skin between the shoulder blades. Animals were allowed to recover 4 to 24 hours prior to microdialysis collection (2.0 microl/min flow rate with isotonic saline). In vivo microdialysis experiments were then conducted to determine (i) the extraction fraction (or clearance) using EtOH no-net-flux (NNF) coupled with the alcohol clamp method, (ii) the dose-response and time-course effects after systemic EtOH administration and to compare with blood EtOH levels, and (iii) the time-course changes in EtOH levels during and after an EtOH drinking episode. RESULTS: In vivo probe recovery (extraction fraction) obtained using the alcohol clamp method was 69 +/- 3%, and was comparable to the in vitro recovery of 73 +/- 2%. For the EtOH dose-response experiment, rats injected i.p. with 0.5, 1.0, or 2.0 g/kg EtOH showed a clear dose-response effect in the s.c. dialysate samples. Peak concentrations (70, 123, and 203 mg%, respectively) were reached by 15 minutes after injection. In an experiment comparing levels of EtOH in s.c. dialysis and arterial blood samples in rats administered 1.0 g/kg EtOH, similar time-course changes in in vivo EtOH concentrations were observed with both i.g. and i.p. EtOH administration. In P rats drinking 15% EtOH during a 1-hour scheduled access period, EtOH levels in s.c. microdialysates rose rapidly over the session and peaked at approximately 50 mg% at 60 to 80 minutes. CONCLUSIONS: Overall, these experiments indicate that s.c. EtOH and blood EtOH concentrations follow a similar time course. Moreover, s.c. microdialysis can be useful as an experimental approach for determining detailed time-course changes in in vivo EtOH concentrations associated with alcohol drinking episodes.

Effects of ethanol drinking on central nervous system functional activity of alcohol-preferring rats.

The [(14)C]-2-deoxyglucose (2-DG) technique was used to assess the rates of local cerebral glucose utilization (LCGU) in key limbic, cerebral cortical, hippocampal, basal ganglionic, and subcortical regions of alcohol-preferring (P) rats following chronic 24-h free-choice ethanol drinking. Adult male P rats were submitted to (1) 8 continuous weeks of two-bottle access to 15% ethanol and water (E-C group); (2) 8 weeks of identical two-bottle access followed by 2 weeks of ethanol deprivation (E-D group); (3) cycles of 2 weeks of two-bottle ethanol access and 2 weeks of deprivation, repeated for four cycles (E-RD group); or (4) water only treatment [ethanol-naive group (E-N group)]. A single pulse of [(14)C]-2-DG (125 microCi/kg) was administered via a venous catheter, and timed arterial blood samples were collected over 45 min and later assayed for plasma glucose and [(14)C]-2-DG concentrations. Quantitative autoradiography was used to determine [(14)C] densities, and LCGU values were calculated. With the exception of a few small differences in the hippocampus, no significant differences were found in any of the central nervous system (CNS) regions examined among the four experimental groups of P rats. Animals in the E-D group had lower LCGU rates in the anterior hippocampal CA1 subregion than animals in the E-N, E-C, and E-RD groups. In the anterior hippocampal CA3 subregion and the anterior hippocampal dentate gyrus, the E-D group had significantly lower LCGU rates than the E-RD group. Overall, the results of this study indicate that 24-h ethanol-drinking experience has little effect on CNS functional neuronal activity in P rats.