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

Abstract

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.

Fig. 1

Total ion chromatograms of the 1^st, 30^th and 58^th injections illustrating that the high quality of the chromatograms did not diminish from the 1^st to the 58^th injection.

Fig. 2

Extracted ion chromatogram for peptide EIYTHFTCATDTK from protein IPI002311733.6 (guanine nucleotide-binding protein Gi, alpha-1 subunit) showing the retention time in min along the X-axis and the intensity along the Y-axis. The heavy solid line represents the AUC for this peptide.

Fig. 3

Abridged Ingenuity® Pathways Analysis of effects of ethanol in the nucleus accumbens shell of NP rats showing up-regulation of calcium/calmodulin signaling pathways. Red indicates up-regulation, green indicates down-regulation, and clear symbol indicates proteins that were not identified as differentially expressed, but were linked to multiple proteins that had changed significantly. Solid lines indicate direct interactions and dashed lines indicate indirect interactions. Abbreviations: CAMK2A – calcium/calmodulin-dependent protein kinase II alpha; CAMK2B – calcium/calmodulin-dependent protein kinase II beta; CAMK2D – calcium/calmodulin-dependent protein kinase II delta; CAMK2G – calcium/calmodulin-dependent protein kinase II gamma; MBP – myelin basic protein; PCP4 – purkinje cell protein 4 or brain-specific polypeptide PEP-19; PI3K – phosphatidylinositol 3-kinase; RAB3B – ras related GTP-binding protein Rab-3B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Abridged Ingenuity® Pathways Analysis of effects of ethanol in the nucleus accumbens shell of NP rats showing up-regulation of G-protein signaling pathways. Red indicates up-regulation, green indicates down-regulation, and clear symbol indicates proteins that were not identified as differentially expressed, but were linked to multiple proteins that had changed significantly. Solid lines indicate direct interactions and dashed lines indicate indirect interactions. Abbreviations: CD81 – Cd 81 protein; GNA12 – guanine nucleotide binding protein alpha 12; GNA13 – guanine nucleotide binding protein alpha 13; GNAQ - guanine nucleotide binding protein alpha q polypeptide; GNAI1 - guanine nucleotide binding protein (Gi) alpha-1 subunit; GNAI2 - guanine nucleotide binding protein (Gi) alpha-2 subunit; ROCK – Roh-associated coiled-coil containing protein kinase; THY1 – thymus cell antigen 1 theta or Thy-1 membrane glycoprotein precursor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)