In our experiments, no significant differences were found in body weight between the alcohol-drinking and control rats, suggesting that the food intake is not affected during the 28 d of 6% alcohol drinking period. Rats stably consumed an average of 4.85±0.79 g/kg/day of alcohol in their home cages for 28 d, which resulted in relatively lower BALs (average: 22.35±1.3 mg/dl). Shah et al. reported that the blood alcohol levels (BALs) in Swiss-Webster mice reached 20.06±8.32 mg/dl on the first day, and 14.75±3.98 mg/dl on the 7th day, by administrating a 7% v/v alcohol solution for 7 d 
. Furthermore, the report by Barson showed that the BALs in SD rats reached 18.9±4.0 mg/dl by administrating an alcohol solution of increasing concentration from 1% to 7% v/v for 16 d 
. In our study, the blood samples were taken from the tail vein at 9
00 AM. The BALs was low because the rats consumed alcohol at a different time points at the dark phase. The rats with lower BAL may consume ethanol in the early period of the dark phase, whereas the rats with higher BAL probably consumed alcohol in the later period of the dark phase 
. Taken together, those results indicate that the alterations in behavior and protein expression in the alcohol-drinking rats resulted from adaptations in brain function rather than insufficient food intake or alcohol accumulation in the blood.
Alcohol consumption inhibits locomotor activity 
. In our hand, locomotor activity started to decrease in alcohol-drinking rats after 7 d. Rearing activity was also decreased. Notably, the distances travelled and rearing were not different compared to control rats at the end of the 28-day alcohol exposure, suggesting adaptation and compensation. Discontinuation of alcohol intake results in the nervous system hyperactivity and dysfunction 
. EWS is the most important evidence indicating the presence of physical alcohol dependence either in humans or in experimental animals 
. Withdrawal symptoms include increases in stereotype behaviors, tail stiffness, hyper-reflexia, agitaion, and anxiety 
. In our study, rats exhibited obvious withdrawal signs after alcohol discontinuation. In line with previous studies 
, the global score of EWS was highest at 6 h. Our results indicated that 28-d of continuous 6% alcohol drinking induces physical dependence in rats.
Activated ERK phosphorylates cellular targets or translocates into the nucleus where it activates specific gene transcription factors 
. By regulating cellular activities and gene transcription, the ERK cascade transduces the activity of a variety of extracellular and intracellular signals into enduring changes in the central nervous system. Several studies have shown that alcohol exposure alters ERK phosphorylation. Moderate doses of acute alcohol (1.5–3.5 g/kg) produce a dose- and time-dependent decrease in phosphorylated ERK in mouse cortex 
. Another study extended these findings by showing that acute alcohol can reduce phosphorylated ERK in the cerebral cortex and Hip in both young and adult rats 
. Forced exposure to chronic ethanol vapor suppressed ERK phosphorylation in the Amy, cortex, cerebellum and CPu in rats 
. Our results showed that chronic or acute exposure to alcohol significantly decreased ERK phosphorylation in the NAc, CPu, Amy, Hip, and PFC. The most intriguing finding of the present study is that ERK phosphorylation also decreased significantly in the NAc, which is inconsistent with the finding of Sanna et al
. wherein they found only minor and mostly non-significant changes 
. This might be the result of the different sample sizes (n
10) used or the duration of the induction periods (12 days v.s.
28 days). Although drugs of abuse possess diverse neuropharmacological profiles, activation of the mesocorticolimbic system, particularly the NAc, Amy, CPu, PFC and Hip via dopaminergic and glutamatergic pathways, constitutes a common pathway by which various drugs of abuse mediate their reinforcing effects 
. The similar alterations of ERK have been thought to contribute to the drug’s rewarding effects and to the long-term maladaptation induced by drug abuse (including cocaine, amphetamine, Δ9
-tetrahydrocannabinol, nicotine, morphine and alcohol) 
. Although long-term alcohol exposure induced significant inhibition of ERK phosphorylation, distinct patterns of p-ERK/ERK ratio were observed in different brain regions. It may reveal that these different brain regions play different roles and have different sensitivity to alcohol. Together, our results demonstrated that chronic or acute alcohol exposure induces decreased ERK phosphorylation in the brain reward circuit. Alcohol inhibits glutamatergic neurotransmission, primarily by acting on ionotropic glutamate receptors (iGluRs). Many reports have demonstrated that acute ethanol exposure inhibits NMDAR channel function in the Hip, cerebellum, cerebral cortex, NAc, Amy and VTA 
. After chronic alcohol exposure, the number of NMDA receptor complexes was increased 
, perhaps indicating an adaption to the chronic presence of ethanol to maintain receptor activity. These results confirmed that the activity of glutamate receptors but not the number of these receptors is inhibited by chronic alcohol exposure. One well-characterized cascade includes the calcium/calmodulin-dependent inhibition of adenylyl cyclase and inhibition of cAMP-dependent protein kinase (PKA). PKA then triggers the inhibition of ERK signaling cascade, resulting in decreased ERK phosphorylation and nuclear translocation and further down-regulation of gene transcription 
. Our recent study has demonstrated that continuous alcohol drinking inhibits the phosphorylation of ERK, and this inhibition is correlated with a decrease in the phosphorylation of CaMKII (Thr286) in hippocampal CA1 and DG subregions 
The GluN1 subunit forms the molecular backbone of functional NMDA receptors and has been hypothesized to play a role in chronic alcohol consumption. To address this issue, we examined the expression and phosphorylation levels of the GluN1 subunit. Long-term alcohol consumption induced significant up-regulation of GluN1 subunits in all five brain regions. Enhanced GluN1 subunit mRNA expression was evident in samples from chronic ethanol-exposed animals in amygdala neurons 
. Previous studies have shown that alcohol consumption is accompanied by increased GluN1 protein levels in the Hip 
, Amy 
, striatum and medial PFC 
. In Kroener et al.
study, they observed that chronic alcohol exposure lead to an increase in expression of GluN1 subunit in the insoluble postsynaptic density fraction, but this increase was more transient and was no longer observed after 1 week of withdrawal 
. The up-regulation of GluN1 subunits caused by chronic alcohol consumption may be a compensatory reaction that leads to recovery of NMDAR functional activity following ethanol-induced GluN1 inhibition 
Protein phosphorylation has been recognized as a major mechanism for the regulation of NMDA receptor function 
. In our hand, the ratio of phospho/total-GluN1 was significantly lower in all brain regions examined. Phosphorylation of GluN1 at Ser897 alters NMDA receptor activity by regulating its sensitivity to glutamate or by activating downstream signal transduction pathways 
. A serine at position 897 in the C1 cassette represents the major PKA phosphorylation site of the GluN1 subunit 
. Dopamine D1 receptors have been shown to directly bind GluN1 and GluN2A subunits 
, these results suggest that the phosphorylation status of the GluN1 S897 site may be a critical factor that regulates the overall sensitivity of NMDA receptors to alcohol. However, a previous study showed that conditions that favor the phosphorylation of S897 in the GluN1 subunit had no effect on the acute alcohol sensitivity of NMDARs 
. Aside from methodological differences between Xu et al.
and the present study, it is also possible that neuronal NMDA receptors exist in a complex intracellular environment characterized by a wide array of signaling proteins that are presumably missing in a heterologous cell expression system like human embryonic kidney cells. Our results confirmed previous observations that continuous alcohol drinking greatly increases GluN1 in both total protein and its phosphorylated version 
. Moreover, our results also showed the ratio of phospho/total-GluN1 subunit was reduced in these brain regions studied, which indicate that total-GluN1 were increased greater than phospho-GluN1 in the continuous alcohol drinking group. it is plausible that chronic alcohol use, by long-term inhibition of NMDA function, triggers compensatory adaptations. Zhao et al.
have demonstrated that decreased phospho/total-GluN1 is consistent with decreased NMDAR function 
. Moreover, we also demonstrated that acute exposure to alcohol induced increases in GluN1 phosphorylation in rat brain. The upregulated phosphorylation of GluN1 may lead to recovery of NMDAR functional activity under the acute effect of alcohol 
. These effects of alcohol on the GluN1 may underlie the mechanisms that compensate for alcohol-induced inhibition of NMDARs. Previous study documented a profound increase in phospho-ERK in response to pharmacological activation of NMDA receptors in hippocampal, cortical and striatal neurons 
. Administration of the NMDARs antagonist MK-801 prevented the increase in ERK phosphorylation in the Amy in alcohol withdrawn rats 
. Thus, it may be speculated that the decreased ERK phosphorylation is associated with the inhibition of phospho-GluN1 by long-term alcohol drinking.
In conclusion, the findings of the present study indicate that alcohol can affects phosphorylation of ERK and GluN1 in brain reward circuits. JNK and p38 phosphorylation in mesocorticolimbic areas were not significantly influenced by this treatment. Our findings support previously reported associations between GluN1-ERK and dependence to alcohol and other substances, and emphasize the relevance of decreased phosphorylation of GluN1 and ERK in the mesocorticolimbic system for chronic alcohol exposure.