epilepsy is one of the most common neurologic conditions , characterized by an enduring predisposition to generate seizures . long - term drug treatments are always required for epilepsy ; however , approximately 30% of the patients with epilepsy are observed to have drug resistance or serious adverse reactions . moreover , multiple risk factors , such as brain injury , stroke , and drug and alcohol abuse , can result in epilepsy , and the relationship between alcohol and epilepsy is reported to be especially strong . however , the molecular mechanisms underlying alcohol - related seizure are largely unknown to date . therefore , a better understanding of the pathogenic mechanism underlying epilepsy will foster the development of new therapeutic strategies . the mammalian target of rapamycin ( mtor ) is a serine / threonine kinase that can control protein synthesis associated with cell growth and proliferation . the mtor signaling pathway has been shown to mediate mechanisms of epileptogenesis in a model of temporal lobe epilepsy , and may serve as a potential therapeutic target . mtor inhibition has significant effects on several major aspects of epilepsy pathophysiology , such as neuronal degeneration , mossy fibers sprouting , and recurrent seizure . in addition , inhibition of mtor is reported to suppress or block the progression of epilepsy . it can therefore be speculated that the mtor pathway may be a key mechanism involved in progression of epilepsy . however , whether the mtor pathway contributes to alcohol - related seizures has not been fully determined . in the present study we used video - electroencephalography ( eeg ) monitoring to assess seizure frequency after long - term alcohol treatment , and sought to determine whether rapamycin could effectively treat alcohol - induced seizures . in addition , we used ng108 - 15 cells to explore in vitro whether alcohol - induced mtor activation is the key mechanism in epilepsy progression . the objective of our study was to determine if the mtor pathway is a key epileptogenic mechanism in the development of alcohol - related seizures . wild - type c57/bl6 mice were housed singly in standard polypropylene cages with free access to food and tap water and a 12 : 12-hour light  dark cycle with an ambient room temperature of 21c . at age 4 weeks , one group of mice continued to receive the control liquid diet and the other group received diet water containing 10% ethanol for 9 weeks . to maintain similar caloric intakes between the groups , the mean of the previous day s consumption by the ethanol group was detected and the mice in the control group after 7 weeks of ethanol treatment , wild - type c57/bl6 mice were injected with rapamycin ( 3 mg / kg / day , 5 days / week , lc labs , woburn , ma ) , an mtor inhibitor , until subsequent analyses were completed at pre - determined time points . notably , rapamycin was first dissolved in 100% ethanol and stored at 20c . before injection care and use of animals were conducted in accordance with an animal protocol approved by the local ethics committee and animal management committee . c57/bl6 mice underwent weekly video - eeg monitoring initiating at 4 weeks of age to assess seizure frequency . mice under isoflurane anesthesia were surgically implanted with 4 epidural screw electrodes and allowed to recover from surgery for more than 24 h before recording . then , once a week , 48-h epochs of continuous eeg data from each mouse were detected using grass p-100 ac amplifiers ( astro - med , west warwick , ri , usa ) , analyzed by axon digidata a  d converters and saved digitally using axoscope software ( molecular devices , sunnyvale , ca , usa ) . simultaneously , digital video was recorded with a sanyo day - night camera and a darim mg-100 mpeg video capture card ( darim vision corp . , pleasanton , ca , usa ) to analyze the behavioral correlate of electrographic seizures . on video analysis , electrographic seizures were identified when a characteristic pattern of discrete periods of rhythmic spike discharges appeared . rhythmic spike discharges were characterized by frequency and amplitude lasting for more than 10 s , ended with repetitive burst discharges , and followed by severe voltage suppression . the behavioral correlate to these seizures was typically characterized by head bobbing , rearing with forelimb clonus , and occasional generalized convulsive activity . mouse neuroblastoma ng108 - 15 cells were cultured in dulbecco s modified eagle s medium ( dmem , gibco ) containing 10% fetal bovine serum in a humidified incubator at 37c with 5% co2 . the cells were treated with 100 mm ethanol for 3 days and subsequently treated with 10 um ly294002 , an inhibitor of akt , for 212 h. the in vitro kinase assay was performed for determining mtor activity as described previously . in brief , ng108 - 15 cells treated with ethanol were first lysed in lysis buffer with protease inhibitor . cell lysate was then incubated with anti - mtor antibody and 120 l of protein a agarose beads at 4c for 3 h. afterwards , beads were washed 3 times with lysis buffer and twice with kinase buffer . the kinase activity for mtor activity was finally determined by monitoring the phosphorylation levels of s6 . western blot analysis was performed to assay the expression levels of p - akt , p - s6k , and p - s6 by standard methods . briefly , neocortex of mice were dissected and sonicated with cell lysis buffer supplement with protease inhibitors ( bestbio , shanghai , china ) to extract total protein . similarly , ng108 - 15 cells were also lysed with cell lysis buffer on ice for 1 h. total protein concentration was determined with the bradford method ( pierce , rockford , il , usa ) . equal amounts of protein extract were separated on 10% sodium dodecyl sulfate - polyacrylamide ( sds - page ) gel and transferred to polyvinylidene difluoride ( pvdf ) membranes ( millipore ) . after blocking , antibodies to p - akt , p - s6k , and p - s6 were from cell signaling technology with a diluted concentration of 1:1000 , and antibody to gapdh ( 1:1000 ) was from genetex . after washing 3 times , the membranes were then incubated with appropriate horseradish - peroxidase - labeled anti - rabbit secondary antibodies ( santa cruz biotechnology , santa cruz , ca , 1:5000 ) at 25c for 1 h. the bands were finally developed by enhanced chemiluminescence kit ( millipore ) and the expression level of these proteins was normalized to gapdh . the data are expressed as mean sd and the differences between groups were compared using student s t - test or one - way anova , as appropriate . wild - type c57/bl6 mice were housed singly in standard polypropylene cages with free access to food and tap water and a 12 : 12-hour light  dark cycle with an ambient room temperature of 21c . at age 4 weeks , one group of mice continued to receive the control liquid diet and the other group received diet water containing 10% ethanol for 9 weeks . to maintain similar caloric intakes between the groups , the mean of the previous day s consumption by the ethanol group was detected and the mice in the control group after 7 weeks of ethanol treatment , wild - type c57/bl6 mice were injected with rapamycin ( 3 mg / kg / day , 5 days / week , lc labs , woburn , ma ) , an mtor inhibitor , until subsequent analyses were completed at pre - determined time points . notably , rapamycin was first dissolved in 100% ethanol and stored at 20c . before injection care and use of animals were conducted in accordance with an animal protocol approved by the local ethics committee and animal management committee . c57/bl6 mice underwent weekly video - eeg monitoring initiating at 4 weeks of age to assess seizure frequency . mice under isoflurane anesthesia were surgically implanted with 4 epidural screw electrodes and allowed to recover from surgery for more than 24 h before recording . then , once a week , 48-h epochs of continuous eeg data from each mouse were detected using grass p-100 ac amplifiers ( astro - med , west warwick , ri , usa ) , analyzed by axon digidata a  d converters and saved digitally using axoscope software ( molecular devices , sunnyvale , ca , usa ) . simultaneously , digital video was recorded with a sanyo day - night camera and a darim mg-100 mpeg video capture card ( darim vision corp . , pleasanton , ca , usa ) to analyze the behavioral correlate of electrographic seizures . on video analysis , electrographic seizures were identified when a characteristic pattern of discrete periods of rhythmic spike discharges appeared . rhythmic spike discharges were characterized by frequency and amplitude lasting for more than 10 s , ended with repetitive burst discharges , and followed by severe voltage suppression . the behavioral correlate to these seizures was typically characterized by head bobbing , rearing with forelimb clonus , and occasional generalized convulsive activity . mouse neuroblastoma ng108 - 15 cells were cultured in dulbecco s modified eagle s medium ( dmem , gibco ) containing 10% fetal bovine serum in a humidified incubator at 37c with 5% co2 . the cells were treated with 100 mm ethanol for 3 days and subsequently treated with 10 um ly294002 , an inhibitor of akt , for 212 h. the in vitro kinase assay was performed for determining mtor activity as described previously . in brief , ng108 - 15 cells treated with ethanol were first lysed in lysis buffer with protease inhibitor . cell lysate was then incubated with anti - mtor antibody and 120 l of protein a agarose beads at 4c for 3 h. afterwards , beads were washed 3 times with lysis buffer and twice with kinase buffer . the kinase activity for mtor activity was finally determined by monitoring the phosphorylation levels of s6 . western blot analysis was performed to assay the expression levels of p - akt , p - s6k , and p - s6 by standard methods . briefly , neocortex of mice were dissected and sonicated with cell lysis buffer supplement with protease inhibitors ( bestbio , shanghai , china ) to extract total protein . similarly , ng108 - 15 cells were also lysed with cell lysis buffer on ice for 1 h. total protein concentration was determined with the bradford method ( pierce , rockford , il , usa ) . equal amounts of protein extract were separated on 10% sodium dodecyl sulfate - polyacrylamide ( sds - page ) gel and transferred to polyvinylidene difluoride ( pvdf ) membranes ( millipore ) . after blocking , antibodies to p - akt , p - s6k , and p - s6 were from cell signaling technology with a diluted concentration of 1:1000 , and antibody to gapdh ( 1:1000 ) was from genetex . after washing 3 times , the membranes were then incubated with appropriate horseradish - peroxidase - labeled anti - rabbit secondary antibodies ( santa cruz biotechnology , santa cruz , ca , 1:5000 ) at 25c for 1 h. the bands were finally developed by enhanced chemiluminescence kit ( millipore ) and the expression level of these proteins was normalized to gapdh . the data are expressed as mean sd and the differences between groups were compared using student s t - test or one - way anova , as appropriate . video - eeg monitoring displayed the effect of ethanol on seizure frequency in c57/bl6 mice ( figure 1a ) . the results showed that long - term ethanol treatment markedly increased the seizure frequency of c57/bl6 mice , and seizure frequency increased with longer duration of ethanol treatment ( from 4 to 7 weeks ) , especially at 7 weeks ( p<0.05 ) , compared with control group . to verify whether ethanol induced seizure frequency via activation of mtor signaling , the expression level of p - s6 in neocortex of mice was determined by western blot analysis ( figure 1b ) . as expected , results showed that ethanol gradually increased the expression level of p - s6 with longer duration of ethanol treatment , indicating the mtor activation in neocortex of mice continuously increased with longer duration of ethanol treatment . to determine whether rapamycin could improve the ethanol - induced seizures , rapamycin was injected into c57/bl6 mice to observe its effect after 7 weeks of ethanol treatment . as shown in figure 2 , compared with the mice only treated with ethanol , the seizure frequency of mice treated with both ethanol and rapamycin gradually decreased with the increase of treatment time , suggesting that ethanol - induced seizures can be reversed by rapamycin . ng108 - 15 cells were used to explore the mechanism of ethanol - induced mtor activation in vitro . the in figure 3 shows that the relative kinase activity gradually increased with longer duration of ethanol treatment . in addition , western blot analysis showed that , compared with ng108 - 15 cells treated without ethanol and an akt inhibitor ly294002 , ethanol treatment increased the expression level of p - akt , p - s6k , and p - s6 in cells only treated with ethanol , whereas ly294002 had the opposite effect on expression levels of these proteins in cells treated with both ethanol and ly294002 ( figure 4 ) , suggesting that ethanol - induced mtor activation is dependent on the akt - mtor signaling pathway . video - eeg monitoring displayed the effect of ethanol on seizure frequency in c57/bl6 mice ( figure 1a ) . the results showed that long - term ethanol treatment markedly increased the seizure frequency of c57/bl6 mice , and seizure frequency increased with longer duration of ethanol treatment ( from 4 to 7 weeks ) , especially at 7 weeks ( p<0.05 ) , compared with control group . to verify whether ethanol induced seizure frequency via activation of mtor signaling , the expression level of p - s6 in neocortex of mice was determined by western blot analysis ( figure 1b ) . as expected , results showed that ethanol gradually increased the expression level of p - s6 with longer duration of ethanol treatment , indicating the mtor activation in neocortex of mice continuously increased with longer duration of ethanol treatment . to determine whether rapamycin could improve the ethanol - induced seizures , rapamycin was injected into c57/bl6 mice to observe its effect after 7 weeks of ethanol treatment . as shown in figure 2 , compared with the mice only treated with ethanol , the seizure frequency of mice treated with both ethanol and rapamycin gradually decreased with the increase of treatment time , suggesting that ethanol - induced seizures can be reversed by rapamycin . ng108 - 15 cells were used to explore the mechanism of ethanol - induced mtor activation in vitro . the in figure 3 shows that the relative kinase activity gradually increased with longer duration of ethanol treatment . in addition , western blot analysis showed that , compared with ng108 - 15 cells treated without ethanol and an akt inhibitor ly294002 , ethanol treatment increased the expression level of p - akt , p - s6k , and p - s6 in cells only treated with ethanol , whereas ly294002 had the opposite effect on expression levels of these proteins in cells treated with both ethanol and ly294002 ( figure 4 ) , suggesting that ethanol - induced mtor activation is dependent on the akt - mtor signaling pathway . alcohol can cause seizures in a variety of ways ; however , the molecular mechanisms underlying alcohol - related seizure remain elusive . in the current study the results showed that long - term alcohol treatment increased the seizure frequency of c57/bl6 mice via activation of mtor signaling . moreover , ethanol also induced mtor activation in ng108 - 15 cells in vitro , and ethanol - induced mtor activation was shown to be dependent on the akt - mtor signaling pathway . all these findings indicate that the akt - mtor signaling pathway is likely to be the key mechanism of ethanol - induced epilepsy . the relationship between alcohol and seizures is reported to be complex   and many studies have been carried out to explore alcohol - related or withdrawal seizures . long - term alcohol abuse can change the seizure threshold with alcohol levels and increase the risks of prolonged or sustained seizure activity . moreover , alcohol withdrawal is shown to increase the risk of sudden unexpected death in epilepsy ( sudep ) . welch and derry also suggested that alcohol misuse might underpin the association between mild traumatic brain injury and epilepsy . demonstrated that mtor activation was required for the anti - alcohol effect of ketamine in alcohol - preferring rats . moreover , the mtor signaling pathway is reported to be activated during status epilepticus ( se ) in a model of temporal lobe epilepsy , as evident by an increase in p - s6 expression . in our study , video - eeg monitoring showed that ethanol induced increased seizure frequency in c57/bl6 mice . moreover , we observed the increased expression level of p - s6 in ethanol - treated c57/bl6 and ng108 - 15 cells . therefore , we speculate that ethanol could induce epilepsy via activating mtor signaling . increased expression of p - s6 is evidence of mtor activation . s6 ribosomal protein is phosphorylated by s6 kinase , which is one of the downstream effectors of the akt pathways . to further explore the mechanism of ethanol - induced mtor activation , ng108 - 15 cells were treated with both ethanol and an akt inhibitor ly294002 . the results showed that ethanol increased the expression level of p - akt , p - s6k , and p - s6 , whereas ly294002 had opposite effects on expression levels of these proteins , suggesting that ethanol - induced mtor activation is dependent on the akt - mtor signaling pathway . phosphoinositide-3-kinase / akt signaling cascade is considered to be a cellular signaling mechanism in the process of liver defects associated with chronic alcohol consumption . in addition , rapamycin / rad001 is shown to have benefit in the treatment of tuberous sclerosis ( tsc ) via having effects on mtorc1 and akt signaling , and tsc in the brain is likely to cause epilepsy , suggesting that mtorc1 and akt signaling may be a key mechanism involved in epilepsy . antiepileptic drugs lead to decreased concentrations of the active forms of akt , suggesting that akt may be a potential target in antiepileptic therapy . therefore , our results are in line with previous studies and suggest that the akt - mtor signaling pathway may contribute to the development of ethanol - induced epilepsy . our findings indicate that long - term alcohol intake increases the risk of epilepsy via activation of mtor signaling . moreover , ethanol - induced mtor activation may be dependent on the akt - mtor signaling pathway . the key molecules involved in the akt - mtor signaling pathway may serve as potential targets in the treatment of epilepsy .
backgroundthe aim of this study was to determine whether activation of mammalian target of rapamycin ( mtor ) is a key epileptogenic mechanism in the development of alcohol - related seizure.material/methodsc57bl/6 mice were administered 10% ethanol in drinking water for 9 weeks . 
 video - electroencephalography ( eeg ) monitoring was then used to assess seizure frequency after alcohol and rapamycin treatment . 
 in addition , mouse neuroblastoma ng108 - 15 cells were treated ethanol for 3 days and subsequently treated with akt inhibitor ly294002 for 212 h. the in vitro kinase assay was performed for determining mtor activity . 
 western blot analysis was used to determine the expression of p - akt , p - s6k , and p-s6.resultlong-term ethanol treatment markedly increased the seizure frequency of c57/bl6 mice over time . 
 moreover , ethanol treatment increased the expression level of p - s6 over time . 
 ethanol - induced seizure can be reversed by rapamycin . 
 in addition , the in vitro kinase assay showed mtor activity was activated by ethanol . compared with ng108 - 15 cells treated without both ethanol and ly294002 
 , ethanol increased the expression level of p - akt , p - s6k , and p - s6 , whereas ly294002 had opposite effects on expression levels of these proteins.conclusionsour findings indicate that long - term alcohol intake increases the risk of epilepsy via activation of mtor signaling . 
 moreover , ethanol - induced mtor activation may be dependent on the akt - mtor signaling pathway . 
 the key molecules involved in akt - mtor signaling pathway may serve as potential targets in the treatment of epilepsy .