the implication of gene  environment interactions in the etiology of psychiatric diseases , such as depression and drug addiction is well established in humans ( caspi et al . 2002 ; nilsson et al . 2007 ) and non - human primates ( barr et al .
is widely used to assess long - term consequences of different early experiences ( ladd et al . 2000 ; pryce and feldon 2003 ) .
ms has been shown to strongly affect the neurocircuitries involved in drug addiction and also cause long - term effects on the propensity to voluntarily drink ethanol ( gustafsson et al .
2008 ; oreland et al . 2009 ; ploj and nylander 2003 ; ploj et al .
2002 , 2003b ) . in the compulsive use of ethanol , neurotransmitter systems such as dopamine , serotonin , noradrenaline and opioids are involved ( koob and volkow 2010 ) .
one common factor affecting the development of these neural systems is the transcription factor activator protein ( tfap ) 2 ( comb et al . 1988 ; damberg 2005 ) .
the tfap-2 may , therefore , be a target for early environmental influences and a mediator of the effects seen after ms .
interestingly , early environmental factors have been associated with altered tfap-2 family gene expression in rats ( meaney et al . 2000 ) . in the present study ,
the aim was to examine possible differences in brainstem tfap-2 levels in animals genetically or environmentally predisposed to prefer high ethanol drinking and high ethanol concentration . besides rats subjected to ms , the ethanol - preferring alko , alcohol ( aa ) and the ethanol - avoiding alko , non - alcohol ( ana ) rats were studied .
the aa and ana lines are among the first rodent lines produced by bidirectional selection for high ethanol preference in a two - bottle choice between ethanol and water ( for review see sommer et al .
three experiments were performed : i comparative analysis of tfap-2 levels in the aa and ana rats , ii assessment of tfap-2 levels in juvenile and adult rats subjected to different early environmental conditions using the ms model , and iii analysis of the impact of long - term voluntary ethanol consumption on adult rats reared in different early environmental conditions .
experiment i twelve alcohol - nave adult male aa and ana rats were obtained from the national public health institute , finland . upon arrival
, the rats were housed with 3 rats per cage in standard macrolon cages ( 59    38    20  cm ) containing wood - chip bedding material .
experiment ii and iii time - mated pregnant wistar rats ( scanbur ab , sweden ) arrived at the animal facility on gestational day 1415 ( experiment ii ) and 1213 ( experiment iii ) . the pregnant rats were singly housed in macrolon cages . on the day of birth , postnatal day ( pnd ) 0 , the litters were divided into litters of 910 pups ( males , n  =  56 ; females ,
the rat pups were subjected to daily 15  min ( ms15 ) or 360  min ( ms360 ) ms or conventional animal facility rearing ( afr ) .
the ms procedures have previously been described in detail elsewhere ( gustafsson and nylander 2006 ) . in adulthood ,
animals in experiment iii were singly housed from pnd 7375 and were then given free access to 5 , 10 and 20% ( v / v ) ethanol solutions and one bottle with water ( ms15e , ms360e and afre ) or just water ( ms15w and ms360w ) for 54  days .
the four - bottle choice paradigm is described in detail elsewhere ( gustafsson and nylander 2006 ) .
the rats exposed to ms in experiment ii were sacrificed at 3 and 10  weeks , respectively , and rats in experiment iii at the end of the ethanol - drinking period .
the brainstems were removed and immediately frozen on dry ice and stored at 80c until further analysis .
all animals had free access to water and r36 labfor food pellets ( lactamin ab , sweden ) in a humidity- and temperature - controlled environment .
all animal experiments were performed in accordance with the european communities council directives ( 86/609/eec ) .
afr animal facility rearing , aa alko , alcohol , ana alko , non - alcohol , e rats having access to ethanol and water , ms15 15  min of maternal separation , ms360 360  min of maternal separation , w rats having access to only water a schematic overview of the experimental design of the three studies .
afr animal facility rearing , aa alko , alcohol , ana alko , non - alcohol , e rats having access to ethanol and water , ms15 15  min of maternal separation , ms360 360  min of maternal separation , w rats having access to only water with some modifications , tfap-2 levels were analyzed using enzyme - linked immunosorbent assay as earlier described ( berggard et al .
50  l of 14  g / ml goat polyclonal tfap-2 primary antibody ( sds biosciences , sweden ) together with 50  l of 1.5  g / ml donkey anti - goat igg ap conjugated secondary antibody ( sds biosciences , sweden ) was added per well .
25  min after adding 50  l of phosphatase substrate solution , an additional 50  l of stop solution ( 3  m naoh ) was added .
the immunoreactive ( ir ) tfap-2 in each sample was deciphered from a standard curve , where known log10 transformed concentrations of ir tfap-2 peptide ( sds biosciences , sweden ) were plotted against optical density using assayzap v3.1 ( biosoft , uk ) . a one - way analysis of variance ( anova ) followed by fisher s protected least significant differences (
plsd ) post - hoc test was performed to compare ir tfap-2 levels between i aa and ana male rats , ii ms15l , ms360l , and afr male rats at 3 and 10  weeks of age , respectively , and iii ms15w , ms360w , ms15e , ms360e and afre male rats .
two - way anova followed by fisher s plsd post - hoc test was used for comparison between the rearing environment ( ms15 or ms360 ) and fluid intake ( water or ethanol ) .
significant differences in immunoreactive ( ir ) tfap-2 protein levels ( f1,21  =  5.136 ; p  < 
no ms - induced effects on ir tfap-2 protein levels were detected in either the juvenile ( f4,44  =  1.379 ; p  =  0.257 ) or adult
=  0.64 ) were seen between the ms15w , ms15e , ms360w , ms360e and afre rats . comparing the water- and ethanol - drinking ms groups , no significant effects of rearing environment ( f1,38  =  0.498 ; p  =  0.48 ) , fluid intake ( f1,38  =  0.036 ; p 
=  0.85 ) or interaction between rearing environment and fluid intake ( f1,38  =  0.021 ; p 
=  0.89 ) with regard to ir tfap-2 protein levels were observed ( table  1 ) .
the voluntary ethanol consumption has been described in detail elsewhere ( gustafsson and nylander 2006).table  1the immunoreactive levels of tfap-2 protein in the brain stem of male ratsrearing environmentageneffect of heredityeffect of rearing environmenteffect of rearing environment    long - term voluntary ethanol intakeaaadult98.76    0.37anaadult116.81    0.34ms15juvenile1027.45    1.20ms360juvenile1027.86    0.73afrjuvenile925.36    0.47ms15adult1028.64    1.18ms360adult1033.52    1.71afradult1031.70    1.37ms15  wadult1014.57    0.95ms360  wadult1015.37    1.10ms15eadult1014.89    0.91ms360eadult1215.42   
0.80afreadult1113.62    0.95results are shown in mean ng ir tfap-2 protein per mg total protein    semafr animal facility rearing , aa alko , alcohol , ana alko , non - alcohol , e rats having access to ethanol and water , ms15 15  min of maternal separation , ms360 360  min of maternal separation , n number of rats , w rats having access to only water * *  p  <  0.01 compared to aa rats ( anova followed by fisher s post - hoc test ) the immunoreactive levels of tfap-2 protein in the brain stem of male rats results are shown in mean ng ir tfap-2 protein per mg total protein    sem afr animal facility rearing , aa alko , alcohol , ana alko , non - alcohol , e rats having access to ethanol and water , ms15 15  min of maternal separation , ms360 360  min of maternal separation , n number of rats , w rats having access to only water * *  p  <  0.01 compared to aa rats ( anova followed by fisher s post - hoc test )
previous clinical studies have suggested an association between a gene variation in tfap-2 and personality ( damberg et al .
2000 ) , severe female alcoholism ( nordquist et al . 2009 ) and depression in adolescents ( nilsson et al .
it has further been proposed that tfap-2 may be a target for factors known to influence psychopathological behavior via its effect on transcription of several monoaminergic genes in the central nervous system ( for review see damberg 2005 ) .
further support for this notion stems from findings of effects of antidepressive drugs on brainstem tfap-2 protein levels in male rats ( berggard et al . 2005 ) , indicating a change in the serotonin network in this brain region .
in addition , the levels of tfap-2 protein in the brainstem area are inversely correlated to monoamine turnover in the male rat forebrain ( damberg et al . 2001 ) .
having these factors in mind , we selected the brainstem region , where the serotonin cell bodies are located .
differences in serotonin , dopamine , noradrenaline and opioid systems between the aa and ana rats have been reported ( for review see sommer et al .
several genes of importance for these neurocircuitries have consensus sites for tfap-2. interestingly , higher levels of ir tfap-2 levels were seen in the predisposed ethanol - preferring aa rats compared to the non - preferring ana rats .
ms affects various brain networks , including the dopamine , serotonin and opioid systems ( arborelius and eklund 2007 ; gustafsson et al .
2008 ; oreland et al . 2009 ; ploj and nylander 2003 ; ploj et al .
, it was hypothesized that the early life environment could affect the levels of tfap-2 and thereby cause alterations in gene expression of various transmitters , receptors and transporters .
in one previous study , it was reported that short ms is associated with altered hippocampal tfap-2 gene expression in rats ( meaney et al .
however , our findings suggest no ms - induced differences in brainstem ir tfap-2 levels in either juvenile or adult male rats .
we also measured the ir tfap-2 brainstem levels in juvenile and adult female rats subjected to litter - wise and individual ms15 and ms360 as well as afr .
also in this case no differences in ir levels were observed ( data not shown ) .
first , the ap-2 gene in the meaney study was measured in another brain area than in our study , thus , making it difficult to compare the results .
the tfap-2 family consists of five known isoforms ( for review see damberg 2005 ) and one of the isoforms other than tfap-2 could have been engaged in the differences observed by meaney and co - workers .
third , alterations in gene expression do not necessarily have to mirror alterations in protein levels .
the early rearing environment has been suggested to be either a protective ( short ms ) or a risk ( prolonged ms ) factor for high voluntary ethanol consumption ( roman and nylander 2005 ) .
in addition , wistar rats subjected to ms were induced to changes in dopamine receptors and opioid peptide levels after long - term , voluntary ethanol self - administration ( ploj et al .
2003a ) . in another study but using the same animals as experiment iii , differences in ethanol consumption and opioid peptide levels between the experimental groups have previously been reported ( gustafsson and nylander 2006 ; gustafsson et al .
therefore , we further examined whether different early life experiences would alter ir tfap-2 levels after long - term voluntary ethanol drinking . however , no differences in ir tfap-2 levels were observed .
furthermore , no ethanol - induced effects on ir tfap-2 levels were observed indicating that tfap-2 is not affected by voluntary ethanol consumption in any of the experimental groups .
it is worth mentioning that the standard curve for the immunoassays between the three experiments did not differ from each other .
the variations seen between the three experiments are speculated to be due to differences in rat lines ( wistar versus aa / ana lines ) or emotional stress level ( due to different housing conditions ) .
the rats in experiment ii , i.e. rats solely exposed to different early rearing conditions , were housed in groups until they were sacrificed .
however , the rats in experiment iii , i.e. having access to either water and ethanol or only water , were housed individually for 8  weeks during the entire ethanol access period .
therefore , we speculate that the differences between the rats in these two experiments might be due to the emotional stress applied in the isolated rats in experiment iii . in conclusion , our data show that the genetically predisposed ethanol - preferring aa rats and ethanol - avoiding ana rats differ in ir tfap-2 protein levels .
no differences in ir tfap-2 protein levels induced by the different early rearing environments were seen in the brainstem of juvenile and adult rats .
furthermore , long - term voluntary ethanol consumption did not alter brainstem tfap-2 protein levels in male wistar rats subjected to ms .
 genes involved in alcoholism have consensus sites for the transcription factor activator protein ( tfap ) 2. in the present study , we investigated tfap-2 protein levels in the ethanol - preferring alko , alcohol ( aa ) and the ethanol - avoiding alko , non - alcohol ( ana ) rat lines . 
 furthermore , basal and ethanol - induced tfap-2 levels were examined in wistar rats exposed to different early postnatal environments that are known to affect later ethanol consumption . taken together 
 , we found differences in brainstem tfap-2 protein between the aa and ana rats . 
Introduction
Materials and methods
Results
Discussion