BACKGROUND
the mouse c <dig> and dba inbred strains differ substantially in many aspects of their response to drugs of abuse, including altered rates of voluntary drug consumption  <cit> .
the c <dig> strain shows increased voluntary consumption of multiple drugs of abuse, including opiates, alcohol, cocaine, and amphetamine, which mirrors the poly-substance nature of some drug-seeking behavior in humans  <cit> .
c <dig> strains of mice have been viewed as a good model for human drug-seeking behavior.
it has been assumed that the poly-substance nature of drug-seeking in c <dig>  as compared to dba, is due, in part, to common mechanisms that operate irrespective of the drug of abuse.
this assumption is fueled by studies showing that there are susceptibility factors that contribute to drug use irrespective of the substance, as well as susceptibility factors that are specific to each pharmacologic class  <cit> .
multiple quantitative trait locus analyses  have been carried in the c <dig> and dba strains for both drug responses and drug-seeking behavior.
qtl analyses for drug-seeking preferences to different classes of drugs might identify regions of the genome associated with drug-seeking across multiple pharmacological classes and, perhaps, more directly target common reward pathways for multiple substances.
for morphine, the strongest evidence for a qtl underlying morphine preference is on the proximal part of chromosome  <dig>  at the position of the mu opiate receptor  <cit> .
in the case of preference for alcohol, the strongest qtl's are on chromosomes  <dig>   <dig>   <dig>  and  <dig>  as determined by meta-analyses of  <dig> studies of alcohol preference  <cit> .
further studies with other substances need to be carried out to determine regions of potential overlap.
the development of microarray analyses represents an additional genome-wide method for measuring differences across strains, focusing on expression differences rather than on sequence differences in the genome.
this is an additional approach to characterize variations between strains as a means of understanding molecular differences that might underlie strain variability in the effect of, and response to, multiple substances of abuse.
in the current study, we have carried out microarray analysis in c <dig> and dba mice in the nucleus accumbens  of naïve and drug-treated animals.
we identified a number of mrnas with altered expression between the two strains.
we validated the mrna expression changes of several such mrnas, as well as the protein activity of one mrna product, catechol-o-methyltransferase .
data mining of expression and behavioral data indicates that comt expression correlates with two aspects of drug response in c57/dba recombinant inbred strains.
similarly, a role for gnb <dig> in responses to multiple drugs of abuse is supported by data from our study and from other studies.
specific pathways were also identified that showed effects of treatment and/or strain.
mrnas with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse.
RESULTS
microarray analysis in the nucleus accumbens
we carried out microarray analysis in the dissected nac from  <dig> vehicle-treated and  <dig> morphine-treated c <dig> and  <dig> dba mice, using a pooling strategy to minimize dissection and other variations.
twelve groups of  <dig> nac were hybridized to the affymetrix mg_u74a microarray and the resultant data analyzed using dchip.
we compared mrna expression between strains, and across treatment groups.
the first analysis was gene oriented, and required a significant  fold change and made use of t-testing to ascribe significance  .
comparing mrna expression between c <dig> and dba mice in the absence of morphine, we identified  <dig> mrnas with altered expression, four  with two different probe sets .
comparing mrna expression between the two strains in the presence of morphine  for  <dig> days, we identified  <dig> mrnas with altered expression, four  1/tcea1) with two different probe sets .
we also compared expression within each strain, contrasting expression in vehicle and morphine-treated mice .
there were fewer morphine-responsive mrnas within each of the two strains, when compared to the number of mrnas that differed between strains.
in the dba strain,  <dig> mrnas were identified as changed with an effect >=  <dig>  fold in response to morphine and we did not identify any mrnas showing a p <  <dig>  and greater than >=  <dig>  fold change in the c <dig> strain in response to morphine.
treatment-invariant mrnas
it was of interest that genes showing greater expression changes in these first analyses were much more common across strains than within strains.
furthermore, there was a very significant proportion of mrnas that showed altered expression between the two strains in both the drug-naïve and the morphine-treated groups .
we examined the correlation of the fold-expression changes of these treatment-invariant mrnas under the two treatments and observed a linear relationship, with a high degree of correlation in the relative expression of these mrnas .
these treatment-invariant mrnas were of particular interest as they represent a molecular environment that is consistently different between the two strains, irrespective of treatment condition.
they also provide evidence that the microarray studies are internally consistent.
two-way anova
the presence of a large proportion of mrnas that showed altered expression irrespective of treatment supported further analysis of the dataset, using two-way anova.
with such an approach, we could identify mrnas showing main effects for strain, main effects for treatment, and interaction effects.
we used more permissive criteria , so as to capture genes and pathways that demonstrated smaller but significant changes in expression.
running such analyses led to the identification of  <dig> genes that showed a main effect of strain,  <dig> genes showing a main effect of treatment, and  <dig> genes showing significant interaction effects .
gnb <dig> showed the most significant  main effect for strain and additional treatment-invariant genes noted above  were also identified in this analysis.
kegg pathway analyses of genes showing main effect for strain identified clusters of genes associated with the ribosome, oxidative phosphorylation/atp synthesis, long-term potentiation/long-term depression, and antigen processing and presentation .
kegg pathway analysis of genes showing a main effect of treatment  identified two significant functional groups, including "axon guidance" .
the axon guidance group of genes identified in these analyses were all related to the semaphorin pathway .
kegg pathway analysis of genes showing significant strain by treatment interactions  identified significant effects for genes in the focal adhesion pathway, particularly genes involved in phosphatidylinositol signaling system .
validation of microarray analyses
we chose a selection of the mrnas from the microarray studies for follow-up using quantitative polymerase chain reaction  to validate the microarray signals.
mrnas were chosen to represent both the up-regulated and down-regulated mrnas, to represent mrnas showing more profound expression changes, to represent mrnas with neuronal expression  and/or potentially relevant neuronal function, and based on other published studies on either varying expression in mouse strains or changes in response to drugs of abuse.
we were able to validate a majority of mrnas for which we carried out qpcr, as shown in table  <dig>  overall validation of the microarray results was reflected in the high correlation of the ratio of mrna expression between the two strains as measured by microarray and by qpcr .
furthermore, by anova there was a significant  main effect of strain with actin related protein 2/ <dig> complex, subunit 5/arpc <dig>  comt, gnb <dig>  phosphodiesterase 9a/pde9a, phosphatidylinositol transfer protein, beta/pitpnb, and secretory granule neuroendocrine protein  <dig>  7b <dig> protein/sgne <dig>  and a significant main effect of treatment with arpc <dig>  and rcn.
there was a trend level  main effect of strain with rcn, and a trend level main effect of treatment with gabra <dig>  finally, there were significant interaction terms with arpc <dig>  ngfi-a binding protein 2/nab <dig>  and rcn.
not all microarray results were unambiguously validated, as an mrna showing great dys-regulation between the two strains by microarray  did not show such profound alterations in expression by qpcr.
failure of validation can occur for several reasons, including differing probes used for the microarray and qpcr experiments , genomic differences between strains that alters the hybridization of the relevant regions of the probes, and false-positive expression changes.
comt levels and morphine
a recent report made the observation that individuals with a met/met genotype  at the val158met polymorphism of the human comt gene required a significantly reduced dose of morphine to achieve pain relief, as compared to individuals with the val/val genotype  <cit> .
these observations are consistent with other studies implicating comt activity with opioid response  <cit> .
since the dba mice showed increased sensitivity to morphine compared to c <dig>  as defined by the tail flick assay, and reduction in comt expression and activity, we sought to determine the degree to which morphine sensitivity varied in c57/dba recombinant inbred strains, as a function of comt expression, making use of the webqtl database  <cit> .
we first noted that in those studies of whole brain homogenates, comt expression was significantly decreased in dba, as compared to c <dig>  consistent with our studies in the nac.
we then examined the indices of morphine effect described above.
slope of the dose-response curve and the response to  <dig> mg/kg of morphine both correlated with levels of comt expression  .
comt expression did not show significant correlation with morphine consumption in a two-bottle choice paradigm .
the correlation of comt expression with two indices of morphine effect suggested a possible relationship between these two measures in mice.
as mrna levels do not always correlate with protein levels and protein activity, we next sought to determine whether comt activity differed in brain extracts from c <dig> and dba mice.
nac, amygdala , and frontal cortex  were dissected from dba and c <dig> mice and comt activity was then determined in homogenates.
comt activity was modestly, but significantly, reduced in nac  and fc  of dba mice, as compared to c <dig> mice .
discussion
we examined mrna expression in the nac in vehicle- or morphine-treated mice, comparing the c <dig> and dba strains.
the nac is part of the mesocorticolimbic dopamine system which originates in the ventral tegmental area  and projects to the nac, fc and other limbic areas  <cit> .
the vta and the nac are two brain regions commonly thought to be involved in the reward system for drugs of abuse.
in studies that compared gene expression changes induced by morphine treatment across multiple brain regions, the nac shows some of the most profound changes in the total number of genes with altered expression in the presence of drug .
in the current study, we made use of an established and well-regarded analytical platform for analysis of microarrays.
in our first analyses, we required reasonable levels of expression, a modest  fold change, and a nominal p <  <dig> .
median false-discovery rate  estimates were between ~1–4% for results contrasting the two strains, but were significantly higher  for contrasts within strains .
of importance to the current study is that kerns et al.
have recently compared mrna expression in the nac in control c <dig> and dba animals  <cit> .
of the  <dig> mrnas showing altered expression in our study of vehicle-treated mice, more than  <dig>  were also observed to show altered expression  in the kerns study, in the same direction for the same probe sets, using different analytical methods .
furthermore, there was a significant agreement in relative mrna expression across the two studies .
this indicates that a significant proportion of mrnas showing altered expression by microarrays can be reproduced across the two studies, irrespective of analytical approach.
it also represents an important validation of such microarray studies.
while the kerns study did not identify changes in comt, our analysis of an independent dataset supported our results.
in addition, microarray studies in the hippocampus of eight mouse strains demonstrated highest expression of comt in c <dig>  amongst all the strains , and a significant inverse correlation of comt expression with aggressive behaviors  <cit> .
this same study identified a decrease in expression of riken cdna 1810037i <dig> in c <dig>  compared with dba, and a positive correlation of expression of this gene with aggressive behaviors.
in the current study we observed increased expression of comt in c <dig>  as compared to dba, and increased expression of 1810037i17rik in dba, with a very significant main effect of strain .
more generally, several genes showing altered expression in controls in our study  also showed similar alterations in that study, when analyzed with both mas <dig> and dchip, even though the focus was on an independent brain area.
microarrays are very important tools for exploratory analyses, but have some important restrictions.
samples run on different platforms , or analyzed with different methods , can produce varying results.
quality of probes, probes directed at minor splice variants, and mis-annotation of probes can also lead to errors in conclusions .
furthermore, for a proportion of mrnas with altered expression, there may not be a corresponding change in protein expression.
for all of these reasons, validation by additional methods is critical.
in recent qpcr analyses of striata from four strains of mice, comt was found to be downregulated in dba, as compared to c <dig>  <cit> , consistent with the microarray, qpcr and enzyme activity results presented here.
similarly, gabra <dig> was observed to be upregulated in dba, as compared to c <dig>  in that study as well.
a common mechanism for many drugs of abuse is the increase of dopamine in the pathway from the vta through the nac and to the fc  <cit> .
in the case of morphine, binding of morphine to gaba interneurons reduces gaba release, thereby reducing the gaba-mediated inhibition of dopamine release in the nac, with a net result of increasing dopamine levels.
in the case of cocaine, blocking of dopamine re-uptake leads to increased dopamine levels, while in the presence of amphetamine, there is increased non-vesicular release of dopamine.
additional studies implicate these pathways in the responses to other substances of abuse.
elevation of dopamine would be inhibited by increased activity of comt.
we, therefore, can hypothesize that animals, or humans, with elevated comt levels would experience reduced rewarding effects to drugs of abuse, while animals or humans with decreased levels of comt may be more sensitive to certain drugs of abuse and perhaps even find them aversive.
alternatively, elevated comt levels may lead to a hypo-dopamine state which drug taking might alleviate.
in several studies in human substance use, there has been evidence for an association of comt genotype with substance use .
while these studies remain somewhat controversial, they are consistent with our hypotheses.
these studies typically focused on the val/met variant in comt.
however, it may be that analysis of other variants within comt, such as those associated with expression  <cit> , may show stronger association with substance use.
a corollary of our hypotheses is that reduction of comt activity, by the use of comt inhibitors, may have two important effects.
in the case of analgesia for severe pain, comt inhibitors may reduce the levels of analgesics required, an hypothesis with some empirical support  <cit> .
in addition, in instances of drug seeking behavior, comt inhibitors may reduce drug seeking by reducing the "hypo-dopamine state," and/or by making the drug aversive or less rewarding.
studies on comt in animals have previously demonstrated that non-specific comt-inhibitors, when given in the presence of morphine, can be lethal  <cit> .
while these results might suggest that there is an important molecular interaction between comt and drugs of abuse, it is important to note that these studies were carried out with less specific, first generation comt inhibitors.
gnb <dig>  one mrna of a group of differentially expressed genes that we identified as also having enriched expression in the brain, showed significant correlation with morphine consumption in a two-bottle choice paradigm.
gnb <dig> was represented by three probe sets on the microarray that showed significant correlation with each other .
correlation of the gnb <dig> probe sets with morphine consumption showed rank order correlations of - <dig>  , - <dig>  , and - <dig>  , for probe sets 94854_g_at, 94853_at, and 97458_at, respectively.
altered expression of gnb <dig> was confirmed by qpcr.
all three probe sets of gnb <dig> were observed to have decreased expression in the dba strain under control conditions in the nac in an independent study  <cit> .
moreover, in that study gnb <dig> also showed decreased expression in the fc in the dba strain.
finally, gnb <dig>  was previously shown to have a five-fold range of expression in bxd recombinant inbred strains, with a heritability of 75%, which is near the maximum observed for any transcript  <cit> .
gnb <dig> is a beta-subunit for the heterotrimeric guanine nucleotide-binding proteins, and is also known to play an important role in the visual system.
in our studies, gnb <dig> expression, as measured by qpcr, showed modestly increased  expression in both strains in the presence of morphine.
interestingly, gnb <dig> expression has been shown to be up-regulated in the nac in response to cocaine administration in mouse  <cit>  and rat  <cit> , and in response to amphetamine  <cit>  and methamphetamine  <cit>  treatment in the mouse.
antisense disruption of gnb <dig> expression blocks cocaine-induced sensitization, but without affecting the acute responses to cocaine  <cit> .
in a recent study examining genes that show altered expression four hours after naltrexone-induced morphine withdrawal, gnb <dig> was downregulated in the locus coeruleus  of c <dig> mice  <cit> .
the lc is the primary site of noradrenergic neurons in the brain, and sends projections throughout the mesocorticolimbic dopamine system, including to the nac, vta, and fc, and is a mediator of physical dependence on opiates  <cit> .
gnb <dig> maps to murine chromosome  <dig>  and gnb <dig> maps to human chromosome 1p <dig> .
a role for gnb <dig> expression in responses to multiple drugs of abuse is supported by the expression data.
by the nature of our experimental design, we were able to consider strain and treatment effects in the same study.
such an approach has not been undertaking previously.
there were multiple genes showing main effects of strain with much more modest numbers of genes showing main effects of treatment or strain by treatment interactions.
one facet of our results is consistent with the hypothesis that an important determinant underlying the differences in morphine response across strains lies in genes that show a main effect of strain and are not necessarily regulated by morphine.
such genes include gnb <dig> and comt, as discussed in detail above.
other genes showing main effects of strain are genes important in plasticity, including those defined in the kegg pathways of long-term potentiation  and long-term depression .
genes in these pathways that show a main effect of strain by anova include an ionotropic glutamate receptor subunit , two isoforms of calcium/calmodulin-dependent protein kinase ii , phosphatases , an inositol  <dig> ,5-triphosphate receptor , ras proteins , and a mitogen activated kinase in the ltp pathway.
in the ltd pathway, kegg analysis identified several guanine nucleotide binding protein  subunits, phospholipase a <dig>  the mitogen activated kinase, protein phosphatase  <dig> and ip3r identified above, as well as insulin-like growth factor  <dig>  these plasticity genes may contribute to multiple differences between the strains, including to drug responses.
in this context, the genes identified in the long-term potentiation pathway are interesting.
the nmda receptor modulates intracellular calcium levels, as does ip3r.
this in turn can modulate calcium dependent protein phosphatases, kinases and ras proteins, followed by changes in activity of mitogen activated kinases.
expression of representatives of all of these classes of enzymes are altered across the two strains, and may thereby contribute to altered drug dependence .
genes showing a main effect of treatment are related to morphine response, irrespective of strain.
these genes fell into a diverse group.
kegg analysis identified two significant groups, identified as "adipocytokine signaling pathway" and "axon guidance."
the axon guidance group of genes identified in kegg analysis were all related to the semaphorin pathway, with semaphorins 3f, 4b, 6c, 6d, and 7a showing treatment effects.
a potential semaphorin receptor, plexin a <dig>  was also identified as showing a main effect of treatment.
this is an interesting finding in light of a recent study looking at the effects of cocaine on semaphorins and other axon guidance molecules in adult rat  <cit> .
many of the semaphorins, including those above, showed significant expression changes in response to cocaine.
although that study made use of cocaine, it may be that changes in axon guidance molecules are a frequent response to drugs of abuse .
another aspect of the drug by strain analysis allows for looking at genes that are differentially regulated across strains in response to morphine.
these genes may also contribute to altered drug response between c <dig> and dba mice.
kegg analysis identified genes within the focal adhesion pathway as showing drug by strain interactions in anova.
several extracellular matrix  genes  were identified, as were two integrins , which can bind to ecm proteins.
genes downstream of the integrins, including phosphatidylinositol 3-kinase , were altered, as well as genes regulated by the phosphatidylinositol signaling system .
these results suggest that the phosphatidylinositol signaling system is of interest in individual variations in drug responses, especially considering that there is recent evidence for a role for this system in morphine withdrawal and cocaine sensitization  <cit> .
qtl analysis, as a means for understanding molecular differences across strains, has proven more difficult than anticipated.
as elaborated in a recent review  <cit> , over the past  <dig> years, there have been more than  <dig>  qtls that have identified in crosses between inbred strains of mice, and  <dig> qtls found in crosses between inbred strains of rats.
however, only about  <dig> genes have been identified from these qtl analyses, many of which have lod scores of over  <dig>  the overall success rate of less than 1% for identifying genes underlying qtls has been explained by various means, and likely reflects a hidden complexity in the qtl analyses.
the use of microarrays to supplement qtl analyses may simplify the identification of quantitative trait genes  that underlie qtls and may also identify additional genes that have not been mapped by qtl analysis.
it is of note that qtl analyses of drug-related behavior in mice will not necessarily identify the comt, gnb <dig> or other loci.
however, it is important to appreciate that genetic factors modulating expression of a given gene can localize either to the gene  or to other loci in the genome .
for example, expression qtl  analysis of murine comt, using the uthsc website  <cit>  demonstrate suggestive  lod scores near the comt locus , but also at murine chromosome  <dig> .
CONCLUSIONS
in summary, there are multiple mrnas that show differential expression between the c <dig> and dba strains.
of them, we highlight comt and gnb <dig> as potentially contributing to altered drug-related behaviors in c <dig> as compared to dba mice.
we suggest that inhibition of comt by pharmacological methods may reduce desire for drugs and perhaps cause them to be more effective in clinical settings and/or less rewarding in non-clinical settings.
pathway analysis highlights important differences between the two strains with regards to plasticity , which may contribute to some of the behavioral differences across the strains, including those associated with drug-related behaviors.
furthermore, pathway analysis supports a role for semaphorin signaling in morphine effects, and indicates that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains.

 the mouse c and dba inbred strains differ substantially in many aspects of their response to drugs of abuse, including altered rates of voluntary drug consumption. the c strain shows increased voluntary consumption of multiple drugs of abuse, including opiates, alcohol, cocaine, and amphetamine, which mirrors the poly-substance nature of some drug-seeking behavior in humans. c strains of mice have been viewed as a good model for human drug-seeking behavior. it has been assumed that the poly-substance nature of drug-seeking in c as compared to dba, is due, in part, to common mechanisms that operate irrespective of the drug of abuse. this assumption is fueled by studies showing that there are susceptibility factors that contribute to drug use irrespective of the substance, as well as susceptibility factors that are specific to each pharmacologic class. multiple quantitative trait locus analyses have been carried in the c and dba strains for both drug responses and drug-seeking behavior. qtl analyses for drug-seeking preferences to different classes of drugs might identify regions of the genome associated with drug-seeking across multiple pharmacological classes and, perhaps, more directly target common reward pathways for multiple substances. for morphine, the strongest evidence for a qtl underlying morphine preference is on the proximal part of chromosome at the position of the mu opiate receptor. in the case of preference for alcohol, the strongest qtl's are on chromosomes and as determined by meta-analyses of studies of alcohol preference. further studies with other substances need to be carried out to determine regions of potential overlap. the development of microarray analyses represents an additional genome-wide method for measuring differences across strains, focusing on expression differences rather than on sequence differences in the genome. this is an additional approach to characterize variations between strains as a means of understanding molecular differences that might underlie strain variability in the effect of, and response to, multiple substances of abuse. in the current study, we have carried out microarray analysis in c and dba mice in the nucleus accumbens of naïve and drug-treated animals. we identified a number of mrnas with altered expression between the two strains. we validated the mrna expression changes of several such mrnas, as well as the protein activity of one mrna product, catechol-o-methyltransferase. data mining of expression and behavioral data indicates that comt expression correlates with two aspects of drug response in c57/dba recombinant inbred strains. similarly, a role for gnb in responses to multiple drugs of abuse is supported by data from our study and from other studies. specific pathways were also identified that showed effects of treatment and/or strain. mrnas with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse.  microarray analysis in the nucleus accumbens we carried out microarray analysis in the dissected nac from vehicle-treated and morphine-treated c and dba mice, using a pooling strategy to minimize dissection and other variations. twelve groups of nac were hybridized to the affymetrix mg_u74a microarray and the resultant data analyzed using dchip. we compared mrna expression between strains, and across treatment groups. the first analysis was gene oriented, and required a significant fold change and made use of t-testing to ascribe significance. comparing mrna expression between c and dba mice in the absence of morphine, we identified mrnas with altered expression, four with two different probe sets. comparing mrna expression between the two strains in the presence of morphine for days, we identified mrnas with altered expression, four 1/tcea1) with two different probe sets. we also compared expression within each strain, contrasting expression in vehicle and morphine-treated mice. there were fewer morphine-responsive mrnas within each of the two strains, when compared to the number of mrnas that differed between strains. in the dba strain, mrnas were identified as changed with an effect >= fold in response to morphine and we did not identify any mrnas showing a p < and greater than >= fold change in the c strain in response to morphine. treatment-invariant mrnas it was of interest that genes showing greater expression changes in these first analyses were much more common across strains than within strains. furthermore, there was a very significant proportion of mrnas that showed altered expression between the two strains in both the drug-naïve and the morphine-treated groups. we examined the correlation of the fold-expression changes of these treatment-invariant mrnas under the two treatments and observed a linear relationship, with a high degree of correlation in the relative expression of these mrnas. these treatment-invariant mrnas were of particular interest as they represent a molecular environment that is consistently different between the two strains, irrespective of treatment condition. they also provide evidence that the microarray studies are internally consistent. two-way anova the presence of a large proportion of mrnas that showed altered expression irrespective of treatment supported further analysis of the dataset, using two-way anova. with such an approach, we could identify mrnas showing main effects for strain, main effects for treatment, and interaction effects. we used more permissive criteria, so as to capture genes and pathways that demonstrated smaller but significant changes in expression. running such analyses led to the identification of genes that showed a main effect of strain, genes showing a main effect of treatment, and genes showing significant interaction effects. gnb showed the most significant main effect for strain and additional treatment-invariant genes noted above were also identified in this analysis. kegg pathway analyses of genes showing main effect for strain identified clusters of genes associated with the ribosome, oxidative phosphorylation/atp synthesis, long-term potentiation/long-term depression, and antigen processing and presentation. kegg pathway analysis of genes showing a main effect of treatment identified two significant functional groups, including "axon guidance". the axon guidance group of genes identified in these analyses were all related to the semaphorin pathway. kegg pathway analysis of genes showing significant strain by treatment interactions identified significant effects for genes in the focal adhesion pathway, particularly genes involved in phosphatidylinositol signaling system. validation of microarray analyses we chose a selection of the mrnas from the microarray studies for follow-up using quantitative polymerase chain reaction to validate the microarray signals. mrnas were chosen to represent both the up-regulated and down-regulated mrnas, to represent mrnas showing more profound expression changes, to represent mrnas with neuronal expression and/or potentially relevant neuronal function, and based on other published studies on either varying expression in mouse strains or changes in response to drugs of abuse. we were able to validate a majority of mrnas for which we carried out qpcr, as shown in table overall validation of the microarray results was reflected in the high correlation of the ratio of mrna expression between the two strains as measured by microarray and by qpcr. furthermore, by anova there was a significant main effect of strain with actin related protein 2/ complex, subunit 5/arpc comt, gnb phosphodiesterase 9a/pde9a, phosphatidylinositol transfer protein, beta/pitpnb, and secretory granule neuroendocrine protein 7b protein/sgne and a significant main effect of treatment with arpc and rcn. there was a trend level main effect of strain with rcn, and a trend level main effect of treatment with gabra finally, there were significant interaction terms with arpc ngfi-a binding protein 2/nab and rcn. not all microarray results were unambiguously validated, as an mrna showing great dys-regulation between the two strains by microarray did not show such profound alterations in expression by qpcr. failure of validation can occur for several reasons, including differing probes used for the microarray and qpcr experiments, genomic differences between strains that alters the hybridization of the relevant regions of the probes, and false-positive expression changes. comt levels and morphine a recent report made the observation that individuals with a met/met genotype at the val158met polymorphism of the human comt gene required a significantly reduced dose of morphine to achieve pain relief, as compared to individuals with the val/val genotype. these observations are consistent with other studies implicating comt activity with opioid response. since the dba mice showed increased sensitivity to morphine compared to c as defined by the tail flick assay, and reduction in comt expression and activity, we sought to determine the degree to which morphine sensitivity varied in c57/dba recombinant inbred strains, as a function of comt expression, making use of the webqtl database. we first noted that in those studies of whole brain homogenates, comt expression was significantly decreased in dba, as compared to c consistent with our studies in the nac. we then examined the indices of morphine effect described above. slope of the dose-response curve and the response to mg/kg of morphine both correlated with levels of comt expression. comt expression did not show significant correlation with morphine consumption in a two-bottle choice paradigm. the correlation of comt expression with two indices of morphine effect suggested a possible relationship between these two measures in mice. as mrna levels do not always correlate with protein levels and protein activity, we next sought to determine whether comt activity differed in brain extracts from c and dba mice. nac, amygdala, and frontal cortex were dissected from dba and c mice and comt activity was then determined in homogenates. comt activity was modestly, but significantly, reduced in nac and fc of dba mice, as compared to c mice. discussion we examined mrna expression in the nac in vehicle- or morphine-treated mice, comparing the c and dba strains. the nac is part of the mesocorticolimbic dopamine system which originates in the ventral tegmental area and projects to the nac, fc and other limbic areas. the vta and the nac are two brain regions commonly thought to be involved in the reward system for drugs of abuse. in studies that compared gene expression changes induced by morphine treatment across multiple brain regions, the nac shows some of the most profound changes in the total number of genes with altered expression in the presence of drug. in the current study, we made use of an established and well-regarded analytical platform for analysis of microarrays. in our first analyses, we required reasonable levels of expression, a modest fold change, and a nominal p <. median false-discovery rate estimates were between ~1–4% for results contrasting the two strains, but were significantly higher for contrasts within strains. of importance to the current study is that kerns et al. have recently compared mrna expression in the nac in control c and dba animals. of the mrnas showing altered expression in our study of vehicle-treated mice, more than were also observed to show altered expression in the kerns study, in the same direction for the same probe sets, using different analytical methods. furthermore, there was a significant agreement in relative mrna expression across the two studies. this indicates that a significant proportion of mrnas showing altered expression by microarrays can be reproduced across the two studies, irrespective of analytical approach. it also represents an important validation of such microarray studies. while the kerns study did not identify changes in comt, our analysis of an independent dataset supported our results. in addition, microarray studies in the hippocampus of eight mouse strains demonstrated highest expression of comt in c amongst all the strains, and a significant inverse correlation of comt expression with aggressive behaviors. this same study identified a decrease in expression of riken cdna 1810037i in c compared with dba, and a positive correlation of expression of this gene with aggressive behaviors. in the current study we observed increased expression of comt in c as compared to dba, and increased expression of 1810037i17rik in dba, with a very significant main effect of strain. more generally, several genes showing altered expression in controls in our study also showed similar alterations in that study, when analyzed with both mas and dchip, even though the focus was on an independent brain area. microarrays are very important tools for exploratory analyses, but have some important restrictions. samples run on different platforms, or analyzed with different methods, can produce varying results. quality of probes, probes directed at minor splice variants, and mis-annotation of probes can also lead to errors in conclusions. furthermore, for a proportion of mrnas with altered expression, there may not be a corresponding change in protein expression. for all of these reasons, validation by additional methods is critical. in recent qpcr analyses of striata from four strains of mice, comt was found to be downregulated in dba, as compared to c, consistent with the microarray, qpcr and enzyme activity results presented here. similarly, gabra was observed to be upregulated in dba, as compared to c in that study as well. a common mechanism for many drugs of abuse is the increase of dopamine in the pathway from the vta through the nac and to the fc. in the case of morphine, binding of morphine to gaba interneurons reduces gaba release, thereby reducing the gaba-mediated inhibition of dopamine release in the nac, with a net result of increasing dopamine levels. in the case of cocaine, blocking of dopamine re-uptake leads to increased dopamine levels, while in the presence of amphetamine, there is increased non-vesicular release of dopamine. additional studies implicate these pathways in the responses to other substances of abuse. elevation of dopamine would be inhibited by increased activity of comt. we, therefore, can hypothesize that animals, or humans, with elevated comt levels would experience reduced rewarding effects to drugs of abuse, while animals or humans with decreased levels of comt may be more sensitive to certain drugs of abuse and perhaps even find them aversive. alternatively, elevated comt levels may lead to a hypo-dopamine state which drug taking might alleviate. in several studies in human substance use, there has been evidence for an association of comt genotype with substance use. while these studies remain somewhat controversial, they are consistent with our hypotheses. these studies typically focused on the val/met variant in comt. however, it may be that analysis of other variants within comt, such as those associated with expression, may show stronger association with substance use. a corollary of our hypotheses is that reduction of comt activity, by the use of comt inhibitors, may have two important effects. in the case of analgesia for severe pain, comt inhibitors may reduce the levels of analgesics required, an hypothesis with some empirical support. in addition, in instances of drug seeking behavior, comt inhibitors may reduce drug seeking by reducing the "hypo-dopamine state," and/or by making the drug aversive or less rewarding. studies on comt in animals have previously demonstrated that non-specific comt-inhibitors, when given in the presence of morphine, can be lethal. while these results might suggest that there is an important molecular interaction between comt and drugs of abuse, it is important to note that these studies were carried out with less specific, first generation comt inhibitors. gnb one mrna of a group of differentially expressed genes that we identified as also having enriched expression in the brain, showed significant correlation with morphine consumption in a two-bottle choice paradigm. gnb was represented by three probe sets on the microarray that showed significant correlation with each other. correlation of the gnb probe sets with morphine consumption showed rank order correlations of -, -, and -, for probe sets 94854_g_at, 94853_at, and 97458_at, respectively. altered expression of gnb was confirmed by qpcr. all three probe sets of gnb were observed to have decreased expression in the dba strain under control conditions in the nac in an independent study. moreover, in that study gnb also showed decreased expression in the fc in the dba strain. finally, gnb was previously shown to have a five-fold range of expression in bxd recombinant inbred strains, with a heritability of 75%, which is near the maximum observed for any transcript. gnb is a beta-subunit for the heterotrimeric guanine nucleotide-binding proteins, and is also known to play an important role in the visual system. in our studies, gnb expression, as measured by qpcr, showed modestly increased expression in both strains in the presence of morphine. interestingly, gnb expression has been shown to be up-regulated in the nac in response to cocaine administration in mouse and rat, and in response to amphetamine and methamphetamine treatment in the mouse. antisense disruption of gnb expression blocks cocaine-induced sensitization, but without affecting the acute responses to cocaine. in a recent study examining genes that show altered expression four hours after naltrexone-induced morphine withdrawal, gnb was downregulated in the locus coeruleus of c mice. the lc is the primary site of noradrenergic neurons in the brain, and sends projections throughout the mesocorticolimbic dopamine system, including to the nac, vta, and fc, and is a mediator of physical dependence on opiates. gnb maps to murine chromosome and gnb maps to human chromosome 1p. a role for gnb expression in responses to multiple drugs of abuse is supported by the expression data. by the nature of our experimental design, we were able to consider strain and treatment effects in the same study. such an approach has not been undertaking previously. there were multiple genes showing main effects of strain with much more modest numbers of genes showing main effects of treatment or strain by treatment interactions. one facet of our results is consistent with the hypothesis that an important determinant underlying the differences in morphine response across strains lies in genes that show a main effect of strain and are not necessarily regulated by morphine. such genes include gnb and comt, as discussed in detail above. other genes showing main effects of strain are genes important in plasticity, including those defined in the kegg pathways of long-term potentiation and long-term depression. genes in these pathways that show a main effect of strain by anova include an ionotropic glutamate receptor subunit, two isoforms of calcium/calmodulin-dependent protein kinase ii, phosphatases, an inositol,5-triphosphate receptor, ras proteins, and a mitogen activated kinase in the ltp pathway. in the ltd pathway, kegg analysis identified several guanine nucleotide binding protein subunits, phospholipase a the mitogen activated kinase, protein phosphatase and ip3r identified above, as well as insulin-like growth factor these plasticity genes may contribute to multiple differences between the strains, including to drug responses. in this context, the genes identified in the long-term potentiation pathway are interesting. the nmda receptor modulates intracellular calcium levels, as does ip3r. this in turn can modulate calcium dependent protein phosphatases, kinases and ras proteins, followed by changes in activity of mitogen activated kinases. expression of representatives of all of these classes of enzymes are altered across the two strains, and may thereby contribute to altered drug dependence. genes showing a main effect of treatment are related to morphine response, irrespective of strain. these genes fell into a diverse group. kegg analysis identified two significant groups, identified as "adipocytokine signaling pathway" and "axon guidance." the axon guidance group of genes identified in kegg analysis were all related to the semaphorin pathway, with semaphorins 3f, 4b, 6c, 6d, and 7a showing treatment effects. a potential semaphorin receptor, plexin a was also identified as showing a main effect of treatment. this is an interesting finding in light of a recent study looking at the effects of cocaine on semaphorins and other axon guidance molecules in adult rat. many of the semaphorins, including those above, showed significant expression changes in response to cocaine. although that study made use of cocaine, it may be that changes in axon guidance molecules are a frequent response to drugs of abuse. another aspect of the drug by strain analysis allows for looking at genes that are differentially regulated across strains in response to morphine. these genes may also contribute to altered drug response between c and dba mice. kegg analysis identified genes within the focal adhesion pathway as showing drug by strain interactions in anova. several extracellular matrix genes were identified, as were two integrins, which can bind to ecm proteins. genes downstream of the integrins, including phosphatidylinositol 3-kinase, were altered, as well as genes regulated by the phosphatidylinositol signaling system. these results suggest that the phosphatidylinositol signaling system is of interest in individual variations in drug responses, especially considering that there is recent evidence for a role for this system in morphine withdrawal and cocaine sensitization. qtl analysis, as a means for understanding molecular differences across strains, has proven more difficult than anticipated. as elaborated in a recent review, over the past years, there have been more than qtls that have identified in crosses between inbred strains of mice, and qtls found in crosses between inbred strains of rats. however, only about genes have been identified from these qtl analyses, many of which have lod scores of over the overall success rate of less than 1% for identifying genes underlying qtls has been explained by various means, and likely reflects a hidden complexity in the qtl analyses. the use of microarrays to supplement qtl analyses may simplify the identification of quantitative trait genes that underlie qtls and may also identify additional genes that have not been mapped by qtl analysis. it is of note that qtl analyses of drug-related behavior in mice will not necessarily identify the comt, gnb or other loci. however, it is important to appreciate that genetic factors modulating expression of a given gene can localize either to the gene or to other loci in the genome. for example, expression qtl analysis of murine comt, using the uthsc website demonstrate suggestive lod scores near the comt locus, but also at murine chromosome.  in summary, there are multiple mrnas that show differential expression between the c and dba strains. of them, we highlight comt and gnb as potentially contributing to altered drug-related behaviors in c as compared to dba mice. we suggest that inhibition of comt by pharmacological methods may reduce desire for drugs and perhaps cause them to be more effective in clinical settings and/or less rewarding in non-clinical settings. pathway analysis highlights important differences between the two strains with regards to plasticity, which may contribute to some of the behavioral differences across the strains, including those associated with drug-related behaviors. furthermore, pathway analysis supports a role for semaphorin signaling in morphine effects, and indicates that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains. 
in addition, we validated alterations in the enzyme activity of one mrna product, catechol-o-methyltransferase. one such mrna is comt and we hypothesize that altered expression of comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. in the current study, we carried out microarray analysis in c and dba mice in the nucleus accumbens of drug-naïve and morphine-treated animals. the development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. data mining of expression and behavioral data indicates that both gnb and comt expression correlate with aspects of drug response in c57/dba recombinant inbred strains. we validated the mrna expression changes of several such mrnas, including gnb which has been observed to be regulated by several drugs of abuse. similarly, a role for gnb in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. mrnas with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains. these analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. the mouse c57bl/ and dba/2j inbred strains differ substantially in many aspects of their response to drugs of abuse.
 the mouse c57bl/ and dba/2j inbred strains differ substantially in many aspects of their response to drugs of abuse. the development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. in the current study, we carried out microarray analysis in c and dba mice in the nucleus accumbens of drug-naïve and morphine-treated animals.  we identified mrnas with altered expression between the two strains. we validated the mrna expression changes of several such mrnas, including gnb which has been observed to be regulated by several drugs of abuse. in addition, we validated alterations in the enzyme activity of one mrna product, catechol-o-methyltransferase. data mining of expression and behavioral data indicates that both gnb and comt expression correlate with aspects of drug response in c57/dba recombinant inbred strains. pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. these analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure.  mrnas with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. one such mrna is comt and we hypothesize that altered expression of comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. similarly, a role for gnb in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains. 