de Jong S et al. BMC Genomics 2010, 11:20 http://www.biomedcentral.com/1471-2164/11/20 differentially expressed between the amygdala and hip- pocampus samples in both strains, i.e. the Red and Pink modules (Figures 2, 3, Additional File 2). Available data- bases were used to validate our brain region specific results in silico (Allen Brain Atlas; http://www.brain- map.org/). In both modules, only ~10% of the probes disagreed in expression direction of amygdala and hip- pocampus, thereby largely validating current brain- region specific findings. Previous studies also found some expression differences between these structures, although they are more similar to each other than most brain regions [41,42]. Functional examination of the genes represented in these two modules was performed using Ingenuity (Ingenuity® Systems, http://www.ingenu- ity.com). This analysis revealed that the Pink module did not contain any known pathways. The module eigengene of this network of co-expressed genes was also shown to be less discriminative between samples from the different brain areas than the Red module. However, the Red module was shown to be enriched with the categories of Neurological Disease, Genetic Dis- order and Psychological Disorder, as well as Behavior and Nervous System Processes. A major advantage of WGCNA is that its module detection does not make use not make use of any prior gene ontology information. This allows the expression data to speak for themselves without biasing the analy- sis. But gene ontology information is very valuable for determining what is known about the modules. While some modules may be highly enriched with genes of a given gene ontology there is no perfect agreement between gene ontology information and module mem- bership. As we show, modules may arise due to non- biological variation. But biologically important modules must not always be enriched with known gene ontolo- gies [8]. While the Ingenuity database is very extensive, GO analyses remain limited by incomplete gene ontol- ogy annotation. Module membership and network con- nectivity may point to a relationship between genes and pathways that was hitherto unknown. Figure 5 indicates genes that are known to fall within these categories, as well as genes that are found to be in the same module based on co-expression profiles but have not been impli- cated in these pathways before. Therefore, this network approach may provide insights into new candidates in already known processes. The connectivity of the genes in this module is an indication of their interactivity with other genes in the same module. As can be seen in the graphical represen- tation of the network of the Red module (Figure 5), sev- eral genes in enriched pathways are highly connected, while others are less related to other genes. For example Tacr1 (tachykinin receptor 1) is a centrally located hub gene in the Red module and appears in all the above- Page 10 of 14 mentioned categories determined by Ingenuity to be sig- nificantly enriched. This gene is more highly expressed in the amygdala compared to the hippocampus in our dataset. This receptor for the substance P is located in the central nucleus of the amygdala. Substance P is a neuropeptide related to pain and it has also been impli- cated in a wide range of behaviors including learning and memory [48], motivational processes, and anxiety [49]. Other genes in the Red module are also of added value since they may complement and/or interconnect our knowledge of pathways without having a known, large effect on neuropsychiatric traits directly. For exam- ple, we observed an enrichment of genes involved in transcriptional processes that distinguished between the amygdala and hippocampus; these genes included Isl1, Fhl2, Tnfrsf25 and Zcchc12. Other examples in the Red module support previous findings from the literature. For example, genes involved in the canonical pathway ‘GABA-ergic signaling’ (Slc32a1, Gabrg1 and Gad1) are closely correlated in the Red module, with increased expression levels in the amygdala. In addition, genes in the subcategory ‘neuro- genesis of nervous system development’ (Nrn1, Cpne6, Dlx6, Enc1, Fgf13, Isl1, Nelf, Neurod2, Olfm1, Sema3a, Sema3f, Sema5a, Tgfb2, Wfs1) were also found in the same module. Adult neurogenesis takes place predomi- nantly in the hippocampus (and olfactory bulb) [50] and most of these genes (except for Dlx6, Hap1, Isl1 and Wfs1) indeed showed an increased expression in the hippocampus [51]. Conclusions We report very distinct patterns of gene expression between brain regions independent of genetic variation due to differences between mouse inbred strains. Differ- ential gene expression patterns associated with strain origin were highly enriched with SNP variation within the probe regions. In our study design we found that tis- sue-specificity is at least as important for variation in gene expression as genetic variation. Functional enrich- ment studies of modules related to the mouse brain regions implicate known human neurological disease pathways, which suggests that effective mouse models can be developed for human disease. Methods Animals For this experiment, male C57BL/6J and A/J mice were used. Initial breeding pairs for these strains were obtained from the Jackson Laboratory and bred in the Rudolf Magnus Institute of Neuroscience animal facility at the UMC Utrecht. All experimental procedures were approved by the ethical committee for animal experi- mentation of the University Medical Center Utrecht,