Assessing the complex nature of behavior

To unravel the molecular pylons of innate anxiety, a well
established animal model has been characterized using
transcriptome- and sequence-based analyses. The animal model –
hyper (HAB) and hypo (LAB) anxious mice – has been created by
selective inbreeding based on outbred CD1 mice using the extreme
values the mice spent on the open arm of the elevated plus-maze, a
test also used to screen drugs for anxiolytic or anxiogenic
effects. These mice proved a robust phenotypic divergence, also for
depression-like behavior and stress-axis reactivity. In a first
assay, brain regions unambiguously involved in regulating
anxiety-related behavior were screened for gene expression
differences between HAB and LAB animals in a microarray experiment
covering the whole genome. This led to the identification of
thousands of differentially expressed transcripts. The highest
significant results were further validated by quantitative PCR or
other techniques focusing either on protein quantification or
enzyme activity. Applying this strategy, differential regulation of
15 out of 28 transcripts could be validated: vasopressin,
tachykinin 1, transmembrane protein 132D, RIKEN cDNA 2900019G14
gene, ectonucleotide pyrophosphatase/phosphodiesterase 5, cathepsin
B, coronin 7, glyoxalase 1, pyruvate dehydrogenase beta,
metallothionein 1, matrix metallopeptidase 15, zinc finger protein
672, syntaxin 3, solute carrier family 25 member 17 and ATP-binding
cassette, sub-family A member 2. Additionally, analysis of
cytochrome c oxidase activity resulted in the identification of
differences in long-term activity between HAB and LAB mice in the
amygdala and the hypothalamic paraventricular nucleus pointing to
an important role of these brain regions in shaping the
anxiety-related extremes in these mice. In a second genome-wide
screening approach, 267 single nucleotide polymorphisms were
identified to constantly differ between HAB and LAB animals (i.e.
to carry the opposite homozygous genotype at these loci) and
subsequently genotyped in 520 F2 mice, the offspring of
reciprocally mated HABxLAB animals. These F2 mice have been
previously phenotyped in a broad variety of behavioral tests and
show – as descendants of intermediate heterozygotes for all
polymorphic genomic loci between HAB and LAB mice – a free
segregation of all alleles, thus allowing genotype-phenotype
associations based on whole-genome analysis. Only focusing on the
most significant findings, associations have been observed between
anxiety-related behavior and loci on mouse chromosomes 5 and 11,
between depression-like behavior and chromosome 2 and between
stress-axis reactivity and chromosome 3. The locus on chromosome 11
is marked by a polymorphism located in the 3’ untranslated region
of zinc finger protein 672, a gene also markedly overexpressed in
LAB mice and expressed at lower levels in HAB mice leading to a
probable causal involvement in shaping the phenotype. Further
associations on chromosome 5 include two functional polymorphisms
in enolase phosphatase 1 that result in a different mobility of the
enzyme in proteomic assays and with a polymorphism located in the
transmembrane protein 132D gene. Furthermore, independently, an
association of a polymorphism in this particular gene, together
with the resulting gene expression differences has been observed in
a group of panic disorder patients, highlighting this gene as a
causal factor underlying anxiety-related behavior and disorders in
both the HAB/LAB mouse model and human patients. The combination of
expression profiling and confirmation by quantitative PCR, single
nucleotide polymorphism analysis and F2 association studies, i.e.
unbiased and hypothesis driven approaches were key to the
identification and functional characterization of loci, genes and
polymorphisms causally involved in shaping anxiety-related
behavior. Thus, it provides an overview of some new promising
targets for future pharmaceutic treatment and will contribute to a
better understanding of the molecular processes that shape anxiety
and thereby also animal and human behavior.