A New Mouse Model for Mania Shares Genetic Correlates with Human Bipolar Disorder

<div><p>Bipolar disorder (BPD) is a debilitating heritable psychiatric disorder. Contemporary rodent models for the manic pole of BPD have primarily utilized either single locus transgenics or treatment with psychostimulants. Our lab recently characterized a mouse strain termed Madison (MSN) that naturally displays a manic phenotype, exhibiting elevated locomotor activity, increased sexual behavior, and higher forced swimming relative to control strains. Lithium chloride and olanzapine treatments attenuate this phenotype. In this study, we replicated our locomotor activity experiment, showing that MSN mice display generationally-stable mania relative to their outbred ancestral strain, hsd:ICR (ICR). We then performed a gene expression microarray experiment to compare hippocampus of MSN and ICR mice. We found dysregulation of multiple transcripts whose human orthologs are associated with BPD and other psychiatric disorders including schizophrenia and ADHD, including: Epor, Smarca4, Cmklr1, Cat, Tac1, Npsr1, Fhit, and P2rx7. RT-qPCR confirmed dysregulation for all of seven transcripts tested. Using a novel genome enrichment algorithm, we found enrichment in genome regions homologous to human loci implicated in BPD in replicated linkage studies including homologs of human cytobands 1p36, 3p14, 3q29, 6p21–22, 12q24, 16q24, and 17q25. Using a functional network analysis, we found dysregulation of a gene system related to chromatin packaging, a result convergent with recent human findings on BPD. Our findings suggest that MSN mice represent a polygenic model for the manic pole of BPD showing much of the genetic systems complexity of the corresponding human disorder. Further, the high degree of convergence between our findings and the human literature on BPD brings up novel questions about evolution by analogy in mammalian genomes.</p> </div

Confirmation of the MSN manic phenotype using an experimental replication of the most robust behavioral measure from previous research on this mouse strain, total locomotor activity.

<p>A) MSN mice display stable heightened locomotor activity relative the outbred strain. ***P<0.001. B) The probability density for MSN mouse total locomotor activity is bimodal, while the probability density for the control strain is unimodal. This leads us to the hypothesis that MSN mice may display behavioral bipolarism, a hypothesis that will be examined in future work.</p

Heatmap of normalized values for the top 100 well-annotated genes from the microarray experiment listed by P-value in order from top to bottom, then left to right.

<p>The lowest P-values are at the top left corner, and the highest are at the bottom right. Blue values represent lower expression and orange values represent higher expression. The names of the genes discussed in the text of this manuscript are highlighted in light orange.</p

Genome enrichment analysis and homology of highlighted enriched clusters to the human genome.

<p>A) Genome enrichment analysis of the MSN phenotype using a novel enrichment algorithm we created for this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038128#s4" target="_blank">Materials and Methods</a>). The y-axis represents the log₁₀ inverse of the corrected binomial probability that a cluster of dysregulated genes would occur by chance. The black horizontal lines demarcate a cluster occurring by chance with a 0.001 corrected probability, consistent with a LOD or NPL score of 3 in a linkage study. Spikes above the black lines indicate dysregulated gene clusters highly unlikely to occur by chance, indicating that the genome region is significantly enriched. Corrected probabilities less than 1×10⁻⁹ are collapsed to 1×10⁻⁹. B) Shared synteny, a similar clustering of orthologous genes, between the clusters on the murine genome highlighted in orange in Fig. 4.A and human genome regions strongly implicated in BPD (see Results for details). Blue lines represent orthologous genes and their positions in the murine (upper) and human (lower) genomes.</p

Interaction plot showing strain differences between back-transformed means and standard errors of MSN and ICR males and females.

<p>A two-way ANOVA found a highly significant strain effect, a significant sex effect, and no significant interaction effect. All significant pairwise tests are summarized in the plot (Tukey HSD: *** <<i>p</i> = 0.001≤ ** <<i>p</i> = 0.01≤ * <<i>p</i> = 0.05≤. <<i>p</i> = 0.10).</p

Diurnal activity plot of mice examined for a month.

<p><b>A</b>) Half-hour in-cage activity averages for each strain with ribbons representing standard error. Diurnal activity panels are double plotted for ease of viewing. MSN mice showed a different diurnal activity pattern than ICR mice, displaying elevated activity just prior to the transition to dark period, highly elevated activity in the early dark period, and a drop in activity midway through the dark period. <b>B</b>) Half-hour in-cage activity averages for each mouse studied with ribbons representing standard error. Panels are double plotted for ease of viewing. MSN mice showed less variability in diurnal activity profile than outbred ICR mice.</p

RT-qPCR confirmation results for seven genes from the microarray.

<p>Ratio distribution is graphed as a box-and-whiskers plot. Ratios greater than 1 represent genes with higher expression in the MSN strain and ratios less than 1 represent genes with lower expression in the MSN strain relative to the ICR strain. *P<0.05; **P<0.01; ***P<0.001.</p

Sexually Dimorphic, Developmental, and Chronobiological Behavioral Profiles of a Mouse Mania Model

<div><p>Bipolar disorders are heritable psychiatric conditions often abstracted by separate animal models for mania and depression. The principal mania models involve transgenic manipulations or treatment with stimulants. An additional approach involves analysis of naturally occurring mania models including an inbred strain our lab has recently characterized, the Madison (MSN) mouse strain. These mice show a suite of behavioral and neural genetic alterations analogous to manic aspects of bipolar disorders. In the current study, we extended the MSN strain's behavioral phenotype in new directions by examining in-cage locomotor activity. We found that MSN activity presentation is sexually dimorphic, with MSN females showing higher in-cage activity than MSN males. When investigating development, we found that MSN mice display stable locomotor hyperactivity already observable when first assayed at 28 days postnatal. Using continuous monitoring and analysis for 1 month, we did not find evidence of spontaneous bipolarism in MSN mice. However, we did find that the MSN strain displayed an altered diurnal activity profile, getting up earlier and going to sleep earlier than control mice. Long photoperiods were associated with increased in-cage activity in MSN, but not in the control strain. The results of these experiments reinforce the face validity of the MSN strain as a complex mania model, adding sexual dimorphism, an altered diurnal activity profile, and seasonality to the suite of interesting dispositional phenomena related to mania seen in MSN mice.</p></div

Results of photoperiod experiment.

<p><b>A</b>) Diurnal activity plots for each photoperiod examined with lights off times aligned. Diurnal activity panels are double plotted for ease of viewing all time periods. <b>B</b>–<b>D</b>) Interaction charts showing back-transformed means and standard errors of: <b>B</b>) total 24-hour in-cage activity, <b>C</b>) mean speed during the dark period, and <b>D</b>) mean speed during the light period. Photoperiod-dependent differences in total in-cage activity were seen in MSN but not ICR mice (<b>B</b>), though photoperiod-dependent alterations to the amount of total activity budgeted in the light and dark periods were seen in both strains (<b>C</b>, <b>D</b>). All significant pairwise tests are summarized in panels <b>B</b>–<b>D</b> (Tukey HSD: *** <<i>p</i> = 0.001≤ ** <<i>p</i> = 0.01≤ * <<i>p</i> = 0.05≤. <<i>p</i> = 0.10).</p

List of all miRNAs found to be significantly linked with upregulated gene expression in postpartum mice using GSEA.

<p>For a given binding site near an upregulated gene, in some cases only one miRNA is identified, but for other sites multiple miRNAs are identified. In the list, if a dash separates two letters, that indicates all miRNAs within those letters were also significant (e.g., MIR-181A-D indicates miRNAs 181A, 181B, 181C, and 181D). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038602#pone.0038602.s002" target="_blank">Table S2</a> provides additional information on the binding sites for the miRNAs and p-values.</p