Reduced cerebellum volume and ataxia-like motoric phenotype in transgenic mouse, carrier of human CYP2C19 gene

Abstract

Introduction: CYP2C19 transgenic mouse (2C19TG) is generated by the insertion of 12 copies of the human CYP2C19 gene into mouse genome. This animal model is a tool to study the neurodevelopmental role of CYP2C19 in vivo, since this enzyme is expressed in the foetal brains of 2C19TG mice and humans. Previous studies [1,2] showed anxiety and depression-like behaviour in these mice, while the aim of this study was to characterize the motoric function of the 2C19TG mouse. Methods: Whole brain dopamine concentration was measured in the brain homogenate of 23 adult mice by the HPLC-MS method. Motoric function in 50 mutant and 43 control mice of both genders was tested by the rotarod and beam walking tests. Beam walking test was repeated after treatment with dopaminergic receptor antagonists, Ecopipam (0.1 mg/kg) and Raclopride (0.25 mg/kg) as a follow-up. The sections of 10 6-month old and 8 15-months old mice were stained with anti-tyrosine hydroxylase antibody and the number of dopaminergic neurons was counted on histological slides under microscope. Next, after transcardial perfusion of 30 2C19TG and 30 control mice of both genders with contrast agent (4% Paraformaldehyde, 0.05M Gadoteridole, 0.01M Phosphate buffered saline, pH=7.4), cranium containing the whole brain was scanned overnight by the 9.4T MRI scanner. The volumes of 39 brain regions were quantified according to the mouse brain atlas [3]. Student's t-test and two-way ANOVA were used to evaluate statistical significance of between-group differences. Results: Adult 2C19TG mice are hyperdopaminergic, as they exhibit 15% increased dopamine concentration (p<0.001). They also show hyperkinetic motoric phenotype with the ataxia-like walking pattern and pathological clasping reflex. In the beam walking test 2C19TG mice had 20% longer beam crossing time (p=0.007) and 60% more paw slips (p<0.001) then the controls, and this motoric impairment could not be improved with antidopaminergic drugs. Both younger and older 2C19TG mice exhibited only a marginal reduction in the number of dopaminergic neurons of both substantia nigra and ventral tegmental area in a subset of coronal sections. This was confirmed by the gadolinium-enhanced neuroimaging that showed no change in substantia nigra volume in 2C19TG mice. On the other hand, significant differences in volume were identified in 11 regions, including cerebellum (-8.3% p<0.001) and striatum (+3.0%, p<0.001), which are the regions connected with the motoric function. The volumetric changes were detected in the hippocampus (-1.3%, p=0.027), amygdala (+2.8%, p<0.001), septum (+3.3%, p=0.014) and nucleus accumbens (+3.5, p=0.004) of 2C19TG mice. These brain regions are involved in emotional and motivational functions. Conclusion: Ataxia-like motoric phenotype in 2C19TG transgenic mice is probably caused by changes in cerebellum, while hyperdopaminergism is most likely the compensatory adaptation, whereas the changes in the hippocampus, amygdala, septum, and nucleus accumbens may be connected with the mutants’ depression-like phenotype and susceptibility to stress. Therefore, CYP2C19 transgenic mouse in potentially useful model of hyperkinetic disorders, and our findings hint at the possible impact of CYP2C19 enzyme on the development of the several brain regions involved in motor and emotional functioning.