Morphological, physiological and behavioural evaluation of a ‘Mice in Space’ housing system

Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called “Mice in Space” (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories

Postnatal training of 129/Sv mice confirms the long-term influence of early exercising on the motor properties of mice

International audienceA previous study showed that motor experiences during critical periods of development durably affect the motor properties of adult C57BL/6j mice. However, dependence on early environmental features may vary with the genetic profile. To evaluate the contribution of the genetic background on external influences to motricity, we performed the same experiment in a 129/Sv mouse strain that show a strongly different motor profile. Mice were subjected to endurance training (enriched environment or forced treadmill), hypergravity (chronic centrifugation), or simulated microgravity (hindlimb unloading) between postnatal days 10 and 30. They were then returned to standard housing until testing at the age of nine months. The endurance-trained mice showed a fast-slow shift in the deep zone of the tibialis. In addition, mice reared in the enriched environment showed a modified gait and body posture, and improved performance on the rotarod, whereas forced treadmill training did not affect motor output. Hypergravity induced a fast-slow shift in the superficial zone of the tibialis, with no consequence on motor output. Hindlimb unloading provoked an increased percentage of immature hybrid fibres in the tibialis and a shift in the soleus muscle. When compared with similarly reared C57BL/6j mice, 129/Sv mice showed qualitative differences attributable to the lower efficiency of early training due to their lower basal motor activity level. Nevertheless, the results are essentially consistent in both strains, and support the hypothesis that early motor experience influences the muscle phenotype and motor output. (C) 2016 Published by Elsevier B.V

Motor Experience Reprograms Development of a Genetically-Altered Bilateral Corticospinal Motor Circuit

<div><p>Evidence suggests that motor experience plays a role in shaping development of the corticospinal system and voluntary motor control, which is a key motor function of the system. Here we used a mouse model with conditional forebrain deletion of the gene for EphA4 (Emx1-Cre:EphA4^tm2Kldr), which regulates development of the laterality of corticospinal tract (CST). We combined study of Emx1-Cre:EphA4^tm2Kldr with unilateral forelimb constraint during development to expand our understanding of experience-dependent CST development from both basic and translational perspectives. This mouse develops dense ipsilateral CST projections, a bilateral motor cortex motor representation, and bilateral motor phenotypes. Together these phenotypes can be used as readouts of corticospinal system organization and function and the changes brought about by experience. The Emx1-Cre:EphA4^tm2Kldr mouse shares features with the common developmental disorder cerebral palsy: bilateral voluntary motor impairments and bilateral CST miswiring. Emx1-Cre:EphA4^tm2Kldr mice with typical motor experiences during development display the bilateral phenotype of “mirror” reaching, because of a strongly bilateral motor cortex motor representation and a bilateral CST. By contrast, Emx1-Cre:EphA4^tm2Kldr mice that experienced unilateral forelimb constraint from P1 to P30 and tested at maturity had a more contralateral motor cortex motor representation in each hemisphere; more lateralized CST projections; and substantially more lateralized/independent reaching movements. Changes in CST organization and function in this model can be explained by reduced synaptic competition of the CST from the side without developmental forelimb motor experiences. Using this model we show that unilateral constraint largely abrogated the effects of the genetic mutation on CST projections and thus demonstrates how robust and persistent experience-dependent development can be for the establishment of corticospinal system connections and voluntary control. Further, our findings inform the mechanisms of and strategies for developing behavioral therapies to treat bilateral movement impairments and CST miswiring in cerebral palsy.</p></div

Unilateral forelimb restraint.

<p>A. Schematic showing a neonate mouse (P1) with unilateral constraint of the right forelimb against the chest using a surgical bandage tape. B. Experimental timeline. C. Experimental design. We traced separately the motor cortex (MCX) contralateral to the constrained right forelimb (termed constrained MCX; blue) and the MCX contralateral to the unconstrained left forelimb (termed unconstrained MCX; red).</p

Forelimb constraint reduces mirror sites in EphA4 conditional knockout mice.

<p>A, B. Color maps plot the occurrence of evoked mirror movements at each MCX site. The percent of mirror sites is represented according to a color scale, from the lowest (blue) to the highest (red). The constrained MCX (A) shows a propensity of sites where mirror movements were not evoked (blue) and no sites were mirror movements were evoked most frequently (red). The unconstrained MCX (B) also showed a substantial reduction in mirror sites and a paucity of mirror movement sites. C. Bar graphs plot the average (n = 8 mice; 25 MCX sites within each of 16 hemispheres) of the percentage of sites from which the microstimulation evoked a mirror response. There was a 79% decrease in mirror sites from the constrained MCX (blue) and a 55% reduction in the unconstrained MCX (red) compared with EphA4 conditional knockout controls (<i>p</i> = 0.0004 Mann-Whitney test). The inset shows no mirror sites found in the WT at the threshold (all ‘‘blue” sites), whereas nearly all sites in EphA4 conditional knockout controls evoked mirror movements (mostly “red” sites); reanalyzed from data in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163775#pone.0163775.ref008" target="_blank">8</a>].</p

Effect of unilateral forelimb constraint in wild type mice.

<p>A, B, Heat maps for WT mice (average of 5 mice) show a significant decrease in density of CST labeling from constrained MCX (A), one-way ANOVA, <i>p</i><0.0009, F_{2, 49} = 8.20; Bonferroni post-test: <i>p</i><0.05). Although the CST projections from unconstrained MCX (B) show a slight expansion, there were no significant changes relative to WT controls (Bonferroni post-hoc: <i>p</i>>0.05). Lines mark contours at the yellow-green boundary. Black line is WT control contour (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163775#pone.0163775.g002" target="_blank">Fig 2A</a>) for comparison with the wild type constrained/unconstrained MCX (gray lines). Constrained WT show both a forelimb placement (C) and grasp capability (D) impairments (p = 0.0078, paired <i>t-test</i>, p<0.0001 paired t-test, respectively). Calibration (A) for heat maps: 250 μm.</p

Topographic distribution of labeled CST axon terminals at C7/8.

<p>Averaged heat maps for mice (n = 5) in each group and condition (A-D). EphA4 conditional knockout controls (B) show extensive ipsilateral misprojections. Constraint of one forelimb caused a significant decrease in the density of CST projections from constrained MCX (C) shifting the distribution toward a pattern similar to WT group (A). CST projections from unconstrained MCX show a bilateral increase (D). Contours enclosing the region of highest density of labeling (the border between yellow and green on the heat maps) are shown for each condition. E. Mediolateral distributions of mean axon density from unconstrained MCX (red) and constrained MCX (blue) within gray matter. The y-axis plots the regional density of CST projections. Note the substantial ipsilateral projection from the unconstrained MCX that is as dense as the contralateral projection from the constrained MCX. Light shading plots ±SEM. The arrow indicates the midline. Inset compares unconstrained distribution (red line) with EphA4 conditional knockout control (black), scaled in amplitude to match that of the unconstrained distribution. Constrained distribution (blue line) with EphA4 conditional knockout control, scaled in amplitude to match that of the constrained distribution. The gray line is the distribution of WT controls. Control data replotted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163775#pone.0163775.ref008" target="_blank">8</a>]. F. Bar graphs plot the average laterality index (measured as ratio of ipsilateral gray matter labeling divided by contralateral labeling for MCX on each side). Data show a robust bilateral projection in EphA4 conditional knockout control mice with experience (black) due to the abundant ipsilateral CST misprojections (one-way ANOVA, <i>p</i><0.0001, F_{3, 71} = 30.18; Bonferroni post-test: <i>p</i><0.05). The unconstrained MCX reveals no significant changes (red), the constrained MCX shows a robust decrease in the aberrant ipsilateral terminations leading to a more contralateral organization (blue) similar to WT group (gray) (Bonferroni post-test: <i>p</i>>0.05). Constr. MCX.: Constrained motor cortex, unconstr. MCX: unconstrained motor cortex, Ctrl.: control. The color bar represents the axon length in micrometers within in each region of interest. Calibration (A) for heatmaps: 250 μm.</p

Forelimb constraint causes dual changes in CS axon terminals morphology.

<p>A-C confocal projection stacked image (30 optical slices). CST axon morphology in EphA4 conditional KO control (A), constrained MCX (B), and unconstrained MCX (C); ipsilateral (left) and contralateral (right) CST. D. Bar graphs plot CST axon branching within ROIs (inset; average of 4–5 mice/group, 4 sections/animal). Contralateral CST axon morphology on the constrained side (blue) of the spinal cord (right) revealed an overall significant difference between the groups (one-way ANOVA, <i>p</i><0.0001, F_2,51 = 37.70). Bonferroni posthoc testing revealed a significant 50% reduction in the mean number of contralateral CST axon branch points per μm originating from constrained MCX (blue) and controls (dark gray, <i>p<</i>0.05). Importantly, the contralateral projections of the unconstrained MCX (red) showed a significant 1.8 times increase in the mean axonal branch points relative to EphA4 control conditional knockout mice (dark gray). There was no difference in the ipsilateral branches for the unconstrained M1 (light red) relative to EphA4 control conditional knockout mice (light gray) (one-Way ANOVA, <i>p</i><0.0001, F_2,51 = 11.71; <i>p</i>>0.05, Bonferroni posthoc) but ipsilateral branching from the constrained MCX (light blue) was significantly less that either in controls (<i>p</i><0.05, Bonferoni posthoc) or from the unconstrained side (<i>p</i><0.05, Bonferoni posthoc). The inset shows the ROIs (95μm x95μm) we analyzed CS axon terminals morphology. Calibration: scale bar: 50μm.</p

Forelimb constraint reduces bilateral reaching movements in EphA4 conditional knockout mice.

<p>A. Stacked bar graphs plot the average forelimb use (n = 10–13 mice/group) during exploratory reaching behavior. Wild type mice show a small incidence of simultaneous use of both forelimbs in this task (black, left bar) In contrast, EphA4 conditional knockout control mice use mirror reaching movements nearly 80% of the time (black, middle bar). Unilateral forelimb constraint caused approximately a 50% reduction in mirror movements while reaching the cylinder wall (black right bar). The overall difference between the groups was highly significant (one-Way ANOVA, <i>p</i><0.0001, F_2.31 = 57.70). Bonferroni post-hoc testing revealed a significant difference between EphA4 constrained and conditional knockout control mice (<i>p</i><0.05), indicating significantly more independent forelimb use during reaching. However, there still was increased mirroring compared with wild type mice (Bonferroni posthoc: <i>p</i><0.05,). B. Bar graphs plot the normalized mean value of independent forelimb use. We found no significant change in right-left forelimb use in the EphA4 constrained mice (Fig 5B) (<i>p</i> = 0.44 paired <i>t-test</i>) and EphA4 conditional knockout control mice (<i>p</i> = 0.37 paired <i>t-test</i>). Thus, the reduction in mirror reaching movements is due to improved independent limb use not a failure to use one limb.</p