Unilateral labyrinthectomy does not affect hindlimb intersegmental coordination during swimming.

<p><b>A</b>. Both UL54 and UL66 juvenile frogs showed a strong caudo-rostral body twisting towards the lesioned side, which was measured as the angle δ (black arc) between the eye (pink line) and knee (orange line) axes. <b>B.</b> Colored markers placed on the three main joints of both hindlimbs and on the back along the axis (upper schematic) enabled bilateral angular variations of the hip, knee and ankle to be measured (lower schematic). Each angular variation was then rectified and plotted against time (traces at extreme right) to allow delay measurements. Color code: blue, left hindlimb, purple, right hindlimb. <b>C</b>: Detail of ankle, knee and hip angular variations on the left side over two consecutive cycles. The knee-ankle delay (d) and hip-ankle delay (d’) were then calculated between maximal angular values (corresponding to the maximal joint aperture) for each cycle, and for both hindlimbs. o: open joint; c: closed joint. <b>D.</b> Knee and hip movement delays relative to maximal ankle excursion expressed as a percentage of cycle duration. No significant differences were found between control (unfilled) and UL54 (shaded) or UL66 (filled) juveniles. Error bars indicate SEM.</p

Cycle periods and burst durations in hindlimb kicking motor patterns <i>in vitro</i>.

<p>UL performed either pre- or post-metamorphosis caused no significant changes in the fictive locomotor cycle period, whereas the durations of extensor bursts were significantly altered following a post-metamorphic UL. Note also a slight increase in ipsilesional flexor burst durations in UL66 juveniles compared to both intact and UL54 animals. The number of animals in each group is indicated in brackets and the power of non-significant ANOVA tests in parentheses.</p

Circular statistical analysis of the phase relations between lumbar and thoracic ventral root motor bursts <i>in vitro</i>.

<p>Coordination patterns between left (L) and right (R)-side flexor and extensor fictive locomotor activities, and with bilateral thoracic (Th2) motor bursts. The Rayleigh (Z) test was used to verify non-uniformity of the circular distributions, and the V-test was used to compare distributions with a selected direction (either 0°, <i>u</i>(0°), for synchrony or 180°, <i>u</i>(180°), for alternation). Bold characters highlight statistically random distribution.</p

Solely a pre-metamorphic UL alters dorsal muscle/limb extensor muscle coordination in the post-metamorphic frog.

<p>Sample left (L) and right (R) electromyographic (EMG) recordings from dorsal back muscle <i>dorsalis trunci</i> (dt) and ankle extensor muscle <i>plantaris longus</i> (pl) in intact (<b>A</b>), UL54 (<b>B</b>) and UL66 (<b>C</b>) juveniles. The sites of the vestibular lesion (UL) and EMG electrode placements are shown at left. The corresponding circular plots (layout equivalent to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071013#pone-0071013-g003" target="_blank">Figure 3</a> except that each dot represents the mean for a single forward rectilinear swim episode) indicate the lack of bilateral <i>dorsalis</i> and right side (ipsilesional) <i>dorsalis</i>/<i>plantaris</i> coordination in the UL54 group only. Scale bars: 1s.</p

A unilateral labyrinthectomy at pre- or post-metamorphosis leads to distinct degrees of locomotor impairment in freely-behaving juvenile frogs.

<p><b>A.</b> Images of the hindlimb extension phase (near its termination) during three consecutive cycles of normal swimming (top) and rolling behavior (bottom) expressed by control and unilateral labyrinthectomy (UL; red dot)-lesioned stage 66 <i>Xenopus</i>. Each cycle consisted of alternate limb flexions (F) and extensions (E). UL-induced rolling behavior consisted of a semi-complete rotation of the animal around its longitudinal body axis during each hindlimb extension. <b>B–D.</b> Swimming behavior of intact control animals (n = 6) before and after metamorphosis (<b>B</b>), and acute and chronic effects of a right-side UL performed at stage 54 before (n = 9, UL54, <b>C</b>) or at stage 66 after (n = 12, UL66, <b>D</b>) metamorphosis. Histograms show the percentage of swim cycles in which normal rectilinear (unfilled), circling (light grey) or rolling (dark grey) trajectories were expressed in each animal group. Error bars indicate SEM.</p

Unilateral labyrinthectomy-induced alterations in static posture of juvenile <i>Xenopus</i>.

<p>Back and limb joint angles on the left (L) and right (R) sides were measured in stationary intact and lesioned juveniles. The number of animals in each group is indicated in parentheses. A unilateral labyrinthectomy performed before metamorphosis caused larger subsequent alterations in static posture than a post-metamorphic UL. Note that large individual variations among the UL54 group for the R hip angle (indicated by high SEM value compared to the two other groups) were responsible for the low ANOVA power (given in parentheses for non-significant ANOVA tests). The number of animals in each group is indicated in brackets. ns: non-significant;</p>***<p>p<0.001;</p>**<p>p<0.01;</p>*<p>p<0.05.</p

Different patterns of actuator activation induce different body distortions and resultant changes in aquatic displacement.

<p><b>A–L:</b> Frontal (A, D, G, J), lateral right (B, E, H, K) and exploded view (C, F, I, L) of the FE model at initial resting state (A–C) and at the end of either a left side (“contralesional”; D–F), symmetrical (Bilateral; G–I) or right side (“ipsilesional”; J–L) activation of the longitudinal actuators. The angle at the end of each simulation and the resulting angular displacement during propulsion (; obtained from the subsequent dynamic simulation) are indicated in panels D, G and J. Arrowhead in <b>B</b> indicates that the left hip marker (l) is on the non-visible side of the model. <b>M</b>: Theoretical explanation of lesioned animal movement according to simulation results. A high angular velocity associated with the strong body torsion of animals with symmetrically-activated postural back muscles (top) induces a complete disequilibrium of the body (red arrow; value taken from panel <b>G</b>) during each hindlimb extension, as observed experimentally in UL66 juvenile frogs. In contrast and in correspondence with UL54 juvenile behavior, a lower angular velocity associated with the much reduced body torsion of animals with an asymmetrical propulsion/posture coupling (bottom) causes only a slight disequilibrium (green arrow; taken from panel <b>D</b>) that is subsequently compensated for by passive water reaction forces (black arrows) during the remainder of the kick cycle. Color code: black, dorsal, white, ventral, and grey, lateral sides of the body.</p

3D model of a UL juvenile with twisted body trunk.

<p>Geometrical model of a lesioned animal’s trunk using the 3D finite element (FE) method, based on anatomical characteristics and the assumption that the main body/water interactive forces occur at the level of the trunk (<b>A</b>). Once the initial FE model body was built, a 37° torsion () was applied in the antero-posterior axis (<b>B</b> right) in order to simulate the mean body twist towards the lesioned side observed in UL juveniles (<b>B</b> left). Red markers n (nose), r (right hip) and l (left hip) indicate model orientation and together with the two dashed lines (see <b>C</b>), illustrate the model’s torsion. <b>C:</b> The two artificial front and rear rigid body components, respectively simulating the scapula and pelvis belts, were linked by an elastic portion to which the torsion was applied. The pink and orange dashed lines indicate the medial plans of the front and rear rigid body components (green plans), respectively, while the l and r red markers correspond to the linear left and right limits of the rear medial plan, and the n marker indicates the front of the anterior plan. <i>Dorsalis</i> muscles were simulated by two actuators (blue dashed lines) placed on both sides of the antero-posterior axis between the two rigid components. <b>D:</b> Lateral (right) view of the twisted FE model corresponding to UL-induced distortion in juvenile frogs. Arrowhead indicates that the left hip marker (l) is on the non-visible side of the model.</p

Summary of changes occurring in spinal locomotor-related networks during metamorphosis and after a right-side labyrinthectomy.

<p><b>A:</b> Normal metamorphic modifications to spinal motor networks responsible for propulsion and dynamic postural adjustments during swimming (see Beyeler et al., 2008). Note the symmetrical left-right organization in the post-metamorphic juvenile frog. <b>B:</b> In already metamorphosed animals, UL causes asymmetry in the activity of descending brainstem commands to the spinal motor networks, producing an over-excitation on the lesioned side that leads to the expression of rolling behavior. This persistent descending imbalance during juvenile-to-adult maturation has no long-term influence on spinal network organization and animals never recover an effective locomotor capability. <b>C:</b> An acute UL in pre-metamorphic tadpoles also produces an asymmetric descending influence that now persists through metamorphosis (see Lambert et al. 2013). In such an unbalanced developmental environment, however, the adult spinal motor networks are built differently from normal through the establishment of a local asymmetry in propriospinal interactions that are somehow able to counterbalance the asymmetry in the descending commands to allow the restoration of swimming in the post-metamorphic frog. Red arrows: post-UL development; Black arrows: normal development; Double arrow: metamorphic development; Simple arrow: post-metamorphic maturation; Red cross: acute UL; Red dot: persistent UL. The widths of vertical arrows, arrowheads and circuit symbols are proportional to levels of activity.</p

Numbers of juvenile frogs analyzed in different experiments.

<p>Distribution of experimental animals. Kin: kinematic analysis during free swimming; EMG: electromyographic recordings during free swimming; ENG: electroneurographic recordings during <i>in vitro</i> fictive swimming; Multi: combination of tests.</p