Ontogeny of mouse vestibulo-ocular reflex following genetic or environmental alteration of gravity sensing

Abstract

The vestibular organs consist of complementary sensors: the semicircular canals detect rotations while the otoliths detect linear accelerations, including the constant pull of gravity. Several fundamental questions remain on how the vestibular system would develop and/or adapt to prolonged changes in gravity such as during long-term space journey. How do vestibular reflexes develop if the appropriate assembly of otoliths and semi-circular canals is perturbed? The aim of present work was to evaluate the role of gravity sensing during ontogeny of the vestibular system. In otoconia-deficient mice (ied), gravity cannot be sensed and therefore maculo-ocular reflexes (MOR) were absent. While canals-related reflexes were present, the ied deficit also led to the abnormal spatial tuning of the horizontal angular canal-related VOR. To identify putative otolith-related critical periods, normal C57Bl/6J mice were subjected to 2G hypergravity by chronic centrifugation during different periods of development or adulthood (Adult-HG) and compared to non-centrifuged (control) C57Bl/6J mice. Mice exposed to hypergravity during development had completely normal vestibulo-ocular reflexes 6 months after end of centrifugation. Adult-HG mice all displayed major abnormalities in maculo-ocular reflexe one month after return to normal gravity. During the next 5 months, adaptation to normal gravity occurred in half of the individuals. In summary, genetic suppression of gravity sensing indicated that otolith-related signals might be necessary to ensure proper functioning of canal-related vestibular reflexes. On the other hand, exposure to hypergravity during development was not sufficient to modify durably motor behaviour. Hence, 2G centrifugation during development revealed no otolith-specific critical period.

Figure 1. Horizontal angular vestibulo-ocular reflex in C57Bl/6J and ied mice.

A–B, Example of eye movements evoked in C57Bl/6J (A) or ied (B) mice during 1 Hz sinusoidal oscillation in horizontal. Shaded areas indicate quick phases. Plots present oculomotor fields. Red points are the eye position of the same traces. The ellipses present 95% of the horizontal and vertical eye positions. θ are horizontal and vertical variance; inclination of the ellipse was computed as the slope of the linear regression between vertical and horizontal eye positions. C–D Averaged oculomotor fields in C57Bl/6J (C) and ied (D) mice across tested frequencies. Line and surface of the ellipses present the mean and standard deviation of the population, respectively. The slopes of the ellipses are the mean slope of the individuals’ ellipses. Green asterisks indicate significantly larger response in ied compared to C57Bl/6J. E–F, Gain (E) and timing (F) of the horizontal component of eye movement responses for C57Bl/6J and ied populations. Asterisk indicates statistical difference with p<0.05. Tp, Table position; EVp, Eye Vertical position; EHp, Eye Horizontal position. In this and following figures, plots present mean ± SD.

Figure 2. Maculo-ocular reflex in C57Bl/6J and ied mice.

A, Left panel: Scheme of spatial displacement of the mouse during counter-clockwise rotation at constant velocity. Mouse was head-fixed 30° nose down; pitch of table was 17°. Roll and Pitch angles variations during rotations are reported for every ¼ cycle. B, Orientation of the gravity in head-fixed coordinates during clockwise rotation. C, Eye movements observed during 50°/s constant velocity off-vertical axis rotation in counter clockwise direction. Vertical eye position changed periodically with table rotation, reaching maximal elevation and depression when head roll was maximal (±8.5°). Horizontal eye movements also consisted in periodical modulation of the eye position on which was superimposed a horizontal nystagmus in compensatory direction (left for CW; right for CCW rotations). D, in ied mice, absence of otoconia resulted in the absence of horizontal and vertical movements during the steady-state. E, F, horizontal bias (E) and vertical gain (F) for C57Bl/6J and ied mice during off vertical axis in all tested conditions. In controls, horizontal bias increased with increasing table velocity. Vertical velocity gains decreased with increasing table velocity. Plots illustrate the absence of modulation of horizontal bias and vertical gain in ied mice. Asterisks indicate significantly larger response in C57Bl/6J compared to ied mice. Tp, Table position; EVp, Eye Vertical position; EHp, Eye Horizontal position.

Figure 3. Scheme of hypergravity groups and timing of the experiments.

A, Adult-HG group was composed of two month old mice centrifuged at 2G during 50 days. Vestibulo-ocular responses were tested 1 and 6 months after return to normal gravity. B, developmental groups. Mice were conceived, born and raised in 2G centrifuge (50 days, group Full), or exposed 20 days to hypergravity during early onset (Pre) or late maturation (Post) periods of vestibular development. For all groups, “+1” and “+6” refer to recording made 1 and 6 months after return to normal gravity, respectively.

Figure 4. Modification of the angular vestibulo-ocular and maculo-ocular reflexes in adult centrifuged C57Bl/6J mice.

A, Averaged oculomotor fields in non-centrifuged (control) and centrifuged (Adult-HG) mice across tested frequencies. Line and surface of the ellipses present the mean and standard deviation of the population, respectively. The slopes of the ellipses are the mean slope of the individuals’ ellipses. Oculomotor fields were not altered in adult-HG compared to control. B, Bode plots of horizontal VOR in dark gain and phase during horizontal sinusoidal rotations. There was no consistent effect of centrifugation on the responses to sinusoidal rotations. C–D, Raw traces showing steady state response of the same adult-HG mouse followed at time +1 month (C) and +6 months (D) after centrifugation. Note the absence of nystagmus on the horizontal traces and the strong reduction in the amplitude of the vertical movements at 1 month. E, horizontal bias (E) for non-centrifuged (control) and centrifuged (Adult-HG) mice in all tested conditions. Horizontal biases (E) were significantly affected in 50°/s CCW condition. Right panel presents individual (small symbols) and mean of the populations (large symbols). Solid lines indicate the evolution of individuals’ bias at +1 and +6 months after centrifugation (black: improved responses; dotted: no or little improvement in the responses). F, vertical gain (F) for non-centrifuged (controls) and centrifuged (Adult-HG) mice in all tested conditions. Tp, Table position; EVp, Eye Vertical position; EHp, Eye Horizontal position. Black or purple asterisks indicate p<0.05 between control and Adult-HG+1 or Adult-HG+6, respectively.

Figure 5. Angular vestibulo-ocular reflex is not modified following developmental exposure to hypergravity.

A, B, C: Averaged oculomotor fields (A), horizontal gain (B) and phase (C) in non-centrifuged (control) mice compared to mice centrifuged between E0–P10 (pre) tested 1 and 6 months after centrifugation. No significant differences were found. D, E, F: Averaged oculomotor fields (D), horizontal gain (E) and phase (F) in non-centrifuged (control) mice compared to mice centrifuged between P10–P30 (post) tested 1 and 6 months after centrifugation. No significant differences were found. G, H, I: Averaged oculomotor fields (G), horizontal gain (H) and phase (I) in non-centrifuged (control) mice compared to mice centrifuged between E0–P30 (full) tested 1 and 6 months after centrifugation. No significant differences were found.

Figure 6. Maculo-ocular reflex in mice centrifuged during development.

A–B, horizontal bias (A) and vertical gain (B) for control and pre mice during off vertical axis rotation in all tested conditions. No significant differences were found at 1 and 6 months. C–D, horizontal bias (C) and vertical gain (D) for control and post mice during off vertical axis rotation in all tested conditions. No significant differences were found at 1 and 6 months. E–F, horizontal bias (E) and vertical gain (F) for control and full mice during off vertical axis rotation in all tested conditions. Asterisks indicate significant differences (p<0.05) between control and full+1 groups.