Animals
Ten male C57BL/6J mice at 9–10 weeks of age weighing 20–26 g were used in this study. The animals were purchased from Japan SLC Inc. (Hamamatsu, Japan). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Osaka University School of Medicine Animal Care and Use Committee approved the protocol of the study (Permit Numbers: 21- 086-0, 27-043-000). All surgery was performed under anaesthesia, and every effort was made to minimize animal suffering and reduce the number of animals used. After the experiments, we euthanized the animals by intraperitoneal injection of sodium pentobarbital (Nembutal; 200 mg/kg body weight).
Surgical procedure
Mice were anaesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) in conjunction with local anaesthesia (1% lidocaine). We made a small incision in the mouse’s head skin and fixed a small metal plate with a screw hole to the centre of the skull using dental cement (Sun Medical, Shiga, Japan). After surgery, mice were isolated and closely observed for 48 h.
Stimulation of linear acceleration during translational motion
The experiment was carried out using 10 mice. The mice were subjected to movement in light and dark conditions. A mouse was placed on a linear sled constructed from a plastic cylindrical container and a computer-controlled motor. The mouse was fixed to the sled with a screw by a metal plate attached to the head, and the mouse’s head remained firmly fixed during movement of the sled. The head position was fixed at a position where the bregma-lambda axis was parallel to the ground. The sled moved on a linear stainless steel rail that was parallel to the ground (Figs.1D, 3D, 5A). The sled and rail were constructed by Bio-Medica Co., Ltd (Osaka, Japan). From the rightmost edge, the sled was accelerated, then decelerated, and travelled 1800 mm to the leftmost edge. The sled was accelerated, then decelerated, and returned to the rightmost edge. The sled made five round trips. At both the rightmost and leftmost edges, the sled was stationary for approximately 0.3 s. Five settings were used, with maximum acceleration and velocity as follows: 1.3 G (3.25 m/s), 0.9 G (3.25 m/s), 0.7 G (3.06 m/s), 0.3 G (2.24 m/s) and 0.2 G (1.69 m/s). The mouse was placed in the sled in transverse and longitudinal orientations. When transverse, the mouse was moved rightward and leftward, and linear acceleration was loaded in the interaural direction (dark grey arrow in Fig. 1D). When longitudinal, the mouse was moved forward and backward, and linear acceleration was loaded in the naso-occipital direction (dark grey arrow in Fig. 3D). The settings were performed in random order. When calculating the maximum shift angle of vertical component of both eyes for making Fig. 2f, the following formula was used:
((maximum shift angle of vertical component of the left eye) – (maximum shift angle of vertical component of the right eye)) / 2
The above value was calculated both during leftward acceleration and during rightward acceleration.
When calculating the maximum shift angle of vertical component of both eyes for making Fig. 4D, the following formula was used:
((maximum shift angle of vertical component of the left eye) + (maximum shift angle of vertical component of the right eye)) / 2
The above value was calculated both during forward acceleration and during backward acceleration.
Static tilt
The experiment was carried out using 10 mice. The experiment was performed in dark conditions. The mouse was placed in a plastic cylinder container and fixed to the device with a screw by a metal plate attached to the head. The container was fixed to a board. The board had a gear with 36 teeth positioned at 10° intervals and meshed at 10° (Fig. 5B). The mouse was fixed to the board in two body positions. In one body position, the mouse was rotated laterally about the roll axis (Figs. 2B, 2E, and 5B). In the other body position, the mouse was rotated back and forth about the pitch axis (Fig. 4C). The board was rotated manually and was held at the 0°, 10°, 20°, 30°, 40°, 50°, 60° and 70° rotated positions for approximately 5 s. The rotated position was changed slowly (3–5°/s) over 2–3 s (Figs. 2C, 4A). The direction of rotation was selected randomly.
When calculating the shift angle of vertical component of both eyes for making Fig. 2F, the following formula was used:
((shift angle of vertical component of the left eye) – (shift angle of vertical component of the right eye)) / 2
When calculating the shift angle of vertical component of both eyes for making Fig. 4D, the following formula was used:
((shift angle of vertical component of the left eye) + (shift angle of vertical component of the right eye)) / 2
Eye movement recording
To record the movement of both eyes during stimulation by translational linear acceleration, a high-speed infrared camera (sampling rate 240 Hz) (STC-CL338A Sentech Co., Ltd, Kanagawa, Japan) was used. The acquisition of images of both eyes was synchronized using a software program (Stream-Pix; NorPix, Montreal, Canada). In light conditions, as a preliminary experiment, we set the angles of the right and left camera axis at 60° to the interaural axis to minimize obstruction of the field of view (Fig. 5C). During the experiment, we set the angles of the right and left camera axis at 30° to the interaural axis to obtain clear images of the eyes (Fig. 5D). By comparing the data of the actual experiment and preliminary experiment, we confirmed that the data were similar in both experiments and confirmed that the camera position did not affect eye movement during the movement. In dark conditions, the cameras were set directly beside the eyes (Fig. 5A). Movements of the eyes during the third and fourth trips were analysed.
The infrared camera (sampling rate 60 Hz) (GR200HD2-IR, Shodensha Co., Ltd, Osaka, Japan) was used to record the movement of both eyes during tilting movement in dark conditions. The acquisition of images of both eyes was synchronized using a colour quad processor (SG-202II; Daiwa, Japan). The cameras were set directly beside the eyes (Fig. 5B).
When recording eye movement in darkness, the pupils were contracted with an ophthalmic solution (1% pilocarpine hydrochloride; Nippon Tenganyaku Kenkyusho, Nagoya, Japan).
Three-dimensional analysis of eye movements
The eye movement images were analysed using an algorithm developed in our laboratory8,24 (see Appendix 1). The eye position is represented by a vector around the axis, of which the length is proportional to the angle of rotation. The reference position was defined as the eye position when remaining stationary. The head coordinates for analysing left and right eye movements, as measured by referencing the centre of the pupil and an iris freckle, were reconstructed in three dimensions and defined as shown in Fig. 2B. The X-, Y-, and Z-components mainly reflect the torsional, vertical, and horizontal components, respectively. For the X-component, “right torsional” and “left torsional” indicate that the superior pole of the eyeball rotated to the right and left, respectively. The rotation vector r describing a rotation of θ around the axis n was given by the formula r = n tan(θ/2), with n being the unit vector, the direction of which represents its axis. The value of the axis angle refers to the Euler angle, and not to tan(θ/2). Accordingly, we used the Euler angle parameter, given as 2 tan−1 (magnitude of rotation vector), to represent eye position as an axis-angle representation25. Because the camera axis was set at 30° to the interaural axis during translational motion in light conditions (Fig. 5D), r of the left eye was calculated using the following formula.
r of the right eye was calculated using the following formula.
Recording and analysis of mouse movement
To record mouse movement during stimulation by translational linear acceleration, the marker was set on the sled and the movement of the marker was recorded using a high-speed infrared camera (STC-CL338A). The acquisition of images of the marker was synchronized with eye images using software (Stream-Pix). The coordinates of the centre of the marker were extracted by binarizing the image of the marker (Fig. 5E). The position of the mouse was calculated from the coordinate.
To record mouse movement during tilting movement in dark conditions, two markers were set on the board and the movement of the markers was recorded using the infrared camera (GR200HD2-IR). The acquisition of images of the markers was synchronized with images of the eyes using a colour quad processor (SG-202II). The coordinates for the centre of gravity of the two markers were extracted (Fig. 5F). The tilt angle of the mouse was calculated from the tilting angle of the line connecting the two centres.