Optokinetic system

A 3D virtual cylinder was simulated with an OptoMotry system^9,10 by projecting sinusoidal vertical stripes in four monitors that formed a square area. The test area consisted of a plexiglass box with rectangular openings in all side walls where the monitors were located. An elevated platform (plexiglass discs of different sizes) was situated at the center of the test area. A video camera was placed perpendicular to the platform to monitor the behavior of the animal from above. The signal was displayed on a computer connected to the system. The apparatus was located in a room with noise isolation and reduced ambient light. See Fig. 4 for details of the setup.

Interior view of the optokinetic system.

The virtual cylinder was projected and their characteristics were modified on the monitors by a computer program. Luminance of the screens was adjusted to equalize the intensity of the stimulus (0.20 cd/m² for black stripes and 150 cd/m² for white stripes). Rotation speed, direction of the cylinder and spatial frequency and contrast of the stimulus could also be controlled by the program.

To start the measurement protocol, the rodent was placed on the platform and allowed to move freely. As the rodent moved on the platform, the experimenter marked the head with a red cross superimposed on the video image. The coordinates of this cross were used to determine the center of the rotation of the cylinder in the viewing position of the animal to maintain the virtual cylinder walls at a constant distance and, thus, its spatial frequency. If a perceptible stimulus by the animal was projected, it tracked the grating with continuous and stable movements of the head and neck in the same direction as the rotation of the cylinder.

A homogeneous gray stimulus was projected at the beginning of each procedure. After the animal was placed on the platform and stopped moving, the gray stimulus was replaced by a cylinder with low spatial frequency (0.042 cycles/deg) and maximum contrast (100%) rotating at a constant speed (12 deg/s). The behavior of the animal was assessed by an experimenter for 5 seconds and the gray stimulus was restored. Thus, rodent adaptation to the stimulus was reduced. If the animal didn’t perform tracking movements or the experimenter had any doubts, the stimulus was shown a second time.

Two different threshold tests were performed to assess visual function in the rodents: VA and CS. The spatial frequency thresholds were measured by systematically increasing (using a staircase method) the spatial frequency of the grating at 100% contrast until the animals no longer tracked. Firstly, a stimulus with low spatial frequency (0.042 cycles/deg) and maximum contrast (100%) was projected. The spatial frequency was progressively increased by 0.150 cycles/deg in each step until the animal didn’t detect the stimulus. Then, system projected a stimulus which spatial frequency is the average between this value and the last frequency detected by the animal. By repeating this sequence, the spatial frequency threshold can be delimited with a method of limits. The value of this threshold was considered the maximum VA. The CS test started with a stimulus at 100% contrast that was consistently reduced (75%, 50%, 25%, 12.5%, 6.2%…) while the spatial frequency was maintained until the contrast threshold was reached, using the same staircase method than VA test. This threshold was calculated with the Michelson equation, using luminance differences of the black and white stripes projected (Lmax−LminLmax+Lmin). Subsequently, the CS value was calculated as the inverse of the threshold. A CS curve was generated by identifying the minimum contrast that generates tracking over a range of spatial frequencies (0.022 cycles/deg, 0.042 cycles/deg, 0.089 cycles/deg and 0.175 cycles/deg). VA and CS were evaluated in both directions of the rotation (clockwise and counterclockwise) that corresponded to the values obtained for each eye separately (left and right eye) because only the movement in the temporal-nasal direction generated tracking¹⁰.