Quantification of visual capabilities

2.4.1 Vir tual-r eality op tomotor system

We assessed visual acuity using the virtual-reality optomotor system (Prusky et al., 2004) (Figure 11 A,B). This test does not require any training of the mice because it is based on the optomotor reflex in response to a moving stimulus.

Figure 11: Scheme of the optomotor testing apparatus (Figure from Prusky et al., 2004). (A) Side view. The mouse can freely move on a platform positioned in the middle of an arena created by four quadratically arranged computer screens. Sine wave gratings on the screens are extended vertically with mirrors on ceiling and floor. To display the animal’s behavior a video camera from above is used. (B) Top view. The mouse is surrounded by 360° of gratings.

In the optomotor setup, the mouse is surrounded by four monitors showing 360° moving sine wave gratings of different spatial frequencies, contrasts and drifting speeds generated by the software OptoMotry 1.4.0 (CerebralMechanics, Lethbridge, Alberta, Canada) like previously reported (Prusky et al., 2004; Lehmann and Löwel, 2008; Goetze et al., 2010b). In the testing arena (39 x 39 x 32.5 cm [L x W x H]), the mouse can move freely on a platform which is placed 13 cm over the floor in the middle of the apparatus (Figure 11 A,B).

Additionally mirrors are placed on the floor and the ceiling while a video camera in the lid of the apparatus records the animals’ behavior. The x-y coordinates of the crosslines were used to center the rotation of the cylinder on the mouse’s eyes (Figure 12 A,C). This guarantees a constant distance of the virtual cylinder from the animal.

31 Figure 12: Virtual cylinder and optomotor response (Figures modified from Prusky et al., 2004). (A) In a 3-D coordinate space a virtual cylinder is projected on all four screens. The center of the rotating cylinder is determined by the head of the mouse. (B) When the cylinder is rotating, the mouse tracks the drifting sine wave grating with neck and head movements. (C) A video camera image of a mouse tracking the moving sine wave grating. The red crosslines are positioned between the eyes of the mouse, and the coordinates are used to center the rotation of the virtual cylinder.

Mice will reflexively track the moving vertical sine wave gratings by head movements as long as they can see the gratings (Figure 12 B). In mice only movement in the temporal-to-nasal direction induces tracking therefore it is possible to measure both eyes separately by reversing the direction of the moving sine wave grating (Douglas et al., 2005). Spatial frequency at 100 % contrast, contrast at six different spatial frequencies (0.031 cycles/degree (cyc/deg), 0.064 cyc/deg, 0.092 cyc/deg,0.103 cyc/deg, 0.192 cyc/deg, 0.272 cyc/deg), and in addition for temporal resolution at 100 % contrast at seven different spatial frequencies (0.064 cyc/deg, 0.103 cyc/deg, 0.150 cyc/deg, 0.192 cyc/deg, 0.200 cyc/deg, 0.272 cyc/deg, and 0.400 cyc/deg) were measured. Contrast sensitivity was calculatedfor each spatial frequency as a Michelson contrast from the screenluminance

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and the reciprocal of the threshold (black mean, 0.22 cd/m²; white mean, 152.13 cd/m²).

Drift speed was usually fixed at 12 degrees/second (°/sec). To investigate whether the loss of the presynaptic protein Bassoon had an effect on temporal resolution we tested different drift speeds (Bsn+/+ and Bsn -/- mice were tested at: 0.064 cyc/deg, 0.103 cyc/deg, 0.150 cyc/deg, and 0.192 cyc/deg, in addition, Bsn+/+ mice at 0.272 cyc/deg and 0.400 cyc/deg and Bsn-/- mice at 0.200 cyc/deg). Because of technical reasons we were not able to measure drift speeds higher than 50 °/sec. Experimenters were blind to the animal’s

32 genotype and thresholdswere regularly validated independently by more than one observer (Prof. Dr. Karl-Friedrich Schmidt and I).

Additionally, we studied the development of visual acuity and contrast sensitivity of individual young mice. Animals were tested from the day of eye opening throughout development and the genotype was determined after visualizing cortical activity maps.

In mice with a monocular deprivation of the right eyes, visual acuity of the open left eyes was tested daily in the virtual-reality optomotor system.

2.4.2 Visua l wa ter task

As a second method to assess visual acuity in mice, we used the visual water task, a visual discrimination task that is based on reinforcement learning (Prusky et al., 2000; Prusky et al., 2004; Prusky and Douglas, 2004) (Figure 13 A-C).

Figure 13: Scheme of the visual water box (Figure from Prusky et al., 2000). (A) View from above illustrating the important components including pool, platform, midline divider, and both monitors. The pool is filled with water (shown in gray). From the release chute, animals learn to swim on the side of the pool on which monitor the grating is projected to find the hidden platform to escape from the water. (B) Front view of both screens, hidden platform and midline divider. (C) Picture of the apparatus from above.

C

33 For this task, animals were initially trained to distinguish a low spatial frequency vertical sine-wave grating (0.086 cyc/deg) from an isoluminant grey generated by the software Vista X 3.5.004 (CerebralMechanics, Lethbridge, Alberta, Canada). Then their ability to recognize higher spatial frequencies was tested. The apparatus consists of a trapezoidal-shaped pool (140 cm long x 80 cm wide x 55 cm high walls, but the pool is wider at one end (80 cm) than the other (25 cm)) with two monitors placed side by side at the wide end (Figure 13 A-C). A midline divider is extended from the wide end into the pool, creating a maze with a stem and two arms. The length of the divider sets the choice point and effective spatial frequency. An escape platform, which is invisible to the animals, is placed below the monitor on which the sine-wave grating is projected. The position of the grating and the platform is alternated in a pseudorandom sequence over the training and test trials. Once mice achieved 90 % accuracy, the discrimination threshold is determined by increasing the spatial frequency of the grating until performance falls below 70 % accuracy. The highest spatial frequency where mice achieved 70 % accuracy is taken as the maximum visual acuity.