Placement of torsion tracking marks

Placement of torsion tracking marks

Ocular torsion changes were measured by tracking the position of small spots of titanium dioxide (TiO₂) paste dots (~300 μm) applied to ventral and/or temporal locations on the cornea as described in van Alphen et al., 2010. The location of the TiO2 spots on the cornea were then incorporated into the eye model, and used to quantify ocular torsion as described in the Methods subsection “Pupil position and pupil torsion tracking”. 

1. Application of the TiO₂ spots was performed with the animal anesthetized with isoflurane (induction: 5% isoflurane, maintenance: 0.5–1.0% isoflurane in air, total time anesthetized 5-10mins). The animal was placed in lateral recumbency, and a small nasal nose mask was used to provide easy access to the eyes while maintaining depth of anesthesia. Respiration rate and depth of anesthesia was monitored throughout the procedure.
2. Anesthesia depth was adjusted so that the eyeball did not rotate in the orbit, and come into contact with the tear film at the eyelid margins, during the period from TiO₂ spot application until the point at which the spot had dried. 
3. The TiO₂ paste consisted of TiO₂ powder (Kronos Titan GmBH, Leverkusen, Germany) mixed with a small quantity of sterile artificial cerebrospinal fluid (ACSF) solution with the following composition (in mM): 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH balanced to 7.2 (300 mOsm/l). 
4. The TiO₂ paste was prepared immediately prior to application in a watch glass, in a 1:1 ratio of powder to diluent (250mg TiO₂ powder to 250 microlitres ACSF). The consistency and adherence of the paste was checked by wiping a small amount on the rim of the watch glass using a spatula (the paste had a meringue-like consistency). 
5. Immediately prior to application of the TiO₂ spot, the corneal region onto which the spot was to be placed was dried using a small ophthalmic cellulose sponge point (Sugi, Kettenbach GmbH & Co. KG, Eschenburg, Germany). When necessary the eyelids were taped open during the application process, using 1.25 cm surgical tape (3M Blenderm surgical tape, 3M Health Care, Neuss, Germany). If present, excess any excess tear film at the eyelid margins was removed using the sponge points. 
6. The small TiO₂ spot was applied using a rounded tipped carbon fibre needle. The end of the tip was loaded with the TiO₂ paste, so that only the point of the needle was covered with paste, and the TiO₂ was applied to form a small circular spot on the cornea. After allowing the spot to dry, a second or third spot was applied directly on top of the first spot and also allowed to dry, creating a single smooth surfaced TiO₂ spot. Care was taken not to create a tall conical spot on the eye, as these had a tendency to come unstuck from the cornea when eyelids closed or the animal groomed itself. 
7. After the spot had dried, the eyelids were manually closed and opened again to check the adherence of the TiO₂ spot to the corneal surface. The animal was then rolled over onto the opposite side, and the process was repeated on the second eye. 
8. Before the end of the procedure the spots in both eyes were rechecked, and if necessary additional TiO₂ paste was re-applied before allowing the animal to recover. 
9. Following application of TiO₂ spots, mice were allowed to recover for >45 min prior to a cricket hunt. 
10. The presence of the TiO₂ marks did not significantly change the animal’s cricket hunting performance as assessed by the average time taken to capture crickets (Figure 7C).

van Alphen B, Winkelman BHJ, Frens MA (2010) Three-Dimensional optokinetic eye movements in the C57BL/6J mouse Investigative Opthalmology & Visual Science 51:623–630.