All procedures were conducted in accordance with guidelines outlined in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were also approved by an internal institutional review committee.
Adult Dutch belted rabbits (Charles River; Wilmington, MA) were anesthetized with intramuscular injection of ketamine (50 mg/kg) in combination with acepromazine (21 mg/kg). Deep anesthesia was then obtained using inhalation of 4.0% isoflurane. The eyes were removed in bright room illumination and placed in room temperature phosphate-buffered saline for approximately 3 minutes. Under dim red illumination, the anterior segment including the lens was removed. A series of radial cuts, extending approximately 5 to 10 mm from the peripheral edge of the eye cup toward the posterior pole, were used to flatten the eye cup. After careful removal of the vitreous, a circular sample of retina-choroid-sclera, 11 mm in diameter and extending inferiorly from the inferior edge of the optic nerve head, was cut from the tissue as shown in
Figure 1A . This tissue sample was then mounted, retina side up, in a custom perfusion chamber, as illustrated in
Figure 1B , and perfused continuously at a rate of 2.5 mL/min with Ames medium (Sigma-Aldrich; St. Louis, MO), which was saturated with a combination of 95% O
2 and 5% CO
2. The tissue was always oriented within the chamber in the same position so that microelectrode recordings were made in the same area immediately inferior to the visual streak. The preparation was maintained at a temperature of 35°C in complete darkness (with the exception of periodic light stimulation) inside a light-tight Faraday cage. Test solutions containing NMDA (Sigma-Aldrich), AP5 (
d-(–)-2-amino-5-phosphonopentanoic acid; Tocris Bioscience, Ellisville, MO), TBOA (
dl-threo-β-benzyloxyaspartic acid; Tocris Bioscience), or memantine (3,5-dimethyladamantane-1-amine hydrochloride; Allergan Inc., Irvine, CA) were prepared by adding the test agents directly to the perfusion medium. Magnesium-free medium was obtained by preparation of a modified Ames medium from which magnesium chloride was omitted. The preparation was dark adapted for approximately 1.5 hours before recording. The results described in this study were obtained from more than 45 retina preparations.