Animals were handled according to the principles of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For isolated retinal preparations (n = 5), animals were dark adapted for at least 12 hours before death, rats were killed by carbon dioxide asphyxiation and rabbits by pentobarbital sodium overdose. Eyes were enucleated with IR-sensitive goggles (AN/PVS-5 Nightvision Goggles; ITT, Roanoke, VA [among others]), eyecups were prepared (Leibovitz L-15 medium; Invitrogen-Gibco Corp., Carlsbad, CA), and neural retinas were teased away from the RPE. All subsequent preparation was performed in the dark with the aid of infrared image converters (Dark Invader; BE Meyers Inc., Redmond, WA) attached to the dissecting microscope eyepieces.
A disc of retina with approximately 6-mm diameter was mounted in a Ussing chamber (World Precision Instruments, Sarasota, FL) modified to receive a fiber optic. The photoreceptor side of the retina faced the fiber optic, whereas the ganglion cell layer (GCL) side was supported by a filter paper. Electrodes connected to the Ussing chamber consisted of Ag/AgCl pellets. The Ussing chamber was attached to a system that perfused warm, bubbled (95% O2-5% CO2) Ames’ medium at a rate of approximately 1 mL/min. Flow was calibrated at the beginning and end of each experiment. Two perfusion lines delivered Ames medium from the heated beakers to the Ussing chamber. One line delivered control medium to the photoreceptor side, and a second line fed the ganglion cell side. This line was configured to deliver either control medium or medium containing gentamicin. The Ussing chamber was perfused with warm solutions heated in water-jacketed beakers (Radnoti, Monrovia, CA) and oxygenated through plastic tubing. The water bath was heated and circulated with a temperature-controlled water pump (Polyscience, Niles, IL). Perfusion lines were heated with a DC power supply (Topward; SpenceTek Inc., Milpitas, CA). The temperature was calibrated at the location of the retina to 37°C. After baseline isolated-tissue ERG responses were recorded (DC to 300 Hz), test medium containing 1 or 10 mg/mL gentamicin was perfused onto the GCL side of the tissue and the responses recorded. Subsequently, the perfusate was switched back to control solution, and washout responses were recorded.
The light source was a 100-W tungsten-halogen lamp focused onto one end of a fiber optic. Stimulus duration was controlled with a shutter with a 6-mm aperture (Uniblitz; Vincent Associates, Rochester, NY). The energy output of the flashes was calibrated daily as the photon flux at the retinal surface in the Ussing chamber. Stimulus strength was controlled by a set of calibrated inconel neutral-density filters that allowed attenuation in steps of approximately 0.3 log units up to a maximum of 6.9 log units attenuation. The unattenuated stimulus was calibrated daily with an optical power meter (Graseby Optronics, Orlando, FL). We used a 505 nm (35 nm bandwidth) stimulus that was determined by a three-cavity interference filter (Andover Co., Salem, NH).
For the isolated-tissue experiments the number of photoisomerizations (R*) is given by the product of the stimulus strength,
i (photons per square micrometer at λ
max), and
A c, the effective collecting area of the outer segment, calculated using the equation by Baylor et al.
16 and Zhang et al.
17 :
\[A_{\mathrm{c}}{=}V_{\mathrm{OS}}Q_{\mathrm{isom}}\ f\ 2.303{\alpha},\]
where
V OS is the volume of the outer segment,
Q isom is the quantum efficiency of photoisomerization (0.67),
18 f is a factor allowing for the use of unpolarized light entering the outer segment perpendicular to its long axis (
f = 1 for end-on stimulus), and α is the specific pigment density (0.016 μm
−1).
19 Based on volume measures from single-cell recordings, rat rods have an
A c of 0.492 (Niculescu D, Kraft TW, personal communication, November 2004). The volume of the rabbit rod outer segments was calculated as 27.2 μm
3 using the length of 15.4 μm reported by Tucker et al.
20 and assuming a diameter of 1.5 μm, giving an
A c of 0.672 for rabbit rods.