RGC recordings were performed 4 weeks after retinal transplantation. At this age (12 weeks), in a non–transplant-recipient
rd mouse, the photoreceptor degeneration was essentially complete. All the rods posterior to the equator had been lost, and only a few scattered cone nuclei (without outer segments) remained.
20 Animals were dark adapted for at least 4 hours and then anesthetized with the same medications and dosages used for transplantation. A 30-gauge needle was used to make a small incision into the cornea just anterior to the limbus. Sodium hyaluronate (Healon GV; Pharmacia, Columbus, OH) was injected into the anterior chamber through the incision site to maintain the intraocular pressure. The cornea was excised and an intracapsular lensectomy was performed. Sodium hyaluronate was instilled to maintain the contour of the posterior pole and enable proper placement of the electrodes on the retinal surface. To remove the vitreous and expose the retinal surface for electrophysiologic testing, sodium hyaluronate was gently injected into the plane between the retina and the vitreous. The vitreous was allowed to slip away from the eye, and great care was taken to avoid traction on the retina. All these preparative procedures were performed under dim red-light illumination. Body temperature was maintained at 35°C with a feedback-controlled heating pad.
For differential RGC recordings, the tips (separated by 200 μm) of two 125-μm-diameter parylene-coated tungsten electrodes (A-M Systems, Everett, WA) were positioned on the retinal surface with the aid of a microscope and a micromanipulator (MX-100; Newport, Irvine, CA). The ground electrode was placed in the animal’s mouth. A halogen light source was used as the light stimulus, and the eyes were directly illuminated (full-field illumination) with a fiber optic positioned at a distance of 1.0 to 1.5 cm from the retina. The onset of the light stimulus was at 0.1 second into the recording cycle. One-second and 200-ms light stimuli were used for the recordings. Ten eyes each were used for RGC recordings in the four experimental groups: (1) the transplant areas of transplant-recipient eyes posterior from the visible injection site; (2) the non-transplant areas of these eyes on the side opposite the transplant (i.e., 6 o’clock position); the corresponding areas (equatorial retina at 12 and 6 o’clock) of (3) control and (4) sham-injected eyes. For each group, three separate locations within a given experimental host retinal area (2 × 2 mm) were sampled. Ten cycles (1 cycle = 1.6 seconds) of RGC responses were recorded at each location with the light turned either on or off (shutter sound only). Animals were excluded if a retinal detachment developed during the preparative procedures for the recordings or during the recordings themselves. Light-driven RGC responses were recorded from a normal B57/Bl6 mouse before each experimental session, to confirm that all recording equipment was functioning properly. The output of the recording electrodes was fed into a differential AC preamplifier (band-pass: 300-2000 Hz; gain: 10-fold; model EX-1; Dagan, Minneapolis, MN) which was connected to another AC amplifier (gain: 2000-fold; model AM502; Tektronix, Beaverton, OR). Total gain was 20,000 fold. A speaker and an oscilloscope were connected to the system to allow both audio and video monitoring of the recording conditions and the RGC responses. The output of the amplifier was fed into in a computer. Data were acquired synchronously to the shutter action with a custom-written software program (Labview; National Instruments, Austin, TX) and analyzed with statistical software (MatLab; The MathWorks, Inc., Natick, MA). Thresholds were then set manually for each file. For the construction of peristimulus time histograms (PSTHs), spikes were grouped into 0.1-second intervals and counted only if the amplitude was at least twice the amplitude of baseline. Total spike counts during the recording interval of 1.6 seconds were compared between the transplant areas, and the various control eyes (nontransplant areas of recipient eyes, sham-injected eyes, and untreated eyes) using a two-tailed unpaired t-test.