Animals were placed in a stereotaxic apparatus where body temperature was maintained at 37°C with a thermostatically-controlled heating blanket, and they were mechanically ventilated. A craniotomy was made above the LGN, and the dura was reflected. All wound margins were infused with lidocaine. A small metal ring was glued to the sclera of each eye to minimize eye movement and to secure the eye for intraocular injections of APB. The pupils were dilated with 1% atropine sulfate and the nictitating membranes were retracted with 10% phenylephrine. The eyes were fitted with contact lenses and focused on a screen located 76 cm in front the animal. Once surgical procedures were complete,

anesthesia was maintained with thiopental sodium (2–3 mg/kg/hr, IV). Animals were then paralyzed with vecuronium bromide (0.2 mg/kg/hr, IV). Proper depth of anesthesia was ensured throughout the experiment by continuously monitoring Selleck PLX4032 the electroencephalogram, the electrocardiogram, and expired CO2. Animals were euthanized at the end of the experiment with an overdose of Euthasol (Virbac, Ft. Worth, TX). Single-unit recordings were made from LGN neurons in layers A and A1, in vivo, using a 7-channel Forskolin manufacturer multielectrode array (Thomas Recording Systems, Marburg, Germany). Neuronal responses were amplified and recorded to a PC equipped with a Power 1401 data acquisition interface and the Spike 2 software package (Cambridge

Electronic Design, Cambridge, England). Spike isolation was based on waveform

analysis and the presence of a refractory period, as indicated by the autocorrelogram. Single-unit recordings were made from RGCs, in vitro, using a 60-channel multielectrode array (MultiChannel Systems, Reutlingen, Germany). Individual electrodes were 30 μm in diameter and arranged on an 8 × 8 rectilinear grid with 200 μm interelectrode spacing. Tissue preparation and recording procedures were similar those previously described (Sun et al., 2008). Briefly, the retinas were isolated and stored in buffered and oxygenated minimum essential medium Eagle (MEME, M7278; Sigma-Aldrich) at room temperature. The retinas were cut into 5–8 mm2 rectangles, Linifanib (ABT-869) placed ganglion cell layer down on the multielectrode array, held in place with a piece of dialysis membrane, and superfused with buffered MEME (2 ml/min) at 37°C. Electroretinograms (ERGs) were recorded using custom-made electrodes. The ERG signal was amplified and low-pass filtered at 100 Hz. One hundred to two hundred trials were averaged to yield the final ERG waveforms. Visual stimuli were produced with a VSG2/5 or a ViSaGe visual stimulus generator for the in vivo and in vitro experiments, respectively (Cambridge Research Systems, Rochester, England). Stimuli were presented on a γ-calibrated Sony Monitor (Sony Corporation, Tokyo, Japan) with a mean luminance of 38 candelas/m2 and a refresh rate of 140 Hz.