The cell suspension was immediately deposited on a concanavalin A–coated glass coverslip that formed the transparent bottom of a recording chamber. The chamber was held on the stage of an inverted microscope equipped with differential interference contrast–enhancement optics and operated under IR illumination with the aid of IR-sensitive video cameras and monitors. After 10 min in darkness, the chamber was vigorously perfused with glucose-Ringer’s solution. Single and twin cones lacking their nuclear and synaptic regions (Miller and Korenbrot, 1993 (link)) remained firmly attached to the coverslip. The chamber was intermittently perfused throughout an experimental session, but not at the time photocurrents were measured.
Tight-seal electrodes were fabricated from aluminosilicate capillary glass (1.5 × 1.1 mm; 1724; Corning) and applied onto the side of the cone inner segment. After forming a giga-seal, whole cell mode was attained by sustained suction while holding membrane voltage at 0 mV. Membrane current drifted continuously toward an outward (positive) value. When this drifting ceased (5–15 s; +40 to +70 pA), holding voltage was shifted to −40 mV, where membrane current was near zero. This method yielded more stable recordings than attaining whole cell mode at −40 mV. Voltage-clamped membrane currents were measured with a patch-clamp amplifier (Axopatch 1D; Molecular Devices). Analogue signals were low-pass filtered below 50 Hz with an eight-pole Bessel filter (Frequency Devices) and digitally acquired at 1 KHz (Digidata 1322A and pClamp 9.2; Molecular Devices). All photocurrents reported and analyzed here were measured within 8 min of the moment whole cell mode was achieved.
Tight-seal electrodes were fabricated from aluminosilicate capillary glass (1.5 × 1.1 mm; 1724; Corning) and applied onto the side of the cone inner segment. After forming a giga-seal, whole cell mode was attained by sustained suction while holding membrane voltage at 0 mV. Membrane current drifted continuously toward an outward (positive) value. When this drifting ceased (5–15 s; +40 to +70 pA), holding voltage was shifted to −40 mV, where membrane current was near zero. This method yielded more stable recordings than attaining whole cell mode at −40 mV. Voltage-clamped membrane currents were measured with a patch-clamp amplifier (Axopatch 1D; Molecular Devices). Analogue signals were low-pass filtered below 50 Hz with an eight-pole Bessel filter (Frequency Devices) and digitally acquired at 1 KHz (Digidata 1322A and pClamp 9.2; Molecular Devices). All photocurrents reported and analyzed here were measured within 8 min of the moment whole cell mode was achieved.
