Mice were killed, the eyes enucleated, and whole retinas removed from eye cups under infra-red illumination. Small pieces of retina were dissected in a drop of chilled Locke's solution (112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, 0.02 mM EDTA, 20 mM NaHCO3, 3 mM Na2-succinate, 0.5 mM Na-glutamate, 10 mM glucose), and placed into a recording chamber. The chamber was continuously refreshed with Locke's solution, pH 7.4, equilibrated with 95% O2/5% CO2, and maintained at 35–37°C with a heating system designed for microscopy (ALA Scientific). Using silanized suction pipettes, we recorded from photoreceptors embedded in 50–100-μm diameter slices of retina exclusively in the “OS out” configuration (Nikonov et al., 2005 (link)); in this effort several nuclei and conjoined “inner segment” tissue were intentionally drawn into the pipette. Once the tissue was drawn into the pipette, responses were evoked with calibrated flashes of light delivered under control of a customized LabView (National Instruments) interface. The optical system in the configuration used for these experiments has two stimulation channels: the light source in one channel is a tungsten-halogen lamp, and in the second a xenon flash lamp that delivers ∼20-μs pulses. Experiments with WT mouse retinal slices required the use of steady illumination to suppress rod activity, and the tungsten-halogen channel was employed for this purpose.
The “inner segment” limb of the rod and cone circulating current is an outward membrane current, carried primarily by K+ channels; light responses recorded from inner segment membranes are thus recorded by the amplifier as negative-going, resulting from the suppression of the outward membrane current as the cell hyperpolarizes toward the K+ reversal potential. Here we will present all photocurrent responses in the conventional manner as positive-going. However, the actual sign (and direction) of the recorded membrane currents will be referred to as needed.
As the expression of mouse M-cone opsin in mice varies in a dorso-ventral gradient (Applebury et al., 2000 (link)), we developed a method that allows the dorsal or ventral region of the retina to be dissected under infrared illumination and used for suction pipette recordings (Nikonov et al., 2005 (link)). This method has played a critical role in the complete characterization of cone function in the WT mouse.
The “inner segment” limb of the rod and cone circulating current is an outward membrane current, carried primarily by K+ channels; light responses recorded from inner segment membranes are thus recorded by the amplifier as negative-going, resulting from the suppression of the outward membrane current as the cell hyperpolarizes toward the K+ reversal potential. Here we will present all photocurrent responses in the conventional manner as positive-going. However, the actual sign (and direction) of the recorded membrane currents will be referred to as needed.
As the expression of mouse M-cone opsin in mice varies in a dorso-ventral gradient (Applebury et al., 2000 (link)), we developed a method that allows the dorsal or ventral region of the retina to be dissected under infrared illumination and used for suction pipette recordings (Nikonov et al., 2005 (link)). This method has played a critical role in the complete characterization of cone function in the WT mouse.
