Whole-cell patch recordings were made from bipolar cell somas in retinal slices by viewing them with an upright microscope (Slicescope Pro 2000; Scientifica, Uckfield, East Sussex, UK) equipped with a charged-couple device camera (Retiga-2000R; Q-Imaging, Surrey, BC, Canada). Recordings were performed in Ames' medium buffered with NaHCO3 (Sigma, St. Louis, MO, USA), which was continuously bubbled with 95% O2 and 5% CO2; the pH was 7.4 at 30°C. Electrodes were pulled from borosilicate glass (1B150F-4; World Precision Instruments, Sarasota, FL, USA) with a P1000 Micropipette Puller (Sutter Instruments, Novato, CA, USA) and had resistances of 8 to 12 MΩ. The intracellular solution contained the following (in mM): 111 K-gluconate, 1.0 CaCl2, 10 HEPES, 10 EGTA, 10 NaCl, 1.0 MgCl2, 5 ATP-Mg, and 1.0 GTP-Na, adjusted to pH 7.2 with CsOH. Liquid junction potentials were corrected after each recording. Clampex and Multi Clamp 700B (Molecular Devices, San Jose, CA, USA) were used to acquire data and to control puff application using a Picospritzer III (Parker Hannifin, Cleveland, OH, USA). A selective α7-nAChR agonist, PNU282987 (30 μM; Tocris, Bristol, UK) was puffed onto recording bipolar cell axon terminals. The data were digitized and stored with a computer using Axon Digidata 1440A (Molecular Devices). Responses were filtered at 1 kHz with the four-pole Bessel filter and sampled at 2 to 5 kHz.
A fluorescent dye, sulforhodamine B (0.005%; Sigma), and Neurobiotin (NB; 0.5%; Vector Lab, Burlingame, CA, USA) were included in the recording pipette; these dyes did not affect the physiological recordings.
15 Sulforhodamine B images were observed after physiological recordings. To visualize NB staining, the slice preparation was fixed with 4% paraformaldehyde for 30 minutes, incubated with streptavidin-conjugated Alexa 488 (1:200; Life Technologies, Carlsbad, CA, USA) and anti-ChAT antibody (1:200; Millipore) overnight, and then incubated with the secondary antibody for 2 hours at room temperature. The preparation was viewed with a confocal microscope (TCS SP8; Leica, Wetzlar, Hesse, Germany) using a water-immersion, ×63 objective. Bipolar cell types were determined based on previous references.
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