Patch-clamp recordings were made from SACs and DSGCs Androgen Receptor Antagonist solubility dmso in flatmount retina in Ames medium (Sigma-Aldrich, Saint Louis, MO), equilibrated with 95% O2 and 5% CO2) at ∼35°C as described previously (Lee and

Zhou, 2006, Zheng et al., 2004 and Zhou and Fain, 1995). Spike responses of DSGCs were recorded with an on-cell loose-patch electrode (3–5 MΩ, filled with Ames medium). Whole-cell patch clamp was made from SACs using a pipette solution containing (in mM) 110 CsMeSO4, 15 CsOH, 5 NaCl, 0.5 CaCl2, 2 MgCl2, 5 ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 adenosine 5′-triphosphate (disodium salt), 0.5 guanosine 5′-triphosphate (trisodium salt), 10 HEPES, and 2 ascorbate (pH 7.2). The same

pipette solution, supplemented with 10 mM lidocaine N-ethyl bromide (QX314), was used Tyrosine Kinase Inhibitor Library in vitro for whole-cell recording from DSGCs. Lucifer yellow (0.1%–0.3%, w/v) was included in the whole-cell pipette solution for morphological confirmation of cell types. The effects of Ca2+ channel blockers, ω-conotoxin GVIA, and ω-agatoxin IVA, which had a slow onset, were tested under perforated patch-clamp recording from SACs using electrodes (6-8 MΩ) filled with (in mM) 145 glutamic acid, 10 HEPES, 10 NaCl, and 500 μg/ml amphotericin B (titrated to pH 7.2 with 147 mM CsOH). Lysozyme (1.5 mg/ml) was added to the superfusate to prevent nonspecific binding of calcium channel toxins to the perfusion system. Flash photolysis of caged Ca2+ (DM-nitorphen) in SACs was done under dual whole-cell voltage-clamp recordings from pairs of SACs and DSGCs,

with the pipette solution for SAC recording containing (in mM) 95 CsMeSO4, 30 CsOH, 5 NaCl, 2 CaCl2, 6 Ca(NO3)2, 2 adenosine 5′-triphosphate (disodium salt), 0.5 guanosine TCL 5′-triphosphate (trisodium salt), 40 HEPES, 2 ascorbate, and 10 DM-nitrophen (pH 7.2). DM-nitrophen (1-(4,5-dimethoxy-2-nitro-phenyl)-1,2-diaminoethane-N,N,N′,N′-tetraacetic acid) was allowed to diffuse into the SAC for 5–7 min before an ultraviolet (UV) light (355 nm in wavelength, 25 ms in duration) was flashed onto the SAC dendrites. The UV light was generated by a UV laser (30 mW average output power, 3 μJ/pulse at 10kHz, Model DPSL355/30, Rapp Opto Electronik, Heidelberg, Germany), which was guided to the epifluorescence port of the microscope via a spot illumination adaptor (OSI BX, Rapp Opto Electronik) by a quartz optic fiber (940 μm in diameter) and focused in a 50 μm diameter spot on the SAC dendrites via a 60× objective lens (NA/09). The uncaging area was positioned by moving the microscope stage on which the recording chamber and micromanipulators were mounted.