Sulforhodamine 101

CypExpress^TM 2C19, 2D6 and 3A4 human kits, quercetin (Q), ticlopidine hydrochloride, quinidine, testosterone, 6β-hydroxytestosterone, ketoconazole, fetal bovine serum (FBS), glutamine, penicillin, streptomycin, pyranine (trisodium 8-hydroxypyrene-1,3,6-trisulfonate), bromosulfophthalein, sulforhodamine 101, sodium orthovanadate, probenecid, 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF), lucifer yellow (LY), and benzbromarone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nicotinamide adenine dinucleotide phosphate sodium salt (NADP⁺), and glucose-6-phosphate barium salt (G6P) were from Reanal (Budapest, Hungary). S-mephenytoin, 4-hydroxymephenytoin, dextromethorphan, and dextrorphan were obtained from Carbosynth (Berkshire, UK). Isorhamnetin (IR) and Ko143 were purchased from Extrasynthese (Genay Cedex, France) and Tocris Bioscience (Bristol, UK), respectively. Quercetin-3′-sulfate (Q3′S), quercetin-3-glucuronide (Q3G), and isorhamnetin-3-glucuronide (I3G) were synthetized as described previously [46 (link)].

Borosilicate glass microelectrodes were filled with intracellular solution containing (in mm): 120 Cs-methanesulfonate, 5 TEA-Cl, 10 HEPES, 10 BAPTA, 3 NaCl, 2 QX 314-Cl, 4 ATP-Mg, 0.4 GTP-Na2, and 10 phosphocreatine-Tris2 (pH 7.3, 280 mOsm), and a red fluorescent dye (Sulforhodamine 101; Sigma-Aldrich). Voltage-clamp recordings were performed at the reversal potential for chloride and cations, respectively, −67 and +15 mV. Membrane potential recordings were obtained in current clamp mode (I = 0 nA). Cs-based solution suppressed potassium channel activity to improve voltage-clamp recordings. While Cs likely also altered the resting potential and amplitude of the recorded membrane voltage response, this was not expected to substantially alter the timing of the stimulus evoked response. Post hoc assessment of dendritic morphology from dye fills was used to confirm α-type identity of all electrophysiologically recorded ganglion cells. To test for inhibitory circuit contributions to phase advancing in ganglion cells we used a loss-of-function approach. Glycine receptors were blocked with strychnine (1 μm; Tocris); GABA_a receptors were blocked with SR95531 (gabazine, 50 μm; Tocris); GABA_a-ρ (former GABA_c) receptors were blocked with the selective, competitive antagonist TPMPA (50 μm; Tocris).

PLGA (poly(lactic-co-glycolic acid), Resomer^® RG503H, 50:50 lactide/glycolide) was obtained from Boehringer Ingelheim (Ingelheim, Germany). PEMA (poly(ethylene-alt-maleic anhydride)), fluorescein sodium salt, sulforhodamine 101, and albumin bovine fraction V (BSA) were bought from Sigma Aldrich (Vienna, Austria). Wheat germ agglutinin (WGA) from Triticum vulgare was purchased from Vector laboratories (Burlingame, USA). BODIPY^® 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) was from Invitrogen (Eugene, USA). All other chemicals were of analytical purity.