Tpmpa

The drug solution was carboxygenated, and bath was applied through perfusion for at least 15 min before the recordings. We used 100 μM meclofenamic acid (MFA; M4531, Sigma-Aldrich) to block gap junctional coupling. In additional experiments (Supplementary Figures), the following drug concentrations were used (in μM): 50 6,7-dinitroquinoxaline-2,3-dione (DNQX, 0189, TOCRIS, Bristol, United Kingdom) and 50 DL-2-amino-5-phosphonopentanoic acid sodium salt (DL-AP5, 3693, TOCRIS) to block ionotrophic glutamate receptors and 50 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA, 1040, TOCRIS) and 25 6-Imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (SR95531, 1262, TOCRIS) to block GABA receptors.

The mGluR1 antagonist JNJ16259685 (Tocris Bioscience) and the GABA_CR antagonist TPMPA (Tocris Bioscience) were added to the bath at 0.5 μM and 100 μM, respectively, using a calibrated syringe pump, as described previously [24 (link)]. Only one drug per retinal preparation was used to avoid possible long-term changes caused by the drug. The effects of a drug were examined only after the drug was bath applied for ~10 min to ensure stable responses.

Pharmaceutical agents were applied via the perfusion system, in the recording chamber for 10 minutes and washed for 20 minutes. We used (in µM): 100 L-AP4 (mGluR6 agonist; L-2-amino-4-phosphonobutyric acid) and 20 NBQX (AMPA/kainate-type GluR antagonist; 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide) obtained from Bio Trend, 75 DL-TBOA (glutamate transporter antagonist DL-threo-β-Benzyloxy aspartic acid); 100 Verapamil (L-type voltage-gated calcium channel blocker), 50 TPMPA (GABA_C receptor antagonist; 1,2,5,6-Tetrahydropyridin-4-yl)met hylphosphinic acid) and 10 Gabazine (GABA_A receptor antagonist; 6-Imino-3-(4-methoxyphenyl)-1(6 H)-pyridazine-butanoic acid hydrobromide) obtained from TocrisBioscience, and 50 carbenoxolone (CBX, gap junction blocker; (3β,20β)-3-(3-Carboxy-1-oxopropoxy)-11-oxoolean-12-en-29-oic acid disodium) obtained from Sigma-Aldrich.

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).

Picrotoxin (50 μM), L-655 708 (11,12,13,13a-Tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1 c][1,4]benzodiazepine-1-carboxylic acid, ethyl ester, 10 μM), SCS (Salicylidene salicylhydrazide, 1 μM), TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl) methylphosphinic acid, 50 μM), etomidate (10 μM), allopregnanolone (100 nM), muscimol (5 μM), bumetanide (10 μM), nifedipine (10 μM), benidipine (10 μM), bay K 8644 (10 μM), bicuculline (50 μM), ATP (150 μM; all from Tocris Bioscience, Bristol, UK), SNAP ((S)-Nitroso-N-acetylpenicillamine, 50 μM), SC (semicarbazide 50 μM), GABA (5 μM), geneticin (10 μM; all from Sigma-Aldrich) and CPCPT (1-(3-Chlorophenethyl)−3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione, 1 μM, Merck Millipore, Darmstadt, Germany) were used at the indicated concentrations, if not differently stated. All pharmacological treatments and live cell stainings were performed in CM at 37°C and 5% CO₂.

All chemicals and reagents, unless otherwise noted, were obtained from Sigma-Aldrich (St. Louis, MO). TPMPA, PTX, bumetanide, muscimol, gabazine, strychnine, amiloride, and CNQX were obtained from Tocris (Ellisville, MO). Drugs and reagents were prepared in double-distilled water either as stock solutions (frozen at −20 °C) or prepared fresh. Superfused drugs normally produced their full effects in approximately 1 min, but in cases in which no response was seen, a limit of 5 min was deemed sufficient to conclude an absence of action.