L ap4

For the experiments requiring the ON-pathway inactivation, we used a metabotropic glutamate receptor agonist (L-AP4, Tocris Bioscience) to block synaptic transmission between the photoreceptors and the ON-bipolar cells. A new Ames medium was preheated at 35–37 degrees with L-AP4 diluted at 5 μM concentration and used to perfuse the retina. To evaluate the effectiveness of L-AP4, we stimulated the retina with full-field flashes at 0.25 Hz and checked that the spiking responses to the onset of the flash were abolished.

ACET, picrotoxin, TPMPA, strychnine, DNQX, W-7, calmidazolium (CMZ), CALP1, ML-9, KN-62, MMPX, ascomycin, BAPTA-AM, thapsigargin (Tg), YM-58483 were obtained from Tocris. L-AP4 was purchased from Tocris or Cayman. Meclofenamic acid (MFA) and I4AA were obtained from Sigma. TTX was purchased from Alomone Labs. Drugs were dissolved in dimethylsulfoxide (DMSO) where appropriate and then diluted into the bath solution. The final concentrations of DMSO were <0.1% (v/v) in all experiments.

The production of IP1 by HEK293 cells was tested using TR-FRET assays, based on Time-Resolved Resonance Energy Transfer. TR-FRET assays were performed in presence of LiCl to block IP1 degradation, using anti-IP1 mouse antibody labeled with Terbium cryptate-conjugate (Tb, Tb-Ab) that will be the donor of energy, and d2-conjugated IP1 (d2, d2-IP1) that will be the acceptor of energy (Cisbio Bioassays, France). After 30 min incubation at 37°C with stimulation buffer alone (≪ Base ≫) or supplemented with a group III mGluRs agonist (L-AP4, 600 μM) or antagonist (CPPG, 100 μM, Tocris Bioscience, France) or the mGlu7-specific NAM ADX71743 (10 μM), d2-IP1 acceptor and Tb-Ab donor were added and cells incubated for 1 h at room temperature. The anti-IP1 antibody competes with native IP1 produced by cells and D2-labeled IP1 from the kit. The fluorescence of Tb and d2 were measured, respectively, at 620 and 665 nm (after 50 μs delay) and divided by the 337 nm excitation signal using a RUBYstar plate reader (BMG, Germany). The resulting signal is inversely proportional to the concentration of IP1 in samples, determined by using a standard curve construction, since the donor–acceptor pair gives a high transfer of energy signal that decreases upon competition with IP1 produced by the cells (Garbison et al., 2012 ).

All compounds were from Sigma-Aldrich, Poole, UK unless otherwise stated. Stock solutions of pregnenolone sulphate, CIM0216 (Tocris Bioscience; Bristol, UK), WIN 55212–2 (Tocris Bioscience; Bristol, UK), AM251 (Tocris Bioscience; Bristol, UK), Gallein (Santa Cruz Biotechnology; Heidelberg, Germany) and BIIE 0246 (Tocris Bioscience; Bristol, UK) were made in DMSO (Calbiochem; Darmstadt, Germany). Stock solutions of morphine and naloxone were made in H₂O and DAMGO (Tocris Bioscience; Bristol, UK), SB205607 (Tocris Bioscience; Bristol, UK), U50488 (Tocris Bioscience; Bristol, UK) and PYY (ABGent; San Diego, CA), were made in physiological extracellular solution. A stock solution of 8-bromo cAMP was made in H₂O titrated with NaOH. Stock solutions of L-AP4 (Tocris Bioscience; Bristol, UK) and (RS)-Baclofen (Tocris Bioscience; Bristol, UK) were made in molar equivalent solutions of NaOH. Stock solutions were aliquoted and stored at −20°C. Stock solutions were diluted in physiological extracellular solution for their use in experiments. PTX (lyophilised powder) was reconstituted in H₂O and was stored at 4°C. PTX was added to cells at a concentration of 200 ng/ml.

L-glutamate was purchased from Sigma. DCG-IV, L-AP4, MNI137 and LY487379 were from Tocris Bioscience. LSP4-2022 was a provided by Dr. F. Acher (Paris, France). Glutamate-pyruvate transaminase (GPT) was purchased from Roche. Lipofectamine 2000 and Fluo-4-AM were from Life Technologies. SNAP-Green was from NEN Biolabs.