D 2 amino 5 phosphonopentanoic acid ap5

TTX and D-(−)-2-amino-5-phosphonopentanoic acid (AP5) were purchased from Tocris (Bristol, UK). 4-Aminopyridine (4AP), Kyn, picrotoxin, and bicuculline (Bic) were purchased from Sigma. Stock solutions of TTX, 4AP, Bic, and AP5 were dissolved in water, kept frozen at −30°C, and then dissolved in ACSF to their final concentration on the day of the experiment.

The D1/D5 receptor agonist SKF‐38393 hydrochloride [(±)‐1‐Phenyl‐2,3,4,5‐ tetrahydro‐(1H)‐3‐benzazepine‐7,8‐diol hydrochloride] (SKF; #D047; Sigma‐Aldrich, Singapore) was stored at −20 °C as a 50‐mm stock in deionised water. The stocks were used within a week. The Zn²⁺ chelator TPEN [N,N,N′,N′‐Tetrakis (2‐pyridylmethyl) ethylenediamine] (Tocris Bioscience, Bristol, UK) was stored as a 25‐mm stock in dimethyl sulfoxide (DMSO) at −20 °C. Emetine dihydrochloride hydrate (Sigma‐Aldrich, Singapore) and d‐2‐amino‐5‐phosphonopentanoic acid (AP‐5) (Tocris Bioscience, Bristol, UK) were prepared as concentrated stock solutions in DMSO and were diluted in ACSF to obtain a final concentration of 20 and 50 μm, respectively. Light‐sensitive drugs were protected from light during storage and bath application. Prior to application, the drug stocks were diluted to the final concentration in ACSF, equilibrated with carbogen, and bath‐applied for specified durations. Whenever the stocks are prepared in DMSO, the final DMSO concentration was kept below 0.1%, a concentration which has been shown to not affect basal synaptic responses (Navakkode et al., 2005).

Ryanodine, 8‐Cyclopentyl‐1,3‐dipropylxanthine (DPCPX), thapsigargin, suramin, and D (−)‐2‐amino‐5‐phosphonopentanoic acid (AP5) were purchased from Tocris Cookson (Bristol, UK). All other drugs were purchased from Sigma (St Louis, MO, USA). Drugs were stored as frozen concentrated stock solutions and dissolved in oxygenated aCSF at the desired concentration immediately before use. If not otherwise mentioned, drugs were added 20 min before caffeine perfusion and were present in the chamber for the entire recording period.

Stock solutions of the following drugs were prepared in water and purchased from the following companies: D(–)-2-Amino-5-phosphonopentanoic acid (AP-5, Cat#3693, Tocris), bicuculline methobromide (B.M.R., Cat#HB0894 Hello Bio), 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX, Cat#ab120046, Abcam), R&D Systems (Minneapolis, MN); tetrodotoxin (T.T.X., Cat#HB1035, Hello Bio); 4-Aminopyridine (Cat # A-0152, Sigma-Aldrich), kynurenic acid Na salt, (Cat. # 3694 Tocris Bioscience), CPG55845 hydrochloride (Cat # 1248 Tocris Bioscience stock concentration 5 mM), and gabazine (SR 95531 hydrobromide, Cat # 1262, Tocris Bioscience, stock concentration 10 mM). Drug-containing stock solutions were diluted to desired concentrations in either ACSF. All drugs were applied via the “Y-tube” application adapted to brain slices (Murase et al., 1989 (link); Hevers and Lüddens, 2002 (link)).