D 2 amino 5 phosphonopentanoic acid d ap5

1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzo–diazepine hydrochloride (GYKI-53655), (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione (UBP-302), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (gabazine) were supplied by Tocris. d-(-)-2-Amino-5-phosphonopentanoic acid (d-AP5) was purchased from Abcam. Drugs were applied by dissolving them into circulating aCSF at the desired concentration. UBP-302 was dissolved in dimethyl sulfoxide (DMSO) supplied by Tocris (DMSO at 0.02% in circulated solution). A wash-in duration of at least 5 min was allowed for all drugs before the effects were evaluated.

4-aminopyridine (4-AP) was purchased from Sigma-Aldrich (St Louis, MO, USA). D-(−)-2-Amino-5-phosphonopentanoic acid (D-AP5) and TTX were obtained from Abcam Plc. (Cambridge, UK). Phrixotoxin-2 was purchased from Alomone Labs (Jerusalem, Israel) and diluted in 0.1% bovine serum albumin (BSA) solution. The dissolved phrixotoxin-2 was added to a perfusion solution containing 0.1% BSA. RuBi-glutamate was obtained from Tocris Cookson (Bristol, UK). To prevent dilution or contamination of re-circulated solutions, the perfusion tubing was drained when switching between drugs. Drugs were dissolved and kept as stock solutions at −20 °C, then diluted freshly before each experiment and applied for at least 10 min.

After whole cell access was established, tetrodotoxin (TTX) (500 nM) (Bio-Techne Corporation, Minneapolis, MN, USA) was applied to the bath for 5 min to eliminate action potential firing. ATP-γ-S (100 μM) was then added to the bath for ~3 min. Continuous recordings of membrane voltage were made using a MiniDigi 1B system and Axoscope 10.0 software (Axon instruments, Molecular Devices) with a sampling frequency of 10 kHz. To determine the involvement of P2X receptors in ATP-γ-S-induced response, the P2XR antagonist (PPADS), was bath-applied for 5 min before ATP-γ-S was added. TTX was present for the whole recording time. To examine the involvement of presynaptic inputs in ATP-γ-S-induced response, the following cocktail of neurotransmitter antagonists was bath-applied with TTX for 5 min before ATP-γ-S was added: the AMPA/Kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Abcam, Cambridge, MA, USA), the NMDA receptor antagonist D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5, 50 µM, Abcam), and the GABA_A receptor antagonist SR95531 (GABAzine, 10 µM, Abcam).