Materials
MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2
phenyl-4-propyl-3-pyridine carboxylate, A3R antagonist),
SKF89976A hydrochloride
(1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride,
GAT-1 blocker) and non-radioactive (cold) GABA (γ-aminobutyric acid)
were purchased from Sigma. Kainic acid, DPCPX
(8-cyclopentyl-1,3-dipropylxanthine, A1R selective
antagonist) and CPA (N6-cyclopentyladenosine,
selective A1R agonist), CCPA
(2-chloro-N6-cyclopentyladenosine, selective
A1R agonist) and picrotoxin (GABAAR
antagonist) were from Tocris (Bristol, England). Pentobarbital
(Euthasol® 400 mg/mL) was from Dechra, Northwick, England). MRS5474
((1R ,2R ,3S ,4R ,5S )-4-(2-chloro-6-((dicyclopropylmethyl)amino)-9H -purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol)
and MRS5698
((1S ,2R ,3S ,4R ,5S )-4-[6-[[(3-chlorophenyl)methyl]amino]-2-[2-(3,4-difluorophenyl)ethynyl]-9H -purin-9-yl]-2,3-dihydroxy-N -methylbicyclo[3.1.0]hexane-1-carboxamide,
A3R agonist) were synthesized at NIH (Jacobson group) as
reported (Tosh et al., 2012a,b, 2015). CPA, CCPA, DPCPX, picrotoxin,
MRS5474, MRS1523 and MRS5698 were prepared as a 5 mM stock solution in
dimethyl sulfoxide (DMSO); and SKF89976A was prepared as a 50 mM stock
solution in DMSO. The DMSO concentration in the working solutions did
not surpass 0.01%. GABA was prepared as a 50 mM (GABA uptake) or 100 mM
(GABA currents) stock solution in deionized water. The stock solutions
were aliquoted and kept at -20°C until use. Fresh dilutions of these
stock solutions to the final concentration were prepared for each
experiment.