Adenosine Measurement in Brain Slices

Slices were kept in oxygenated artificial cerebral spinal fluid (aCSF), as described previously.^{51 (link)} aCSF was comprised of 126 mM NaCl, 2.5 mMKCl, 1.2 mM NaH₂PO₄ monohydrate, 2.4 mM CaCl₂ dihydrate, 1.2 mM MgCl₂ hexahydrate, 25 mM NaHCO₃, 11 mM glucose, and 15 mM tris(hydroxymethyl)aminomethane and was adjusted to pH 7.4 immediately prior to experimentation. A 10 mM stock solution of adenosine was prepared in 0.1 mM HClO₄ and this was diluted daily in aCSF to 1 μM for post-calibration of the CFMEs. One mM stock solutions of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, Sigma-Aldrich) were prepared in dimethyl sulfoxide (DMSO) and kept frozen until used. Stock DPCPX was added to perfusion aCSF to make a 100 nM solution and slices were perfused for 15 min before the electrode was implanted and adenosine measurements made while DPCPX was perfused. Tetrodotoxin (TTX, Tocris) was reconstituted to 3 mM in 0.2 M citrate buffer, diluted to 50 μM aliquots, and kept frozen until used. 50 μM aliquots were added to perfusion aCSF to make a 200 nM working solution and perfused over slices in the same manner as DPCPX. After each experiment all solutions and surfaces that came into contact with TTX were treated with 10% bleach to deactivate residual TTX.