The external recording solution consisted of (in mM): tetraethylammonium hydroxide 145, methanesulphonic acid 140, HEPES 10, glucose 15, CaCl2 2.5, tetraethylammonium chloride 15 and TTX 0.0003. The pH was adjusted to 7.40 with tetraethylammonium hydroxide. The pipette solution contained (in mM): N-methyl-D-glucamine 110, tetraethylammonium hydroxide 25, HCl 20, EGTA 11, HEPES 10, CsCl2 20, CaCl2 1, MgATP 4, Na2GTP 0.3 and Na-Creatine phosphate 7. The pH of this solution was adjusted to 7.20 with tetraethylammonium hydroxide. Both stock solutions of morphine and fentanyl (both from Sigma Aldrich) were prepared in water and diluted in the external recording solution to their final concentration. The agonists were applied to the neuron under study with a custom-designed gravity-fed microperfusion system.
