Figure 1.
Experimental protocol of the superfusion experiments of the present study. (A) The majority of experiments lasted for 110 min and two periods of electrical field stimulation (flash symbols) starting after 40 (S1) and 90 min of superfusion (S2) were administered. Nociceptin, related peptides or tetrodotoxin were added to (and Ca2+ ions were omitted from) the medium from 62 min of superfusion onward (i.e. from 28 min before S2 onward). In few experiments, nociceptin was added from 72 or 82 min of superfusion onward (i.e. from 18 or 8 min before S2 onward, respectively). Other drugs (desipramine, indalpine, metitepine, naloxone, naloxone benzoylhydrazone, L-NAME, naproxen, rauwolscine, 8-(p-sulphophenyl)theophylline and/or talipexole) were present in the medium throughout superfusion. In most of the experimental series, the superfusion medium contained desipramine 1 μM, rauwolscine 1 μM plus naloxone 10 μM (‘standard medium') and the stimulation parameters were 0.3 Hz, 50 mA, 2 ms (duration of 2 min) (‘standard stimulation'). (B) The experiments shown in Figure 3 lasted for 210 min and four 2-min periods of electrical field stimulation (0.3 Hz, 50 mA, 2 ms) starting after 40 (S1), 90 (S2), 140 (S3) and 190 min of superfusion (S4) were administered. Nociceptin was added to the medium either from 62 min to 112 min of superfusion or from 62 min until the end of superfusion. Other drugs (desipramine, rauwolscine plus naloxone) were present in the medium throughout superfusion.