5A with 4I and K), and simply consisted in a time-independent reduction of the current. In order to test the physiological relevance of our voltage-clamp results, experiments were also performed in current clamp in the presence of the synaptic blockers mentioned above, but in the absence of TEA. Action potentials were evoked by a short (3-ms) depolarizing pulse (50–400 pA). Under these conditions, only ω-conotoxin (1 μm) was able to reduce the amplitude of the BMI-sensitive mAHP (by 79.7 ± 15.7%; n = 6). Mibefradil (30 μm) was devoid of any effect (n = 5). When

we co-applied the two blockers, Enzalutamide nmr the reduction in the amplitude of the mAHP amounted to 79.5 ± 14.4% (Fig. 6A; n = 5). The three experimental conditions (mibefradil alone, ω-conotoxin alone and co-application of the two agents) induced a differential block of the mAHP ( = 7.47, P = 0.0077, Kruskal–Wallis test). Both ω-conotoxin alone and co-application of ω-conotoxin and mibefradil produced selleckchem a significantly larger effect than mibefradil alone (U = 2.74, P = 0.006 and U = 2.6, P = 0.009, respectively; Mann–Whitney test).

We next performed intracellular recordings in the current-clamp mode in DR neurons from adult rats to test the sensitivity of the mAHP to blockers. Concentration–inhibition curves were first constructed with apamin (n = 5). The IC50 of the peptide was 2.5 ± 0.7 nm (not shown) with a mean Hill Dichloromethane dehalogenase coefficient of 2.5. In addition, we tested the sensitivity of the mAHP to tamapin, whose IC50 was found to be 9 and 17 nm (n = 2; mean Hill coefficient was 3.6; not shown). Taken together, these results suggest (but do not prove) that SK3 subunits are the main components of the SK channels underlying the mAHP of DR neurons. We then performed the same pharmacological experiments as above using Ca2+-channel blockers. Superfusion of ω-conotoxin (1 μm) also markedly reduced the amplitude of the mAHP in adult DRN serotonergic neurons (n = 6; mean inhibition 83 ± 3%). Its effect developed progressively to reach a stable maximum after 8 min. In contrast, no modification of the mAHP was observed with either mibefradil (30 μm; n = 4) or nifedipine (20 μm;

n = 4; Fig. 6B and C). In addition, the effect of ω-conotoxin was again not increased by the co-application of mibefradil (n = 4; not shown). A mixed anova showed a highly significant interaction between time and groups (F = 5.46, P < 0.001). The effect of ω-conotoxin was significantly higher than that of the two other blockers (P < 0.001 in both cases). The previous results show that N-type channels are the major source of Ca2+ that activates SK channels underlying the mAHP. However, these results were obtained in neurons which were silent (i.e. action potentials were induced by depolarizing current injection). In vivo, 5-HT neurons are known to have a slow pacemaker-like firing, at least in anaesthetized animals (Jacobs & Fornal, 1991).