Figure 7A shows the typical slow firing rate that is observed in these conditions. A single action potential can be seen on a faster time scale in Fig. 7B. We have previously shown that apamin, at a concentration that completely blocks SK channels (100 nm), increases the firing rate of MK0683 5-HT neurons by ~30% in slices (Rouchet et al., 2008). If N-type channels are also the most important source of Ca2+ that activates SK channels involved in
the mAHP in slowly firing cells, an effect similar to that of apamin should be observed with ω-conotoxin, but not with the blockers of other Ca2+ channels. This is exactly what we found (Fig. 7C). For these experiments, we chose Anti-infection Compound Library mouse to use first TTA-P2 (3 μm, a concentration that completely blocks T-type currents in slices; Dreyfus et al., 2010) instead of mibefradil to block T-type channels because of its higher selectivity for these channels. Thus we compared the effect of ω-conotoxin, nifedipine and
TTA-P2. The control firing rates in the three groups (n = 8 in each) were 2.32 ± 0.62, 2.22 ± 0.41 and 1.26 ± 0.23 spikes/s, respectively. As can be seen in Fig. 7C, a clear increase in firing was observed in the ω-conotoxin group but not in the other groups, although a very slight excitation was seen in the nifedipine group. A mixed anova test demonstrated a significant interaction between time and groups (F = 11.49, P < 0.001). In addition, the effect of ω-conotoxin
was significantly larger than that of the two other blockers (P < 0.001 for both comparisons). The percentage increase in firing (~30%) produced by ω-conotoxin was similar to the effect of apamin found previously (from 2.32 ± 0.62 to 2.96 ± 0.69 spikes/s for conotoxin and from 1.7 ± 0.02 to 2.2 ± 0.03 spikes/s for apamin, n = 18; Rouchet et al., Fossariinae 2008), showing that N-type channels are the only significant source of Ca2+ that activates the mAHP channels when these neurons fire spontaneously. Finally, because we had used mibefradil to block T-type channels in patch-clamp and intracellular experiments, we also tested this blocker at the same concentration (30 μm) during extracellular experiments (not shown). Mibefradil had no effect on the spontaneous firing rate of 5-HT neurons; firing rates were 1.42 ± 0.1 and 1.40 ± 0.15 spikes/s (n = 4) during the control period and after 10 min superfusion of the blocker, respectively. Our findings can be summarized as follows: we found that both N- and T-type channels can provide a source of Ca2+ needed to activate SK channels in DRN serotonergic neurons. However, physiologically it appears that only N-type channels are providing the Ca2+ ions which generate the opening of SK channels during the mAHP. Importantly, this was true in neurons from both juvenile and adult rats.