Supplementary Figure 2: LC neurons have functional expression of Ca²⁺ channels.

(a) Somatic voltage clamp experiments were performed using the command action potential waveform obtained from a pacemaking LC neuron. Outward K⁺ currents were blocked using 5 mM TEA in the bath and recordings were performed under low external Na⁺ (50 mM), and Cs⁺-based internal solution supplemented with 5 mM TEA. (b) Ionic current measurements in response to the action potential waveform were sampled during normal external [Ca²⁺], following TTX to remove Nav1 component, and following equimolar substitution of Ca²⁺ with cobalt (Co²⁺). Co²⁺-sensitive currents were calculated by subtracting ionic current obtained with TTX and 2 mM Co²⁺ from ionic currents obtained in the presence of TTX and 2 mM Ca²⁺. (c) LC neurons display a subthreshold inward current during the inter-spike interval). (d) LC neurons also display a significant inward spike-associated current. (e) Box-plot quantification of charge for subthreshold and spike currents. (f) Box-plot quantification demonstrating that subthreshold inward currents carry approximately 70% of charge and the remaining 30% carried by the spike Data represents n=4 neurons from 4 mice, p=0.0143, Mann-Whitney).