Supplementary Figure 2: Decrease in spike probability and changes in spike shape from somatic HCN channel block arise from postsynaptic membrane hyperpolarization

(a) Stimulus protocol (top) and example responses (bottom) to a train of 10 synaptic stimuli delivered at 100 Hz. A glass pipette (~50 µm diameter) was used to stimulate contralateral glutamatergic inputs while glycinergic inhibitory inputs were blocked by bath application of 1 µM strychnine. Stimulus amplitude was adjusted to trigger ~50% suprathreshold responses. AAQ treated cells were field illuminated at 380 nm (left), after which the soma was scanned at 488 nm (center), hyperpolarizing the cell. Synaptic responses were again elicited after the resting potential was restored to control values with direct somatic current injection (right) (b) Phase plane analysis of the first action potential triggered in the train show that voltage threshold and spike shape are not altered with HCN channel block in with somatic 488 nm illumination. Dotted lines: spike threshold. (c-f). Group data showing that the decrease in spike probability is eliminated when the resting potential is restored to control values during somatic scanning at 488 nm (p = 0.02 and 0.56 for soma and soma + DC; n=4). Spike shape parameters are unchanged (soma and soma+DC respectively: Vrest, p=0.01 and 0.4; Max dV/dt, p=0.19 and 0.76; spike threshold, p=0.73 and 0.19; n=4). One-way repeated measures ANOVA. Error bars indicate SEM.