Procedure 4: Determination of the light effect on AP amplitude and latency.
Cells were recorded in current-clamp mode at their resting membrane potential. In cases where spontaneous firing was present, a hyperpolarization of the membrane potential was produced by injecting negative current. Two consecutive trains of 50 APs, separated by 10 s, were generated with 5 ms depolarizing steps applied at the frequency of the light stimulation pattern chosen for that specific neuron. In half of the recorded neurons, the first train was generated in the absence of light and the second train generated during light stimulation, with the depolarizing steps concomitant to the last 5 ms of the illumination. In the other half of the neurons, light stimulation train preceded the no light train. After 10 s, a single depolarizing step (test step) was generated at the end of a 1 s continuous light stimulation. Since the latter procedure is expected to produce a consistent modification of AP amplitude and latency (Ait Ouares et al. , 2019), the AP generated by the test step was used as positive control. In the protocols for which the total illumination time (T.I) during patterned stimulation was different from 1 s, one AP was also generated at the end of the continuous T.I. step. This protocol was repeated 10 times for each neuron. For each of the ten acquired traces, the effect of light was assessed trough a step-by-step comparisons of AP in the absence and in the presence of light (i.e., 1st AP no LED vs 1st AP LED, 2th AP no LED vs 2th AP LED……. 50th AP no LED vs 50th AP LED). The AP of the test step and T.I. step was compared to the 1st AP generated in the no LED condition. For each AP, the latency was quantified as the time of the AP peak relative to the beginning of the depolarizing step (Ait Ouares et al. , 2019). AP amplitude was calculated as the difference between AP peak and the median membrane potential calculated in the 200 ms preceding the first depolarizing step. Steps presenting AP failures, or AP for which the time of AP peak exceeds the step duration, were excluded from the analysis. In half of the recorded neurons, the steps of current in the no-light condition preceded the steps of current in the light condition and vice versa for the other half of the neurons. The effect of light on each step was expressed as the average difference in AP amplitude and latency, between the light and no light conditions for that specific step. Only one out of the ten stimulation patterns was applied on each MC.