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.