Hypothesis 1 was tested as follows:
For each light pulse, the effect of light on AP amplitude was calculated
as the difference in AP amplitude between the LED condition and the no
LED condition (i.e., 1st AP amplitude LED – 1st AP amplitude no LED;
…….; 50th AP amplitude LED –
50th AP amplitude no LED). For each MC the median
effect was calculated. This resulted in the median light effect for each
of the 50 pulses (i.e., effect of light on the amplitude of the 1st AP;
……..; effect of light on the amplitude of the
50th AP). The values calculated for all recorded cells
were used for descriptive statistics by calculating the average
population effect (± 95% CI) as well as the Cohen’s d ES (average of
population effect /STD of population effect). Similar analyses were
performed for the positive control (test LED) and the total continuous
illumination exposure (T.I. LED), for which the effect was evaluated as:
LED AP amplitude - 1st AP amplitude no LED. In order to reduce the
probability of Type I error produced by multiple comparisons correction,
statistical tests were made only for three light pulses (the 1st,
25th, and 50th) by using a
unilateral one-sample Wilcoxon signed rank test (tested hypothesis,
median effect < 0). The σ risk level was set at 0.05 and
Bonferroni-Holm correction was applied. Experiments were conducted on 24
neurons for each pattern of light stimulation and cell type (336 neurons
in total). This sample size was determined based on a previously
published experiment on MCs where continuous light stimulation reduced
AP amplitude with ES = -1.2 (Ait Ouares et al. , 2019) and takes
into account the Bonferroni-Holm correction for 30 statistical
comparisons. As a consequence, the statistical power to observe an
effect as small as that produced by 1 s continuous light stimulation is
estimated between 98% et 99% depending on the level of correction
(calculated with G*Power 3.1.9.2, (Faul et al. , 2007). The 24 MCs
were recorded from 57 mice. The homogeneity of the effect produced by
the “Test LED” between the groups was assessed by one-way
Kruskal-Wallis test. Exploratory analysis was performed to evaluate a
possible difference between the average AP amplitude (over the 50
depolarizing steps) in the presence and in the absence of patterned
light stimulation.