Hypothesis 3 was tested as follows:
Light effect on membrane current was calculated on the median trace of
the 15 recorded traces. For each of the 50 light pulses, the effect of
light on membrane current was calculated as the difference between the
average membrane current in the 1 s preceding the beginning of patterned
light (the CTR period) and the average membrane current during the light
period. For each of the three powers used, this resulted in the median
light effect for each of the 50 pulses (i.e., effect of the
1st light pulse; ……..; effect of the
50th light pulse). The values calculated for all
recorded neurons 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 (T.I. LED), for which the effect was evaluated as the
difference between the average membrane current during light stimulation
and the average membrane current in the respective CTR period.
Statistical test was performed 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
was set at 0.05 and a Bonferroni-Holm correction was applied. The
experiment weas conducted on 24 neurons for each pattern of light
stimulation and cell type (240 neurons in total). This sample size was
determined based on a previously published experiment showing an effect
size (ES) of continuous light stimulation on MC membrane current equal
to 1 (Ait Ouares et al. , 2019) and takes in account the
Bonferroni-Holm correction for 30 statistical comparisons. Therefore,
the statistical power to observe an effect as small as that produced by
1 s continuous light stimulation is between 91% et 99%, depending on
the level of correction (calculated with G*Power 3.1.9.2). The 24 MCs
were recorded from 51 different mice (i.e., different patterns were
applied on slices coming from the same animal). The homogeneity between
the groups produced by the “Test LED” was assessed by a one-way
Kruskal-Wallis test.