3.2 Patterned light stimulation produces an outward current in MCs (Procedure 3)
The effect produced by the ten different patterns on MC membrane current was studied using the protocol depicted in figure 2A. Whatever the pattern, light intensities of 1mW had no effect, while for 5mW, the light tended to produce an outward current that did not reach statistical significance (Figure 2B and supplementary Figures 1-2). This lack of effect appears mainly to result from the low statistical power produced by the correction for multiple comparisons (see p-values before correction https://osf.io/e6brs). Moreover, the exploratory analysis performed by calculating the average effect produced over all 50 light pulses produced a small but significant outward current for all patterns (supplementary table 2). When the light power was set at 13mW, the light induced an outward current that tended to increase with pulse repetition (Figure 2B, C and supplementary Figure 1). A significant effect was consistently observed at the 50th pulse for most of the patterns (Figure 2C). Similar to the effects of light on tissue temperature, the amplitude of the light-induced outward current increased with duty cycle. The effect of the pulse duration was less pronounced (figure 2C). The difference between patterns cannot be attributed to sample variability since the effect produced by the test LED was the same for all patterns (Supplementary figure 1, Kruskal-Wallis test, H=0.79, p= 0.612). For patterns with a high duty cycle, the effect produced by the continuous stimulation (TI LED) was not different from the effect produced by patterned light. However, for lower duty cycles, discontinuous stimulation produced a lower effect than continuous ones (Figure 2C). Interestingly, exploratory analysis showed a covariation between the effect produced by each pattern on tissue temperature and the generation of outward current (Figure 4A). These results suggest a causal link between the light effect on MC electrophysiological properties and the light-induced modification of tissue temperature, supporting previous findings (Ait Ouares et al. , 2019; Owenet al. , 2019).