3.2 Patterned light stimulation reduces AP amplitude without affecting AP latency in MCs (Procedure 4)
The effect produced by the different patterns on AP properties was studied using the protocol depicted figure 3A. Most of the recorded neurons were previously submitted to the voltage-clamp protocol shown in figure 2A. Light stimulation induced a reduction in AP amplitude that reached a significant effect only for patterns 3, 4, 6, 7 and 8 (Figure 3B-C and supplementary figure 3). The lack of effect of the other patterns is likely due to the low statistical power that follows the Bonferroni-Holm correction. Indeed, with the exception of pattern 1, all patterns had a p-value lower than the α risk (0.05) before the correction (see https://osf.io/h7g8p/ ). Moreover, exploratory analyses for which the effect was assessed on all 50 APs showed a significant decrease in AP amplitude for all tested patterns (supplementary table 3). Again, the light effect was higher for patterns that have a longer duty cycle. However, the covariation between the duty cycle and the AP amplitude reduction was less pronounced than the covariation between the duty cycle and the light-induced outward current shown in figure 2. In addition, no clear relationship emerged between step duration and AP amplitude reduction. The effect of light on AP amplitude had a moderate covariation with the tissue temperature increase produced by the different patterns (Figure 4B). Patterned light did not alter AP latency (Figure 3 C, bottom, and supplementary figure 4). We further performed exploratory analysis to look for other eventual modifications produced by light on other AP parameters. In particular, we looked at the full width at half maximum (FWHM) and at the AHP amplitude. Both of these parameters were reduced by patterned stimulation (supplementary table 3). To test whether light also alters APs during more physiological stimuli, we induced neuronal firing with longer pulses of current (2.5 s). We used only the pattern and power that produced the largest overall effect in previous tests (pattern 4 at 13mW). As shown in figure 5, light stimulation had a heterogeneous action on MC population. The firing activity was mildly reduced or unaffected in around 80% of the recorded neurons and strongly reduced in the remaining cells. The average frequency modification was -16 ± 30 Hz (N= 15, p=0.013).
Effect of patterned light stimulation on other neuronal types (procedures 3 and 4)
We next determined whether discontinuous light stimulation also affects the activity of GCs, PYRs, FSIs and MSNs by using the pattern that produced the largest effect on MCs (pattern 4 at 13mW power). While continuous light stimulation induced an outward current in all cell types (Ait Ouares et al. , 2019), discontinuous stimulation had this effect only in PYRs, GCs and MC (figure 6A). Interestingly, in one of the recorded GC, we observed a clear inward current upon light stimulation (supplementary figure 6) suggesting that light can have an excitatory action on a small fraction of neurons. Patterned light reduced AP amplitude in all neuronal types, but this effect was less pronounced than that produced by continuous stimulation (Figure 6B). Similar to our observation for MCs, we did not find any effect of patterned light on AP latency (supplementary figure 7).