Introduction
The continuous light stimulation of brain tissue has been shown to affect the electrophysiological properties of several neuronal subtypes,in vitro and in vivo , by producing a modification of firing rate associated with a hyperpolarizing outward current (Stujenskeet al. , 2015; Ait Ouares et al. , 2019; Owen et al. , 2019) . The sensitivity of neurons to light can therefore represent a confounding factor for those assays that use light as a tool to investigate neuronal processes such as optogenetics and fluorescent imaging. Because the effect of light is primarily mediated by the increase in tissue temperature and discontinuous light has a weaker heating action than continuous light, it can be assumed that the use of specific patterns of discontinuous light stimulation is preferable to avoid non-specific effects. In particular, theoretical models (Stujenskeet al. , 2015) and experimental measurements (Senova et al. , 2017) have demonstrated a decrease in light-induced heating concurrent with decreasing power, frequency and duty cycle of light stimulation. For example, 10 mW of discontinuous light stimulation at 10 Hz with a duty cycle of 10 % is responsible for a temperature increase equivalent to that produced by 1 mW of continuous light stimulation (Stujenske et al. , 2015). As we previously showed that the latter protocol had no impact on neuronal activity (Ait Ouares et al. , 2019), 10 mW of discontinuous light stimulation at 10 Hz should also be free of artifactual effect assuming that light effects are exclusively due to temperature increase. However, light has also been shown to be responsible for temperature independent effects (Ait Ouares et al. , 2019; Tyssowski & Gray, 2019), including the activation of encephalopsins (Friedmann et al. , 2015; Wang et al. , 2019) or cytotoxic processes (Duke et al. , 2020). It is therefore important that theoretical predictions about the “safety” of a specific pattern of optical stimulation be confirmed by empirical evidence. Based on a literature review using the PubMed query “Optogenetics”, we selected 10 different patterns of discontinuous light stimulation commonly used in optogenetic research and we tested their impact on several electrophysiological properties of mitral cells (MCs). More specifically, we analyzed the effects of light on the generation of outward hyperpolarizing current as well as on the amplitude and latency of action potentials (APs) induced by short steps of positive current, as well as on neuronal firing produced by continuous depolarization. All these parameters were consistently modified by continuous light stimulation (Ait Ouares et al. , 2019). We therefore tested whether patterned light stimulation would be responsible for: 1) the generation of an outward current, 2) a decrease in AP amplitude, 3) an increase in AP latency and 4) a reduction in firing frequency. Finally, the pattern producing the most pronounced effects in MCs was also applied to cortical pyramidal neurons (PYRs) and fast-spiking interneurons (FSIs), in striatal medium-sized spiny neurons (MSNs) and in hippocampal granular cells of the dentate gyrus (GCs).