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).