Discussion
The main conclusion of this study is that a significant number of
discontinuous light stimulation patterns, commonly used in optogenetic
experiments, can affect neuronal electrophysiological activity. However,
their impact is, in general, smaller than that produced by continuous
light stimulation. It also highlights how the different parameters of
the stimulation impact the light action, providing several guidelines to
minimize the artefactual effects associated with the use of light in
neuroscientific investigations. Indeed, a bibliographic analysis done on
the first 50 discontinuous patterns retrieved when using the query
“Optogenetics” on PubMed showed that the light parameters investigated
here are among the most commonly used in optogenetic experiments
(https://osf.io/anvsj). We used the change in AP amplitude and latency
and the generation of an outward current as a read-out for light action,
all of which were modified by continuous light stimulation (Ait Ouareset al. , 2019). We found that patterned light consistently reduced
AP amplitude without significantly altering AP latency in all cell types
and produced an outward current only in MCs, PYRs and GCs. The lack of
effect on AP latency is likely related to the low statistical power of
the pre-registered analytical method, since exploratory analysis shown
in supplementary table 3 suggest that AP latency does indeed slightly
increase during patterned light stimulation. The effects of light on
membrane current and AP amplitude are small, with a change of only a few
pA for membrane current and a ~0.5 mV reduction in AP
amplitude for the pattern with the highest effect. However, even small
variations in AP amplitude can consistently modify synaptic transmission
(Rama et al. , 2015). Moreover, discontinuous light produced a
strong reduction in firing frequency for a subpopulation of MCs when
neurons where depolarized to a membrane potential that allow spontaneous
activity. A possible reason for the heterogeneity of this effect might
be explained by a diversity of biophysical properties among different
MCs (Padmanabhan & Urban, 2010; Fourcaud-Trocmé et al. , 2022).
However, the effects of discontinuous light where in general lower than
those produced by continuous stimulation showing that patterned
stimulation can reduce the artifactual effect of light. Both the
induction of the outward current and the reduction in AP amplitude
induced by discontinuous light occur rapidly after few pulses and
quickly reach a plateau. Therefore, reducing the number of stimuli does
not appear to be an optimal strategy to reduce the artefactual effect of
light. On the other hand, the light effect can be attenuated by reducing
the stimulation power, the duty cycle and, in some cases, the pulse
duration. Note that varying the frequency of the light stimulation does
not change the artefactual effects as long as the duty cycle is the
same. This is particularly important for experiments whose goal is to
induce high frequency firing by optogenetic stimulation. As a rule, we
recommend adapting the pulse duration to the stimulation frequency in
order to keep the duty cycle at or below 5%. Using a light power of 1
mW or less also appears to be an effective means of preventing
unspecific effects during discontinuous stimulation compared to the
higher powers, which induce an outward current (see supplementary table
1). The reduction in AP amplitude induced by pattern 4 is observed in
all cell types, whereas an outward current is induced only in FSIs and
MSNs, suggesting a cell-specific effect of light. The greater reduction
in the discharge frequency of FSIs induced by light compared to
pyramidal neurons (Owen et al. , 2019) also suggests a
differential effect of light across cell types. However, we did not
observe such a difference here where all experiments were done in the
presence of ionotropic glutamatergic receptor antagonists. Since light
appears to reduce glutamatergic synaptic transmission (Ait-Ouares et
al., 2019), the pronounced decrease in FSI firing observed by Owen et
al., (2019) may be related to a reduction in their excitatory inputs.
However, we cannot rule out the possibility that the weaker effect
observed here is due to the presence of the tomato fluorescent protein
in the recorded FSI. The covariation between the change in tissue
temperature and the effect of light suggests a causal relationship
between the two factors, as previously reported (Stujenske et
al. , 2015; Senova et al. , 2017; Ait Ouares et al. , 2019;
Owen et al. , 2019). The light-induced temperature changes
observed in this study (between 0.1 and 0.8 °C) remain below the
threshold for tissue damage but may modulate a set of voltage-dependent
channels involved in neuronal discharge (Podgorski & Ranganathan, 2016;
Ait Ouares et al. , 2019). Our results suggest that light is able
to modulate different type of K+ channels beside the
inwardly rectifying potassium channels (Kir) (Owen et al. , 2019).
For instance, the reduction in AP duration suggests an increase in Kv
conductance’s responsible for AP repolarization (Bean et al., 2007)
while the reduction in AHP amplitude suggests an impact of light on the
calcium- and voltage-dependent potassium channels (Sah & Faber, 2002).
The light-induced reduction in AHP amplitude after a single AP contrasts
with the increase in AHP measured at the end of a long depolarization
that we observed previously (Ait Ouares et al. , 2019). This
result suggests that light may oppositely modulate the
K+ channels involved in the fast-medium and slow AHP.
However, because the AHP in MCs is modulated by recurrent glutamatergic
transmission (Duménieu et al. , 2015), we cannot rule out that the
light-induced reduction in AHP amplitude is partially due to the
presence of ionotropic glutamatergic receptor antagonists. Finally,
because AP initiation in MCs is promoted by hyperpolarizing events
(Fourcaud-Trocmé et al. , 2022), the light-induced decrease in AHP
could also contribute to the reduction of the firing frequency of these
cells.