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\begin{document}

\title{Patterned light stimulation, does it affect neuronal activity?}



\author[1]{Nicola Kuczewski}%
\author[1]{Anistasha Lightning}%
\author[2]{Corinne Beurrier}%
\author[3]{Marie Bourzeix}%
\affil[1]{CRNL, UCBL, UJM, INSERM, CNRS, U1028, UMR5292, NEUROPOP, Lyon, France}%
\affil[2]{Aix Marseille Univ, CNRS UMR 7289, Institut de Neurosciences de la Timone (INT), Marseille, France}%
\affil[3]{Aix Marseille Univ, CNRS UMR 7289, Institut de Neurosciences de la Timone (INT), Marseille, France.}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Neuronal sensitivity to light stimulation can be a significant
confounding factor for assays that use light to study neuronal
processes, such as optogenetics and fluorescent imaging. While
continuous light stimulation has been shown to be responsible for a
decrease in firing activity in several neuronal subtypes, discontinuous
light stimulation commonly used in optogenetic experiments is supposed
to have a negligible action. In the present report, we experimentally
test this theoretical prediction by evaluating the effect produced by
ten of the most commonly used patterns of discontinuous light
stimulation under several electrophysiological parameters.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The continuous light stimulation of brain tissue has been shown to
affect the electrophysiological properties of several neuronal
subtypes,\emph{in vitro} and \emph{in vivo} , by producing a
modification of firing rate associated with a hyperpolarizing outward
current (Stujenske\emph{et al.} , 2015; Ait Ouares \emph{et al.} , 2019;
Owen \emph{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 (Stujenske\emph{et al.} , 2015) and experimental measurements
(Senova \emph{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 \emph{et al.} , 2015). As we previously
showed that the latter protocol had no impact on neuronal activity (Ait
Ouares \emph{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 \emph{et al.} , 2019; Tyssowski \& Gray, 2019), including
the activation of encephalopsins (Friedmann \emph{et al.} , 2015; Wang
\emph{et al.} , 2019) or cytotoxic processes (Duke \emph{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 \emph{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).

\textbf{Methods}

\textbf{Animals.}

Male C57Bl6/J mice (Charles River Laboratories, France) aged between 60
and 90 days were used. To identify FSIs, mice expressing the fluorescent
tomato protein in parvalbumin neurons were generated by crossing
B6;129P2-Pvalbtm1(cre)Arbr/J strain with
B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J strain from Jackson
Laboratory11The initial proposal planned to use wild type mice. The use
of transgenic mice expressing tomato protein in parvalbumin neurons was
introduced to identify the fast spiking interneurons in cortical slices.
This changes in the protocol received editorial approval prior to data
collection, on 9 Feb 2022.. All procedures were in accordance with
European Union recommendations for animal experimentation (2010/63/UE).
Mice were housed in groups of up to five in standard laboratory cages
and were kept on a 12-hour light/dark cycle (at a constant temperature
of 22\selectlanguage{ngerman}°C) with food and water ad libitum.

\textbf{Electrophysiology.}

\textbf{Protocols for slice preparation and recordings of olfactory
bulb, hippocampal and cortical neurons.}

Mice were anaesthetized with an intra-peritoneal injection of ketamine
(50 mg/ml) and decapitated. The head was quickly immersed in ice-cold
(2-4°C) artificial cerebrospinal fluid (CutACSF) of the following
composition: 125 mM NaCl, 4 mM KCl, 25 mM NaHCO3, 0.5 mM CaCl2, 1.25 mM
NaH2PO4, 7 mM MgCl2 and 5.5 mM glucose (pH = 7.4 oxygenated with 95 \%
O2/5 \% CO2). The osmolarity was adjusted to 320 mOsm with sucrose.
Horizontal olfactory bulb (OB) slices (400 \selectlanguage{greek}µ\selectlanguage{english}m thick), cortical coronal
slices (400\selectlanguage{greek}µ\selectlanguage{english}m thick) and hippocampus coronal slices (350 \selectlanguage{greek}µ\selectlanguage{english}m thick) were
prepared with a vibratome (Leica). Slices were then incubated in a
recovery chamber at 30 \selectlanguage{ngerman}± 1°C using an ACSF solution with a composition
similar to the Cut ACSF, except for changes to CaCl\textsubscript{2} and
MgCl\textsubscript{2} concentrations (2 mM and 1 mM, respectively).
Slices were transferred to a recording chamber mounted on an upright
microscope and continuously perfused with oxygenated ACSF (4 ml/min) at
30 ± 1°C. Neurons were visualized using a microscope (Zeiss axioscope)
with a 40X objective (Zeiss Plan-APOCHROMAT). Data were acquired with
the amplifier RK 400 BioLogic at full sampling frequency of 25 kHz using
a 12-bit A/D-D/A converter (Digidata 1440A, Axon Instruments) and PClamp
software (PClamp10, Axon Instruments). Patch-clamp whole-cell recordings
were achieved with borosilicate pipettes having a resistance 4-8 M\selectlanguage{english} and
filled with: 126 mM K-gluconate, 5 mM KCl, 10 mM HEPES, 1 mM EGTA, 1 mM
MgCl2, 2 mM ATP-Na2, 0.3 mM GTP-Na3, and 10 mM phosphocreatine (pH =
7.3, 290 mOsm). The membrane potential was corrected for the junction
potential (-15 mV). All experiments were performed in the presence of
ionotropic receptor antagonists (NBQX 10 \selectlanguage{greek}µ\selectlanguage{english}M, APV 40 \selectlanguage{greek}µ\selectlanguage{english}M and Gabazine
10\selectlanguage{greek}µ\selectlanguage{english}M).

\textbf{Protocol for slice preparation and recordings of striatal
neurons} . Mice were deeply anesthetized with ketamine/xylazine (100/10
mg/kg, i.p.) and transcardially perfused with an ice-cold N-methyl
D-glucamine (NMDG)-based solution containing (in mM): 93 NMDG, 2.5 KCl,
1.2 NaH\textsubscript{2}PO\textsubscript{4}, 30 NaHCO\textsubscript{3},
20 HEPES, 20 glucose, 10 MgCl\textsubscript{2}, 93 HCl, 2 Thiourea, 3
sodium pyruvate, 12 N-acetyl cysteine and 0.5
CaCl\textsubscript{2}(saturated with 95\% O\textsubscript{2} and 5\%
CO\textsubscript{2}, pH 7.2-7.4). The brain was then removed from the
skull and glued to the stage of a vibratome (Leica, VT1000S) where it
remained submerged in ice-cold oxygenated NMDG-based solution. Coronal
slices (250 \selectlanguage{greek}µ\selectlanguage{english}m thick) containing the striatum were collected. Slices
were immediately transferred to recover in NMDG-based solution at 35\selectlanguage{ngerman}°C
for 5 min and then stored for at least 1h at room temperature in normal
artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5
KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 25 NaHCO3 and 11 glucose, to
which 250 \selectlanguage{greek}µ\selectlanguage{english}M kynurenic acid and 1 mM sodium pyruvate had been added. For
the recordings, slices were transferred one at a time to a
submersion-type chamber and perfused continuously with warm ACSF
(32-34\selectlanguage{ngerman}°C) at a rate of 3 ml/min. Solutions were continuously
equilibrated with 95\% O2 / 5\% CO2. All experiments were performed in
the presence of ionotropic receptor antagonists (NBQX 10 \selectlanguage{greek}µ\selectlanguage{english}M, APV 40 \selectlanguage{greek}µ\selectlanguage{english}M
and Gabazine 10\selectlanguage{greek}µ\selectlanguage{english}M). MSNs were visualized on an upright microscope (Nikon
Eclipse FN1) equipped with DIC optic using an IR 40x water-immersion
objective (Nikon). Patch-clamp electrodes (4-6 M\selectlanguage{greek}Ω\selectlanguage{english}) were prepared from
filamented borosilicate glass capillaries (PG150T-7.5, Harvard
Apparatus) using a micropipette puller (PC-10, Narishige) and were
filled with an intracellular solution containing (in mM): 126 KMeSO4, 14
KCl, 3 MgCl2, 0.5 CaCl2, 5 EGTA, 10 HEPES, 2 NaATP and 0.5mM NaGTP, 10
Na-Phosphocreatine, pH adjusted to 7.25 with NaOH and osmolarity
adjusted to 270-280 mOsm/L. Recordings were obtained using motorized
micromanipulators (MP-85, Sutter Instrument), a Multiclamp 700B
amplifier, a Digidata 1550B digitizer, and pClamp 10.7 acquisition
software (Molecular Devices, San Jose, CA, USA). Signals were low-pass
filtered at 10 kHz online and sampled at 10 kHz. Electrode capacitances
were compensated electronically during recording. In current-clamp mode,
the bridge was continuously balanced and input resistances were
monitored. Cells showing more than 20\% input resistance variation were
excluded from the analysis.

\textbf{Optical stimulation and fluorescence detection.}

\emph{Olfactory bulb, hippocampal and cortical experiments.} Blue light
stimulation at 470 nm (430-495 nm) was performed using a Dual Port
OptoLED (CAIRN, UK) dichroic mirror 495 nm at a power of 13, 5 or 1 mW
measured at the output of the x40 objective. Power loss at the working
distance (2.5 mm from the objective), due to the presence of ACSF, was
empirically estimated at 13\%. The average power density in the tissue
was estimated by dividing the power at the working distance by the
empirically measured illumination area (\textasciitilde{} 7
mm\textsuperscript{2} ) which gives 1.61 mW/mm\textsuperscript{2} for a
output power of 13 mW , 0.62 mW/mm\textsuperscript{2} for 5 mW and 0.12
mW/mm2 for 1 mW. The excitation light for the detection of tomato
expressed in cortical parvalbumin interneurons was produced by a white
light LED (Dual Port OptoLED, CAIRN, UK) filtered at 545/25 nm and
reflected on the sample by a dichroic mirror of 570 mn (Zeiss). The
emission light was filtered at 605/70 nm (Zeiss filter). To avoid an
effect of the light for tomato detection on neuronal activity, only
short light pulses (\textless{}100 ms) at~power \textless{} 0.2 mW were
used. The illumination frequency was lower than 0.5 Hz, the total time
of green led illumination was lower than 2s and a minimum of 3 min was
applied between green and blue light stimulation.

\emph{Striatal experiments.} Light was delivered under the control of
the acquisition software via the x40 microscope objective lens using
wide-field 475 nm LED illumination (Spectra Light Engine, Lumencor,
Optoprim).

\textbf{Temperature measurement.}

Bath temperature in the recording chamber was measured and controlled by
a ThermoClamp-1 device (Atomate Scientific).

\textbf{Experimental procedure.}

For MC recordings, light stimuli consisted of fifty pulses delivered
according to one of the ten different patterns showed in table 1 (N=24
MCs for each pattern). For experiments in MSNs, GCs, PYRs and FSIs, only
the pattern producing the maximal effect on MCs was tested (pattern 4,
N= 15).

\emph{Procedure 1: determining the profile of temperature modification
produced by light patterns}

The thermal modifications produced by the different stimulation patterns
was determined by placing the thermal probe on the surface of OB slices
at the center of the objective field. Each pattern was repeated 10 times
(interstimulus time: 10 s) and the median normalized temperature trace
was used to calculate the temporal profile of temperature variation for
each pattern in each slice. This procedure was repeated on 10 different
slices and the comparison between patterns was performed on the average
trace (\selectlanguage{ngerman}± 95\% confidence interval).

The following experimental procedures 2, 3 and 4 were applied to each
recorded neuron.

\emph{Procedure 2: Calculation of the membrane resistance (Rm)}

Cells were held at -60 mV in voltage-clamp mode. Ten steps to -65 mV (1s
duration @ 1 Hz) were generated and recorded on fifteen separate
electrophysiological traces and a median trace was calculated. The Rm
was calculated on the median trace according to ohm's low R=\selectlanguage{greek}Δ\selectlanguage{english}V/\selectlanguage{greek}Δ\selectlanguage{english}I where
\selectlanguage{greek}Δ\selectlanguage{english}V=-5mV and \selectlanguage{greek}Δ\selectlanguage{english}I was the difference between the median current in the last
100 ms of the hyperpolarization and the median current in the 200 ms
pre-step period. Access resistance A\textsubscript{R} was calculated as
A\textsubscript{R}=\selectlanguage{greek}Δ\selectlanguage{english}V/\selectlanguage{greek}Δ\selectlanguage{english}I\textsubscript{A} where \selectlanguage{greek}Δ\selectlanguage{english}I\textsubscript{A} is
the difference between the peak of transient current and the median
current in the 200 ms pre-step period.

\emph{Procedure 3: Determination of light effect on membrane currents}

Cells were held at -60 mV in voltage-clamp mode. The stimulation pattern
was repeated three time at 10 s intervals for the three powers (1mW,
5mW, and 13mW). The discontinuous light patterns were followed, after 10
s, by 1 s of continuous light stimulation. Since 1 s stimulation was
expected to produce a consistent outward current (Ait Ouares \emph{et
al.} , 2019), the effect of continuous stimulation was used as positive
control. In the protocols for which the total illumination time (T.I)
during patterned stimulation was different than 1s, a continuous T.I.
light step was also applied. Fifteen electrophysiological traces were
recorded for each neuron, these were aliened on the pre-light control
period and a median trace was calculated. The light effect was
quantified on the median trace by subtracting, for each of the 50 light
steps, the average membrane current in the 1 s preceding the application
of light (the CTR period) from the average membrane current during light
period. Only one of the ten stimulation patterns was applied on each MC.
Procedures 2 and 3 were performed on the same electrophysiological
acquisition.

\emph{Procedure 4: Determination of the light effect on AP amplitude and
latency.}

Cells were recorded in current-clamp mode at their resting membrane
potential. In cases where spontaneous firing was present, a
hyperpolarization of the membrane potential was produced by injecting
negative current. Two consecutive trains of 50 APs, separated by 10 s,
were generated with 5 ms depolarizing steps applied at the frequency of
the light stimulation pattern chosen for that specific neuron. In half
of the recorded neurons, the first train was generated in the absence of
light and the second train generated during light stimulation, with the
depolarizing steps concomitant to the last 5 ms of the illumination. In
the other half of the neurons, light stimulation train preceded the no
light train. After 10 s, a single depolarizing step (test step) was
generated at the end of a 1 s continuous light stimulation. Since the
latter procedure is expected to produce a consistent modification of AP
amplitude and latency (Ait Ouares \emph{et al.} , 2019), the AP
generated by the test step was used as positive control. In the
protocols for which the total illumination time (T.I) during patterned
stimulation was different from 1 s, one AP was also generated at the end
of the continuous T.I. step. This protocol was repeated 10 times for
each neuron. For each of the ten acquired traces, the effect of light
was assessed trough a step-by-step comparisons of AP in the absence and
in the presence of light (i.e., 1st AP no LED vs 1st AP LED,
2\textsuperscript{th} AP no LED vs 2\textsuperscript{th} AP
LED\textsuperscript{\ldots{}\ldots{}.} 50\textsuperscript{th} AP no LED
vs 50\textsuperscript{th} AP LED). The AP of the test step and T.I. step
was compared to the 1st AP generated in the no LED condition. For each
AP, the latency was quantified as the time of the AP peak relative to
the beginning of the depolarizing step (Ait Ouares \emph{et al.} ,
2019). AP amplitude was calculated as the difference between AP peak and
the median membrane potential calculated in the 200 ms preceding the
first depolarizing step. Steps presenting AP failures, or AP for which
the time of AP peak exceeds the step duration, were excluded from the
analysis. In half of the recorded neurons, the steps of current in the
no-light condition preceded the steps of current in the light condition
and vice versa for the other half of the neurons. The effect of light on
each step was expressed as the average difference in AP amplitude and
latency, between the light and no light conditions for that specific
step. Only one out of the ten stimulation patterns was applied on each
MC.

\emph{Procedure 5: Determination of the light effect on average firing
frequency on MCs.}

The protocol was the same as that described in procedure 4 except that
the current steps for AP induction were replaced by a long-lasting
depolarization. The average firing frequency under light and no light
condition was calculated during a 2.5-s current-induced depolarization
(N=15 MCs).

\textbf{Statistics:}

Since the normality of the data was not known and normality tests have
low statistical power for a sample size of 24 (\selectlanguage{ngerman}Öztuna \emph{et al.} ,
2006), non-parametric one-sample Wilcoxon signed rank test and one way
Kruskal-Wallis test were used for the analysis. Type I error probability
was kept at 5\% by using Bonferroni-Holm method for correction of
multiple comparisons (stats.multitest.multipletests function from
Statsmodels module, Python, Holm method).

\textbf{Data analysis and statistics:}

For each of the ten patterns, the hypotheses tested were:

\emph{Hypothesis} \emph{1} : patterned light stimulation is responsible
for a decrease in AP amplitude.

\emph{Hypothesis 2} : patterned light stimulation is responsible for an
increase in AP latency.

\emph{Hypothesis 3} : patterned light stimulation is responsible for the
generation of an outward current.

\emph{Hypothesis 4:} patterned light stimulation is responsible for a
decrease in the average firing frequency.

The sample sizes required to have a statistical power \textgreater{} 90
\% were estimated based on the Cohen'd effect size observed in Ait
Ouares et al. (2019) (ES = mean of light effect /standard deviation of
light effect).

\emph{Hypothesis 1 was tested as follows:}

For each light pulse, the effect of light on AP amplitude was calculated
as the difference in AP amplitude between the LED condition and the no
LED condition (i.e., 1st AP amplitude LED -- 1st AP amplitude no LED;
\ldots{}\ldots{}.; 50\textsuperscript{th} AP amplitude LED --
50\textsuperscript{th} AP amplitude no LED). For each MC the median
effect was calculated. This resulted in the median light effect for each
of the 50 pulses (i.e., effect of light on the amplitude of the 1st AP;
\ldots{}\ldots{}..; effect of light on the amplitude of the
50\textsuperscript{th} AP). The values calculated for all recorded cells
were used for descriptive statistics by calculating the average
population effect (± 95\% CI) as well as the Cohen's d ES (average of
population effect /STD of population effect). Similar analyses were
performed for the positive control (test LED) and the total continuous
illumination exposure (T.I. LED), for which the effect was evaluated as:
LED AP amplitude - 1st AP amplitude no LED. In order to reduce the
probability of Type I error produced by multiple comparisons correction,
statistical tests were made only for three light pulses (the 1st,
25\textsuperscript{th}, and 50\textsuperscript{th}) by using a
unilateral one-sample Wilcoxon signed rank test (tested hypothesis,
median effect \textless{} 0). The \selectlanguage{greek}σ \selectlanguage{english}risk level was set at 0.05 and
Bonferroni-Holm correction was applied. Experiments were conducted on 24
neurons for each pattern of light stimulation and cell type (336 neurons
in total). This sample size was determined based on a previously
published experiment on MCs where continuous light stimulation reduced
AP amplitude with ES = -1.2 (Ait Ouares \emph{et al.} , 2019) and takes
into account the Bonferroni-Holm correction for 30 statistical
comparisons. As a consequence, the statistical power to observe an
effect as small as that produced by 1 s continuous light stimulation is
estimated between 98\% et 99\% depending on the level of correction
(calculated with G*Power 3.1.9.2, (Faul \emph{et al.} , 2007). The 24
MCs were recorded from 57 mice. The homogeneity of the effect produced
by the ``Test LED'' between the groups was assessed by one-way
Kruskal-Wallis test. Exploratory analysis was performed to evaluate a
possible difference between the average AP amplitude (over the 50
depolarizing steps) in the presence and in the absence of patterned
light stimulation.

\emph{Hypothesis 2 was tested as follows:}

The general procedure was the same as for testing the hypothesis one
except that AP latency was measured instead of AP amplitude

Exploratory analysis was performed to evaluate whether light stimulation
modifies other parameters of the AP such as AHP amplitude and action
potential full width at half maximum (FWHM).

\emph{Hypothesis 3 was tested as follows:}

Light effect on membrane current was calculated on the median trace of
the 15 recorded traces. For each of the 50 light pulses, the effect of
light on membrane current was calculated as the difference between the
average membrane current in the 1 s preceding the beginning of patterned
light (the CTR period) and the average membrane current during the light
period. For each of the three powers used, this resulted in the median
light effect for each of the 50 pulses (i.e., effect of the
1\textsuperscript{st} light pulse; \ldots{}\ldots{}..; effect of the
50\textsuperscript{th} light pulse). The values calculated for all
recorded neurons were used for descriptive statistics by calculating the
average population effect \selectlanguage{ngerman}± 95\% CI as well as the Cohen's d ES (average
of population effect /STD of population effect). Similar analyses were
performed for the positive control (Test LED) and the total continuous
illumination (T.I. LED), for which the effect was evaluated as the
difference between the average membrane current during light stimulation
and the average membrane current in the respective CTR period.
Statistical test was performed only for three light pulses (the
1\textsuperscript{st}, 25\textsuperscript{th}, and
50\textsuperscript{th}) by using a unilateral one-sample Wilcoxon signed
rank test (tested hypothesis, median effect \textgreater{}0). The \selectlanguage{greek}σ \selectlanguage{english}risk
was set at 0.05 and a Bonferroni-Holm correction was applied. The
experiment weas conducted on 24 neurons for each pattern of light
stimulation and cell type (240 neurons in total). This sample size was
determined based on a previously published experiment showing an effect
size (ES) of continuous light stimulation on MC membrane current equal
to 1 (Ait Ouares \emph{et al.} , 2019) and takes in account the
Bonferroni-Holm correction for 30 statistical comparisons. Therefore,
the statistical power to observe an effect as small as that produced by
1 s continuous light stimulation is between 91\% et 99\%, depending on
the level of correction (calculated with G*Power 3.1.9.2). The 24 MCs
were recorded from 51 different mice (i.e., different patterns were
applied on slices coming from the same animal). The homogeneity between
the groups produced by the ``Test LED'' was assessed by a one-way
Kruskal-Wallis test.

\emph{Hypothesis 4 was tested as follows:}

For each of the 10 repetitions on each recorded neuron, the effect of
light on AP frequency was calculated as the difference between the
firing frequency in the LED condition and the firing frequency in the no
LED condition. For each neuron, the median effect of light was
calculated. The values calculated for all recorded neurons were used for
descriptive statistics by calculating the average population effect \selectlanguage{ngerman}±
95\% CI as well as the Cohen's d ES (average of population effect /STD
of population effect). Statistical tests were performed using unilateral
one-sample Wilcoxon signed rank test (tested hypothesis, median effect
\textless{}0). The \selectlanguage{greek}σ \selectlanguage{english}risk level was set at 0.05. The experiment was
conducted on 15 neurons. This sample size was determined based on a
previously published experiment on MCs and MSNs, in which continuous
light stimulation reduced firing frequency with an ES=-1 at -1.6,
respectively (Ait Ouares \emph{et al.} , 2019). Therefore, the
statistical power to observe an effect as small as that produced by 1-s
continuous light stimulation was between 97\% and 99\% (calculated with
G*Power 3.1.9.2).

See supplementary table 1 for a summary of statistical analysis.

The results obtained from procedure 1 were used for exclusion criteria
based on Rm (see later) as well as for exploratory analysis aimed at
correlating the eventual effect of light stimulation with Rm.

Analyses were performed with custom scripts (Python 3.7).

\textbf{Data sharing.}

All raw electrophysiological traces, scripts for the analysis and raw
data are accessible via the Open Science Framework website
(https://osf.io/fe6t3/)

\textbf{Exclusion criteria.}

Cells were excluded from experiments when i) their resting membrane
potential (Vrest) was above -50 mV, ii) the membrane resistance was
higher than 1000 M, and iii) no AP could be elicited in the no light
condition. Traces were excluded from the analysis when: i) the starting
Vm (calculated in the 100 ms preceding the depolarizing current step) in
the no light condition differed by more than 5 mV from the starting Vm
in the light condition, ii) there was an instability in membrane
potential, and iii) a degradation of neuronal whole-cell recording
(depolarization of resting potential \textgreater{} 10 mV, AP overshot
below 0mV, or access resistance \textgreater{} 60 m 11Revised from
initial proposal of access resistance \textgreater{} 40 m, after
editorial approval on 13 Sept 2021. Analysis using 40 m criteria lead
to qualitatively similar results that are available at
https://osf.io/amvkq).

\textbf{Competing Interests:} the authors declare no competing financial
interests.

\textbf{Results}

3.1 Tissue temperature increases with light duty cycle and pulse
duration during patterned light stimulation (procedure 1)

We first evaluated the modification of tissue temperature when fifty
blue light pulses were delivered to OB slices following the stimulation
patterns described in table 1. The temperature of the tissue gradually
increased over the fifty pulses for all patterns (Figure 1, inset). The
higher the power, the duty cycle, and pulse duration, the more
pronounced the temperature increase (Figure 1). Interestingly,
temperature change did not appear to be correlated with the frequency of
the stimulation within each duty cycle. At 13mW, most of the patterns
induced a temperature increase that overcame 0.1 \selectlanguage{ngerman}°C, a thermal condition
that has been previously associated with modifications in neuronal
activity (Ait Ouares \emph{et al.} , 2019; Owen \emph{et al.} , 2019).

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 \emph{et al.} , 2019;
Owen\emph{et al.} , 2019).

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 \selectlanguage{greek}α \selectlanguage{english}risk (0.05) before the
correction (see \emph{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 \selectlanguage{ngerman}± 30
Hz (N= 15, p=0.013).

\textbf{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 \emph{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).

\textbf{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
Ouares\emph{et 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
\textasciitilde{}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 \emph{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 \emph{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 \emph{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 \emph{et al.} , 2015; Senova \emph{et al.} , 2017; Ait Ouares
\emph{et al.} , 2019; Owen \emph{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 \emph{et al.} , 2019). Our results suggest
that light is able to modulate different type of K\textsuperscript{+}
channels beside the inwardly rectifying potassium channels (Kir) (Owen
\emph{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 \emph{et al.} , 2019). This result suggests that light may
oppositely modulate the K\textsuperscript{+} channels involved in the
fast-medium and slow AHP. However, because the AHP in MCs is modulated
by recurrent glutamatergic transmission (Duménieu \emph{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 \emph{et al.} , 2022), the
light-induced decrease in AHP could also contribute to the reduction of
the firing frequency of these cells.

\textbf{Conclusions:}

Prevention of off-target effects during optogenetic stimulation is
fundamental for reducing misinterpretation of experimental results. The
use of discontinuous light at low power levels and/or duty cycle can
achieve this goal. However, such conditions could reduce the efficiency
of optogenetic protocols in neurons expressing first-generation opsins,
a limitation that can be overcome by using new-generation opsins such as
ChRmine or ChroME (Marshel \emph{et al.} , 2019; Sridharan \emph{et al.}
, 2022).

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