Artifact-free, high-temporal-resolution in vivo opto-electrophysiology with microLED optoelectrodes

The combination of in vivo extracellular recording and genetic-engineering-assisted optical stimulation is a powerful tool for the study of neuronal circuits. Precise analysis of complex neural circuits requires high-density integration of multiple cellular-size light sources and recording electrodes. However, high-density integration inevitably introduces stimulation artifact. We present minimal-stimulation-artifact (miniSTAR) µLED optoelectrodes that enable effective elimination of stimulation artifact. A multi-metal-layer structure with a shielding layer effectively suppresses capacitive coupling of stimulation signals. A heavily-boron-doped silicon substrate silences the photovoltaic effect induced from LED illumination. With transient stimulation pulse shaping, we reduced stimulation artifact on miniSTAR µLED optoelectrodes to below 50 µVpp, much smaller than a typical spike detection threshold, at optical stimulation of > 50 mW mm-2 irradiance. We demonstrated high-temporal resolution (< 1 ms) opto-electrophysiology without any artifact-induced signal quality degradation during in vivo experiments. MiniSTAR µLED optoelectrodes will facilitate functional mapping of local circuits and discoveries in the brain.

Introduction A brain is made up of densely populated neurons. Analysis of neuronal communication requires simultaneous high-resolution recording and neuron-specific perturbation of circuit components under controlled conditions. The combination of genetic engineering-assisted optical stimulation and massively parallel electrical recording of neuronal activities (opto-electrophysiology) is a promising tool for studying neuronal circuits in behaving animals 1 . A number of devices [2][3][4][5][6][7][8][9][10][11] have been introduced for the past few years for in vivo opto-electrophysiology. For high-resolution in vivo opto-electrophysiology, a micromachined silicon multi-electrode-array structure also known as the Michigan Probe 12,13 has been widely utilized [6][7][8][9][10][11] . These silicon optoelectrodes take advantage of the planar profile of the Michigan Probe platform and accommodate multiple light sources in the vicinity of high-density recording electrode arrays. This compact configuration provides the capability to electrically record activity of sets of neurons at high spatial resolution while optically stimulating a portion of the recorded neurons.
An undesirable feature of many of these devices is stimulation artifact. With its magnitude often an order of magnitude larger than those of underlying neuronal signals, the stimulation artifact may mask neuronal signals and prevent temporally precise recording of neuronal responses 14,15 . In order to enable precise detection of neuronal activities, the magnitude of the stimulation artifact should be reduced to lower than a threshold voltage level for neuronal activity detection. Typically, the threshold level is set as a few integer multiples (often 5 ×) of the root-mean-square value of background noise 16,17 .
To keep the artifact magnitude lower than the threshold level, optical stimulation had been limited to slowly-changing, low-frequency pulses, such as slow (< 10 Hz) sine waves 11 or trapezoidal pulses with a long ( > 10 ms) rise time 18 . These slowly-changing optical stimulation protocols, however, are not suitable for many neuroscience experiments in which high-speed neuromodulation is required, such as those in closed-loop experimental setups 19 . An ideal optoelectrode should therefore provide optical stimulation with temporal resolution higher than the duration of the neuronal activities while keeping the stimulation artifact magnitude lower than a spike detection threshold.
Additionally, transient pulse shaping control reduces the magnitude of residual stimulation artifact on all recording channels to < 50 V peak-to-peak (Vpp) without compromising the temporal resolution of optical stimulation. With an in vivo experiment using a miniSTAR optoelectrode implanted in a mouse brain, we demonstrate the absence of distortion in the recorded neuronal signals during precise in situ optical stimulation.

Fabrication and characterization of miniSTAR LED optoelectrodes
We fabricated miniSTAR LED optoelectrodes (Fig. 1a) using microfabrication techniques adapted from those used for the fabrication of the family of Michigan optoelectrodes including onemetal-layer LED optoelectrodes 11 . Figure 1b describes the simplified device fabrication flow. MiniSTAR optoelectrodes were fabricated using gallium-nitride-on-silicon (GaN-on-Si), gallium nitride/indium gallium nitride multi-quantum-well (GaN/InGaN MQW) LED wafers with heavily boron-doped silicon (p + -Si, NA ≈ 1 × 10 20 cm -3 ) substrates. In order to reduce EMI-induced stimulation artifact, metal traces for LED drive signals (LED interconnects) and those for recorded neuronal signals (recording electrode interconnects) were placed in two different metal layers separated from each other by a groundconnected shielding layer (Fig. 1c, top), forming a multi-metal-layer structure. A heavily-boron-doped substrate was chosen to suppress diffusion of optically generated electron-hole pairs and, as a result, to reduce PV-induced stimulation artifact (Fig. 1c, bottom). First, LED mesa structures were formed on GaN/InGaN MQW layer, and the LED interconnects were defined on the first metal layer. After passivating the surface of the LEDs, the EMI shielding layer was defined on the second metal layer and the recording electrode interconnects were defined on the third metal layer. Neural signal recording electrodes were then formed by depositing electrode material (iridium) on top. Finally, the entire wafer was thinned down to 30 m and the miniSTAR optoelectrodes were released from the silicon wafer.
Released miniSTAR optoelectrodes were assembled on printed circuit boards (PCBs) that provide connections to a neuronal signal recording IC and an LED driver (Fig. 1d). Figure 1e shows a microphotograph of a tip of the fabricated miniSTAR optoelectrode. The dimensions of exposed surface area of each LED and recording electrodes are 10 × 15 m and 11 × 13 m (W × L), respectively.
After fabricating the miniSTAR optoelectrodes, we characterized performance of the LEDs and recording electrodes and confirmed that they are suitable for in vivo opto-electrophysiology. Optical power equivalent to greater than 1 mW mm -2 of irradiance is considered a threshold for activation of channelrhodopsin-2 (ChR2) 9,11 . LEDs on miniSTAR optoelectrodes generated a radiant flux of 150 nW, equivalent to an irradiance of 1 mW mm -2 at the surface when voltage of 2.86 ± 0.02 V (mean ± SD, n = 22) was applied across their terminals. The LEDs were capable of generating 50 mW mm -2 at the surface (7.5-W radiant flux) at 3.46 ± 0.10 V, which is more than sufficient for activation of ChR2-expressing cells further away from the LED surface. We confirmed that the effect of substrate doping density on the electrical and optical characteristics of the fabricated LEDs is not as significant as the die-to-die variation in a wafer (Supplementary Figure 1). The impedance magnitude and phase of the recording electrodes were measured as 1.15 ± 0.07 M and -68.33 ± 5.11 ° at 1 kHz (n = 54, mean ± SD), respectively, acceptable for high-quality in vivo extracellular recordings 30 .

Reduction of EMI-induced artifact
EMI is inevitable in a system where a source of high-voltage, fast-changing signal is located in close proximity to a signal-carrying trace connected to a high-impedance load. Previous LED optoelectrodes 30 contained only one metal layer on which all the interconnects that carry optical stimulation signals as well as those carrying recorded neural signals were densely integrated. Therefore, mutual capacitances between the traces of two signal types were high, and, in turn, the recording interconnects were highly susceptible to EMI from LED drive signals. Moreover, the n-GaN layer that forms the common cathode of all the LEDs on the optoelectrode was directly underneath the interconnects and acted as another significant source of EMI. We observed significant suppression of EMI-induced stimulation artifact with the integration of a shielding layer. We implemented the triple-metal-layer structure on LED optoelectrode and dedicated a layer between the stimulation and recording interconnects as a shielding layer (Supplementary Figure 2d). Triple-metal-layer (shielded) LED optoelectrodes were fabricated on the same GaN-on-Si LED wafer on which one-metal-layer LED optoelectrodes were fabricated, which had a lightly boron-doped silicon substrate (NA ≈ 5 × 10 16 cm -3 ). We compared stimulation artifacts between one-metal-layer LED optoelectrodes and shielded LED optoelectrodes while turning on and off LEDs in vitro. Figure 2 shows the magnitude of the transient artifact (peak-to-peak) and the wideband and highpass filtered waveforms of artifacts resulting from optical stimulation. One-metal-layer LED optoelectrodes showed a high transient magnitude (> 1 mVpp) in most recording sites regardless of the amount of optical power generated from the LEDs (Fig. 2b). The shape of wideband stimulation artifacts

Elimination of PV-induced artifact
Although EMI-induced artifact was greatly suppressed with introduction of the shielding layer, the magnitude of the residual artifact was still high and should be further eliminated below that of typical neuronal spikes (~ 100 Vpp). Interestingly, we noticed that the polarity of stimulation artifact at the rising and falling edges of LED drive pulses were inverted on the shielded LED optoelectrodes. As can be seen in Fig. 2c, the transient artifact on one-metal-layer LED optoelectrodes has the same positive polarity as that of the LED drive signal, forming an inverted-'v' (or '^') shaped waveform.
However, on the shileded LED optoelectrodes ( Fig. 2f and h), the polarity of the transient artifact was inverted, making a v-shaped waveform. Inversion of the polarity of the transient artifact suggested that the residual artifact could result from a different source other than EMI.
We hypothesized that the source of v-shaped stimulation artifacts is photovoltaic (PV) effects in the silicon substrate and confirmed our hypothesis with a few experiments. First, we observed the waveform of signals recorded on electrodes while exposing the LED optoelectrodes to external optical illumination. Using a focused beam at a wavelength similar to that of the light generated from LEDs (peak ≅ 470 nm), we illuminated tips of the shielded LED optoelectrodes. The shape of induced voltage signals was identical to that of the stimulation artifact observed on the optoelectrodes (Supplementary Figure 3). The identical shape suggested that the artifact is truly optically induced, not resulting from EMI. We repeated the experiment using electrode arrays fabricated on non-silicon substrates: GaN-onsapphire wafer and soda lime glass. We did not observed any v-shaped stimulation artifacts on electrodes on both substrates (Supplementary Figure 4), verifying that the artifact is due to neither photoelectrochemical (PEC) effects on the electrodes nor PV-induced artifact on the GaN layer. With the exclusion of PEC effects and PV effect from the GaN layer, the only remaining source of potential lightinduced artifact is the PV effects from the silicon substrate.
A few experimental studies in the past reported that light-induced noise on silicon electrode arrays can be reduced with use of heavily-doped substrate 31,32 . Heavy doping of semiconductor greatly reduces carrier lifetimes 33,34 and diffusion lengths of free carriers, which supposedly contributes to the amount of dipole-induced voltage 31 . Therefore, PV-induced stimulation artifacts should be suppressed with heavy doping of the silicon substrate. We conducted FEM simulations of optically induced voltage generation in silicon substrates and verified that the voltage is reduced with heavy substrate doping. We  Figure 3c shows the waveforms of stimulation artifact measured on the optoelectrodes of each group. It can be seen that even with high-intensity illumination (11.5 W, 75 mW mm -2 ), the mean magnitude of stimulation artifact was below 200 Vpp, suggesting that PV-induced stimulation artifact was effectively reduced by use of heavily-boron-doped silicon substrate.
We confirmed elimination of the PV-induced stimulation artifact by inspecting the shape and the magnitude of stimulation artifact waveforms recorded from electrodes at different locations on LED optoelectrodes (Fig. 4). Figure 4b shows the magnitude of stimulation artifact recorded from channels that correspond to the electrodes marked in Fig. 4a. The artifact waveform recorded from each channel is presented in Fig. 4c. It is interesting to note that, while the v-shaped waveform in stimulation artifact was observed in the recordings from optoelectrodes with FZ-Si and p --Si substrates, we no longer observed the v-shape in the optoelectrodes with p + -Si substrates. The absence of the characteristic v-shaped waveform confirms that PV-induced stimulation artifact has been eliminated on the optoelectrodes with p + silicon substrate.

Suppression of residual EMI-induced artifact by transient stimulation pulse shaping
Considering great reduction of both EMI-and PV-induced stimulation artifact, we refer to the shielded LED optoelectrodes fabricated using LED wafer with p + silicon substrate as minimalstimulation-artifact (miniSTAR) LED optoelectrodes. We quantified the amount of reduction in stimulation artifact from the implementation of shielding layers and the replacement of substrate with highly-boron doped silicon in miniSTARoptoelectrodes (Supplementary Figure 6). The magnitude of artifact was reduced by a factor of 5.2 in average only from the shielding layers (from 2477.75 ± 1733.83 to 474.59 ± 146.26 Vpp, at 11.5 W, mean ± SD), and by a factor of 17 in average from both shielding and substrate replacement combined (to 146.05 ± 143.40 Vpp, at 11.5 W, mean ± SD). However, the magnitude of stimulation artifact in a couple of recording sites (sites 1 and 2) was still high, as large as 200 -300 Vpp, while those on some other sites (sites 7 and 8) were less than 50 Vpp (Fig 4e).
Location dependence of residual stimulation artifact revealed that the residual artifact is due to EMI resulting from imperfection in the shielding layer. The shieling layer on miniSTAR optoelectrode contains openings (or optical windows) on top of LEDs for illumination. However, the optical windows allow the electric field generated from the LEDs to exit the shielding layer and makes the interconnects susceptible to EMI. Once the PV-induced artifact was removed in miniSTAR optoelectrodes, we observed emergence of ^-shaped waveforms (Fig. 4c), which is especially pronounced on sites 1 and 2. The magnitude of ^-shaped waveform is inversely proportional to the distance between the interconnect for each site and the optical window on the shielding layer ( Fig. 4c-e). The polarity and the distance dependence of stimulation artifact waveforms suggest that this residual artifact is due to EMI originating from the LEDs that are exposed through optical windows on the shielding layer.
Additional suppression of residual artifact was achieved by transient pulse shaping of LED drive signal. We modified the slew rate of voltage pulses by changing the rise and fall times of the pulses.
With sufficiently long rise time (trise > (2Fs) -1 ), the magnitude of higher-order harmonics of the coupled signal that contributes to the artifact ((πtrise) -1 < f < Fs/2) is reduced by additional -20 dB/decade (Supplementary Figure 7). Figure 5 shows the peak-to-peak magnitude and waveforms of stimulation artifact recorded from the channels corresponding to the bottom two electrodes (sites 1 and 2) on the tip of miniSTAR optoelectrodes, which show the worst residual EMI-induced artifact. We observed significant reduction in stimulation artifact as we increased the rise time longer than 100 s. At 50 mW mm -2 irradiance, the artifact magnitude was reduced below 200 Vpp (173.99 ± 55.76 Vpp, mean ± SD, for 1-ms rise time). In order to further reduce the slew rate of the voltage driving signal, we adjusted the low-level (or off-state) voltage in the stimulation pulse signals. We increased the low-level voltage to 2.8V, just below the lowest turn-on voltage of LEDs. The voltage required for irradiance of 50 mW mm -2 (radiant flux of 7.5 W) is approximately 3.5 V. By adjusting the low-level voltage from 0 V to 2.8 V, we reduced the voltage swing from 3.5 V to 0.7 V and the slew rate by a factor of 5. We confirmed that the artifact magnitude can be reduced to 111.92 Vpp (SD = 55.76 Vpp) even without adjusting the rise time (Fig. 5b, Vlow = 2.8 V, blue). With a 1-ms rise time and 2.8-V low-level voltage, the mean artifact magnitude was reduced to 46.53 Vpp (SD = 11.33 Vpp). In typical in vivo extracellular measurements, 100 Vpp is used as a spike detection threshold due to biological and environmental noise. Therefore, stimulation artifact with less than 50-Vpp magnitude can be considered nearly artifact-free.
Validation of stimulation-artifact-free in vivo opto-electrophysiology Following in vitro characterization, we demonstrated successful elimination of supra-threshold stimulation artifact in vivo. We implanted a miniSTAR LED optoelectrode in the brain of a mouse and positioned its tips in the CA1 region of the hippocampus (Fig. 6a). Once spontaneous spikes and the characteristic high-frequency oscillations (ripples) were detected from the recording electrodes on a shank, each LED on the shank was turned on with varying powers to identify the optimal intensity of optical stimulation to alter the spiking activity of neurons ('localized effect') without inducing highfrequency oscillations due to synchronized firing of neuron populations 11 Fig. 6d). As shown in Fig. 6d, the series of optical pulses did not generate noticeable stimulation artifacts that would prevent either online detection of spikes or their offline spike sorting.
With offline spike sorting 35 , we identified 6 putative pyramidal neurons with distinct spike waveforms in the vicinity of the shank on which the LEDs were activated. Further analysis of processed data identified a neuron which was clearly detected at the time stimulation offset of LED 1 and onset of LED 3 (Fig. 6f).
No noticeable distortion of the spike waveform was present due to optical stimulation (Fig. 6g).

Discussion
Results of this study the demonstrated the capability of high-spatiotemporal-resolution in vivo opto-electrophysiology with miniSTAR LED optoelectrodes. We validated that the implementation multi-metal-layer structure, high-density boron doping of silicon substrate, and transient pulse shaping of stimulation waveform can effectively suppress stimulation artifact. A few non-ideal features in the fabricated miniSTAR optoelectrodes prevented the magnitude of the stimulation artifact from being further reduced. One imperfection is existence of optical windows on the shielding layer, which allow EMI generated from LEDs to reach their neighboring recording electrode interconnects. The other nonideal factor is that the shielding layer has non-zero resistance. The shielding layer, especially near the tips of the shanks, is not strictly an ideal ground due to a resistive voltage drop through the thin-film metal layer it is made of. This resistive voltage drop would make the voltage of the shielding layer fluctuate as the voltage of LED interconnect changes, and the shielding layer itself could have acted as a source of EMI. These non-ideality resulted in less efficient reduction of EMI-induced artifact than that FEM electrostatic simulation predicted. Residual EMI artifact might be able to be further suppressed with a few additional techniques.
First, techniques to reduce the mutual capacitance between the recording electrode interconnect and the LED anode interconnect can be utilized. Increasing the distance between the recording electrode interconnects and the optical windows on the shielding layer (Fig. 4d) results in reduction of the mutual capacitance. Alternatively, a pair of ground-connected traces serving as shielding guards could have been placed between the optical windows and the recording electrode interconnects. However, these measures to reduce the mutual capacitance would inevitably increase the width of the shanks. Shank width is the main limiting factor for high-density scaling of the device which is required for larger-scale recording applications. Therefore, these options might not be considered optimal.
Methods to reduce electrode impedance might also be utilized for further reduction of EMIinduced artifact. The current carrying the capacitively coupled signal is divided between two branches in the signal recording circuit each of which is terminated with the amplifier load and the electrode (Supplementary Figure 8a). Therefore, lowering the electrode impedance would result in less current flowing through the amplifier load and thus reduction in the magnitude of the recorded voltage. In the data we collected using fabricated miniSTAR optoelectrodes, however, no obvious correlation between the electrode impedance and the magnitude of the stimulation artifact was observed (Supplementary With nearly-zero amplitude artifact, miniSTAR optoelectrodes can be readily utilized for applications that require real-time event detection and closed-loop perturbation of neural circuits 1,19 . Still, the tradeoff between the temporal precision of optical stimuli and the amount of reduction in stimulation artifact should be taken into consideration. Most opsins have slow kinetics and will not provide submillisecond-precision responses regardless of the precision of optical stimulus. Short-pulse optical stimulation protocols, similar to electrical protocols utilizing a train of sub-millisecond pulses 41 , might be found useful in combination with some fast-responding opsins 42,43 . Generation of such short (< 2 ms) optical pulses may result in discernable signatures (> 50 Vpp) in the recordings. These potential suprathreshold-amplitude artifacts, however, can easily be subtracted 44 from the recorded trace since they occur at predetermined times and display identical waveforms. Therefore, residual stimulation artifact would not significantly compromise the quality of recording.
In some applications, current-based LED driving might be more desirable than voltage-based driving. When driven with current pulses, the voltage changes across the two terminals of an LED would follow the I-V characteristics of the LEDs. Therefore, current driving allows setting the non-zero off-state voltage across an LED, typically just below the LED turn-on voltage (≈ 2.8 V). We validated that the effect of current-based driving of LEDs is similar to that with voltage-based driving of LEDs with non-zero lowlevel voltage (Supplementary Figure 9). We further tested pulses with three different rise-and fall-time shapes: trapezoidal, sinusoidal and sigmoidal. The shape of current pulses during on-to off-state transition did not significantly affect the magnitude of stimulation artifact. This result suggests that, if stimulation pulses have sufficiently low slew rate, smoothening of pulse edges does not necessary provide additional reduction in stimulation artifact magnitude.
Overall, our work demonstrates that stimulation artifact can be successfully suppressed using miniSTAR LED optoelectrodes. This new device will allow performing high temporal resolution in vivo opto-electrophysiology for in-depth understanding of the interactions among the multiple components of neuronal circuits.

Setup for in vitro characterization of LED-drive-induced artifacts
In vitro characterization was conducted in 1 × PBS solution in AMAC 530C-CRY container. An LED optoelectrode was lowered into the container until the bottom halves of the shanks were submerged in PBS. The exposed stainless steel tips at the loose ends of the ground and the reference wires were also submerged in the PBS.
An Intan RHD2000 neuronal signal recording system, in combination with an Intan RHD2132 Characterization of the effect of the boron doping of the silicon substrate on the magnitude of in vitro

LED-drive-induced artifact
Six shielded LED optoelectrodes fabricated using LED wafers with FZ, p -, and p + silicon substrate (two optoelectrodes from each wafer) were used. LED drive signals identical to those used for characterization of the effect of the shielding layer were used.
Characterization of the effect of the transient stimulation pulse shaping on the magnitude of in vitro LED-drive-induced artifact Two miniSTAR LED optoelectrodes (shielded LED optoelectrodes fabricated using LED wafers p + silicon substrate) were used. The low-level voltage and the rise time of the LED drive pulse signal were varied, while the high-level voltage was fixed as 3.5 V. Low-level voltages of 0 V and 2.8 V were used, and rise and fall times (10 -90 %) between 5 ns and 1 ms were used.

Recording of in vitro LED-drive-induced artifact and data processing
For each experimental condition for each LED, signals from the input channels of the neural signal amplifier IC were recorded for 30 seconds, so that artifact signals from longer than 100 pulses can be recorded. Average artifact signal was calculated by first highpass filtering the signal to remove lownoise fluctuations (with filters with 10 Hz and 250 Hz cutoff frequencies for wideband signal and highpass filtered signals, respectively) and calculating the average of the fifty 200-ms long segments in the middle of the 30 second period after the first 5 s of the recorded signal. Transient artifact magnitude was calculated from the difference between the maximum and the minimum values of high-pass filtered signal during the first 5-ms period from the point when the voltage changed from the off-level voltage.
The mean transient artifact magnitude was calculated by taking the mean of the values from electrode whose impedance magnitudes are between 500 k and 2 M and the phases are between -80 ° and -55 ° at 1 kHz. Two LED optoelectrodes from each cohort were used, and at least 21 electrodes per optoelectrode (out of 32 total, 25.83 in average) contributed to calculation of the mean artifact magnitude. The mean 1 kHz magnitude and phase of the electrode impedance of the electrodes which contributed to calculation of the mean artifact magnitude were 1.09 ± 0.09 M and -68.2 ± 4.9 ° (mean ± SD, measured at 1 kHz).

In vivo characterization and demonstrations of stimulation-artifact-free opto-electrophysiology
The animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Michigan (protocol number PRO-7275). One male C57BL/6J mouse (32 g) was used for in vivo characterization. The mouse was kept on a regular 12 h -12 h light -dark cycle and housed in pairs before surgery. No prior experimentation had been performed on this animal. Atropine (0.05 mg/kg, s.c.) was administered after isoflurane anesthesia induction to reduce saliva production.
The body temperature was monitored and kept constant at 36 -37 °C with a DC temperature controller On the day of recording, the mouse was anesthetized with isoflurane, the craniotomy was cleaned, and a shielded LED optoelectrode with p + silicon substrate was lowered to the CA1 region of the hippocampus. Baseline recording was performed (30 min), after which simultaneous recording and stimulation were done using three LEDs from one shank (as described in Results in more details). 0.46 W power, equivalent to 3 mW mm -2 irradiance at the surface of each LED, was used to characterize the light induced artifact in vivo and to alter the activity of neurons (more details are provided in Results). For characterization of stimulation artifact and confirmation of optical induction of neuronal activities, pulsed optical stimulation (100-ms long, 2 Hz, 100 pulses) was generated from each LED. The (10 -90 %) rise and the fall times of each voltage pulse were set as 1 ms. After collecting sufficient data using optical stimulation from each LED, a 500-ms long optical stimulation sequence involving switching on and off all the three LEDs on the shank (whose details are provided in Results) were repeated 100 times. RHD2000 recording system with RHD2132 miniature neural signal amplifier headstage was used for acquisition of data from all the recording electrodes (n = 32, 20 kS/s sampling rate). Keysight 33220A function generator provided voltage pulses for LED driving.
A custom MATLAB (MathWorks, USA) script was used to calculate average stimulation artifact.
Wideband traces were first high-pass filtered with a first-order filter with 250 Hz cutoff frequency to remove low-noise fluctuations. The average artifact signal from each recording channel was then obtained by averaging the middle 500-ms long segments (90 total segments out of 100).
The recorded data were then further analyzed for identification and clustering of action potentials. No manipulation in data (e.g. trimming of 1-ms long segments before and after the beginning and the ending of each pulsed optical stimulation) other than high-pass filtering (at 500 Hz) of the baseband signal was conducted. Spikes were first detected and automatically sorted using the Kilosort algorithm 35 and then manually curated using Phy to get well-isolated single units (multi-unit and noise clusters were discarded). To measure the effect of LED stimulation on neuronal activity, peristimulus time histograms (PSTHs) were built around stimulus onset (spike trains were binned into 10-ms bins).
Baseline and light-induced firing rate were calculated for each single unit, in which the baseline was defined as light-free epochs (400 ms) between trials and the stimulation period as the light-on (100 ms).
Wilcoxon-signed rank test was used to compare the mean firing rate per trial (n = 100 trials) during baseline and LED stimulation.