Non-invasive, opsin-free mid-infrared modulation activates cortical neurons and accelerates associative learning

Neurostimulant drugs or magnetic/electrical stimulation techniques can overcome attention deficits, but these drugs or techniques are weakly beneficial in boosting the learning capabilities of healthy subjects. Here, we report a stimulation technique, mid-infrared modulation (MIM), that delivers mid-infrared light energy through the opened skull or even non-invasively through a thinned intact skull and can activate brain neurons in vivo without introducing any exogeneous gene. Using c-Fos immunohistochemistry, in vivo single-cell electrophysiology and two-photon Ca2+ imaging in mice, we demonstrate that MIM significantly induces firing activities of neurons in the targeted cortical area. Moreover, mice that receive MIM targeting to the auditory cortex during an auditory associative learning task exhibit a faster learning speed (~50% faster) than control mice. Together, this non-invasive, opsin-free MIM technique is demonstrated with potential for modulating neuronal activity.

The authors show that optical pulsatile mid infrared stimuli (MIM) delivered directly on mice cortex or thought a thinned skull increase both c-fos expression in cortical cells and neuronal activity. They also provided evidences that MIM delivered during an associative training task accelerate the mice learning. The work is very interesting and present original results important for the neuroscience field interested on brain optical stimulation. However sometime it lacks of detailed methodological information and some of the statistical analysis are not yet sufficiently robust to support authors conclusions. Moreover, some assertions, such as that MIM effect is independent of the modification of tissue temperature, needed to be supported by further investigation.
Major concerns: c-fos experiments: 1-For the illumination duration of 20 s the authors calculate the proportion of cortical C-fos positve cells over the total cells (line 53), however they do not give any information about the method used to count the C-fos negative cells. Does the latter were labeled by florescence dyes or counting was done on brightfield images? 2--The authors assessed the relation between c-Fos activation and illumination time by quantifying the total number of c-Fos positives cells in the different illuminating conditions (figure 1d). Since these measurements could be potentially affected by the cell density it would be more correct to calculate the proportion of c-fos positive cell over the total number of cell as done for the condition 20s. Please mention the number of animals used in the different conditions.
3-Based on c-fos experiment the author conclude that "MIM application through opened skull could reliably induce neuronal activation"( lines 58-59). However, they did not provide evidences of the fact that positive cells are indeed neurons. c-fos can also be expressed in glial cells and astrocytes, as the same authors suggest when they consider that c-fos positives cell found in layer I are not neurons (lines 68-69) . A double labeling with a neuronal marker is required to determine the proportion of neurons among the c-fos labeled cells.
4-When discussing about the cellular mechanisms responsible of the effect produced by MIM the authors seems to exclude that it could be due to light induced increase of tissue temperature (lines 73-77). They justified their conclusion by citing two works (Owens et al 2019 and Ait Ouares et al 2019) on brain slices showing that light-induced increase of temperature reduces firing activity in all tested neuronal types. However, a precedent work had already shown an increase of firing activity of cortical neurons when stimulated by visible light in vivo condition, un effect that was proposed to be induced by temperature increase (Stujenske et al 2015;Cell Reports). Such apparent contradiction between the effect observed in vivo et in vitro has been explained by a stronger inhibitory effect of light on cortical interneurons than on pyramidal neurons (Owens et al 2019). Therefore, is still plausible that the effect observed in the present study is also due to temperature increase. To support the claim that MIM effect is temperature independent the authors should measure temperature in layer II/III before and during MIM stimulation, both using IR and visible light, and shown that: 1-temperature modification is below the limits reported to affect neuronal activity ( ex. Ait Ouares et al figure 2) or 2-Temperature modification produced by visible light is equal or higher than that produced by IR.
Loose patch experiments: 5-Please provide evidences that the current deflections on electrophysiological traces (fig 1j) are real spikes and not an electrical artefacts produced by the MIM procedure. Does this activity can be blocked by TTX ?
6-Statistical analysis shown in figure 1J should be performed considering all recorded neurons (9) and not by selecting only the five on which un apparent effect of MIM is observed. Alternatively, a neuron by neuron analysis could be performed taking in account the MIM effect over the different trials. A supplementary figure showing the raster plot of all the recorded neurons would be also informative.
7-Please give information on how was calculated the normalized firing rate. A more direct method to evaluate light effect on firing would be to compare the absolute firing frequency in the 3 conditions (20 s before MIM, during MIM and 20s after). In any case a paired statistical test should be used.
Behavioral experiment: 8-The p values of figure 3d and 3e need to be corrected by thanking in account multiple testing. Since three statistical comparisons were made for each of the 6 sessions (Ctr Vs MIM thinned, Ctr vs MIM opened and MIM opened Vs MIM thinned) by using a Bonferroni correction no significant effect are observed. In order to make the statistical analysis more informative (does the data support the absence of the MIM effect on success rate?) I suggest to performed Bayesian statistic (see Keysers et al Nature Neuroscience 2020). 9-Please detailed the method used to fit the learning curve.
Minor points: 10-Lines 20-22 "transcranial magnetic stimulation4 and transcranial direct-current stimulation5, have also been extensively practiced in healthy subjects with similar expectations" .Looking at the quoted references and the sentence that follow ( lines 22-24), I suppose that "healthy subjects" should be replaced with "patients".
11-Please give wavelength used for visual light stimulation 12-Could the author explain the reason to choose this quite atypical pattern of light stimulation ( 0.3 µs duration @ 100KHz)?
13-The fact that continuous visual light has also been reported to increase cortical firing activity (Stujenske et al 2015) should be mentioned.

Nicola Kuczewski
We would like to express our deepest appreciation to the reviewers for their constructive comments and suggestions on our manuscript entitled "'Non-invasive, opsin-free mid-infrared modulation activates cortical neurons and accelerates associative learning" (NCOMMS-20-35406-T). We completely agree with these comments and suggestions. In order to fully address their comments, we have performed extensive new experiments and new analysis that resulted in the following changes.
(1) We carried out a systematic measurement of tissue temperature, mapping the spatial and temporal profiles of cortical tissue temperature in vivo under the mid-infrared light (MIR) stimulation as well as those under the visible light (VIS) control stimulation. We present the new data in Fig. 1i and Extended Data Fig. 1.
(2) We performed a double labeling by using a neuronal marker NeuN together with c-Fos, and confirmed that the c-Fos positive cells emerged after MIR stimulation were dominantly neurons. We present a representative image in Fig. 1c and report the number in the main text.
(3) As requested by the reviewer we improved the data reporting format in Fig. 1 and relevant texts, including: using c-Fos positive cell proportions instead of simple counts, and using absolute spike firing rate instead of normalized firing rate.

(4) We performed additional loose-patch recordings and with tetrodotoxin (TTX) control experiments to confirm that the recorded spiking waveforms were neuronal firing signals but not optoelectrical artefacts. In addition, we performed the statistical analysis of all loose-patch recorded cells and also performed a neuron-by-neuron analysis over trials and the raster plot of all recorded cells.
We present the new data in Fig. 1l, m and Extended Data Fig. 2, 3, 4.
(5) We performed a new set of statistical analysis on the behavior data (Fig. 3) by using the Bayes Factor Hypothesis Testing as suggested by the reviewer. The results further confirmed our conclusion and are more informative, thus we also updated the main text accordingly.
(6) We included the reference papers as suggested by the reviewers.

Reviewer's comments
Reviewer #1 (Remarks to the Author): This paper uses a pulsed 5.6 µm laser to carry out optical stimulation in the mouse brain and in patchclamped neurons. We thank the reviewer for mentioning the thermal effect induced by the infrared light in the wavelength range of 1-3 µm and the possible mechanism involving TRPV4 channels for this effect.
However, our new experiment on measuring tissue temperature in vivo showed that the maximum temperature elevation was less than 2°C with the 5.6 µm light stimulation, which was much lower than the activation temperature measured in that reference paper (

µm is in the mid-infrared spectrum, as defined by the ISO20473 standard, and far from the near-infrared (NIR 0.78-3 µm wavelength) spectrum as previously used by others.
4 In addition to stating the average power of the laser (9mW) they should also state the peak power (300 mW) We thank the reviewer for mentioning this key parameter. Following the suggestion, we now explain the peak power in detail in the methods section, as well as the rationale of why such a configuration was used (also as requested by reviewer #2).

Reviewer #2 (Remarks to the Author):
The

authors show that optical pulsatile mid infrared stimuli (MIM) delivered directly on mice cortex or thought a thinned skull increase both c-fos expression in cortical cells and neuronal activity. They also
provided evidences that MIM delivered during an associative training task accelerate the mice learning.
The work is very interesting and present original results important for the neuroscience field interested on brain optical stimulation. However sometime it lacks of detailed methodological information and some of the statistical analysis are not yet sufficiently robust to support authors conclusions. Moreover, some assertions, such as that MIM effect is independent of the modification of tissue temperature, needed to be supported by further investigation.
We sincerely thank the reviewer for mentioning the importance of our study and for suggesting those important additional experiments. We completely agree with these suggestions and performed all the requested experiments, which greatly helped improving our manuscript.

1-For the illumination duration of 20 s the authors calculate the proportion of cortical C-fos positve cells over the total cells (line 53), however they do not give any information about the method used to count the C-fos negative cells. Does the latter were labeled by florescence dyes or counting was done on brightfield images?
We thank the reviewer's suggestion. The c-fos negative cells were counted by the nucleus labelling by DAPI. We now mentioned it in the revised manuscript.  We thank the reviewer's suggestions. We performed additional loose-patch experiments that local TTX application blocked all the recorded spikes. We also analyzed the waveforms of the recorded signals to confirm that they were real neuronal spikes. We now present the new data in Extended Data Fig. 2 and 3. figure 1J should be performed considering all recorded neurons (9)   Many thanks to this valuable suggestion. Following the suggestion, we applied the same Bayesian statistics as introduced in this important paper (Keysers et al, Nat Neurosci 2020) and obtained consistent results that more rigorously supported the original conclusion. The corresponding figure panels and texts have been updated with the new analysis results accordingly, and the main conclusion (that MIM application accelerates learning speed) still holds.

6-Statistical analysis shown in
9-Please detailed the method used to fit the learning curve.
Added in the Methods.

Minor points:
10-Lines 20-22 "transcranial magnetic stimulation4 and transcranial direct-current stimulation5, have also been extensively practiced in healthy subjects with similar expectations" .Looking at the quoted references and the sentence that follow ( lines 22-24), I suppose that "healthy subjects" should be replaced with "patients". Corrected.

11-Please give wavelength used for visual light stimulation
Provided.

12-Could the author explain the reason to choose this quite atypical pattern of light stimulation (0.3 µs duration @ 100KHz)?
We appended the detailed explanation in the methods section. In short, this MIR pulsing laser was initially designed for the purpose of infrared spectroscopy; when we used it for this study, all the parameters were configured to maximize the total average output power at the 5.6 μm wavelength.
13-The fact that continuous visual light has also been reported to increase cortical firing activity (Stujenske et al 2015) should be mentioned.
We thank the reviewer for mentioning this reference paper, which is now included.