Peer review of: Rhythmic light flicker rescues hippocampal low gamma and protects ischemic neurons by (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This study aims to determine the main mechanism underlying the benefit of 40 Hz flickering light 
in promoting neuroprotection of CA1 neurons, synaptic plasticity and cognitive function following 
cerebral ischemia using the 2-vessel occlusion (2VO) mouse model of global cerebral ischemia in 
susceptible mouse strain C57BL/6 with low PCom patency. 

In brief, the investigators applied visual stimulation to mice with rhythmic LED light in 40Hz 
frequency, which entrained low gamma oscillation (30-50Hz) in the CA1. Light treatment was 
provided 2 sessions/day, one hour per session, commenced at 2 hrs after 2VO and lasted for up to 
14 days for the longest survival group. The initial observation was that it restored CA1 low gamma 
power in exploring mice with 2VO, reduced CA1 cell death (assessed by FJB staining), improved 
locomotion, reduced anxiety (open field test) and improved memory performance (water maze 
test) compared to the 2VO group without light stimulation. The effect on low gamma entrainment 
was specific, since normal light or random frequency LED didn’t have an effect in freely behaving 
mice. 40Hz LED light didn’t affect high gamma in exploration. For the neuroprotection study, they 
found light frequencies of 30 and 50 Hz were also effective, but not 10, 80 Hz or random 
frequency. If light treatment was delayed till 3 ds after 2VO, there was much more cell death. 
Microglia were reduced in the CA1 and DG of the light treated 2VO group, but GFAP remained 
unchanged compared to the non-treated 2VO group. 

In order to investigate the physiological mechanism of this light stimulation on the hippocampus, 
synaptic plasticity through fEPSP was determined in acute hippocampal slices. Both postsynaptic 
synaptic plasticity LTPNMDA and LTPL-VGCC was reduced by 2VO but restored by light treatment. 
Data for all synaptic experiment were presented from the 3-day time point (after 2VO) in the main 
figures. Parallel experiments provided evidence that 40-HzLED light also improved presynaptic 
short-term plasticity with data in presynaptic vesicles readily release probability (PPR) determined 
with paired-pulse ratio at 40 ms and the kinetics of presynaptic vesicles release with fluorescent 
tracer FM1-43 by multi-photon microscopy. They also provided additional structural evidence 
showing a reduction of spines in apical and basal dendrites in CA1, but not in CA3, by 2VO and 
restored by light treatment. 

Then proteomic analysis was performed on the whole hippocampus to search for candidate 
molecular targets. The authors decided to focus on RGS12, a G protein that regulates the N-type 
voltage gated calcium channel for neurotransmitter release, of which the level of expression was 
reduced by 2VO but restored by light treatment. By using a synthetic decoy peptide that blocks 
the interaction between RGS12 and N-VGCC via i.c.v delivery, the authors were able to 
demonstrate an abolishment of the effect of 40Hz light on presynaptic vesicles release and LTP. 
Similarly annulment was observed with knock down of RGS12 with an AAV siRNA vector. This is 
clearly the crescendo of the study. Both experiments were well controlled. The AAV knock down 
also reduced low gamma but not high gamma power in freely exploring mice. With the converging 
evidence, the authors claimed that the 40HzLED light improved synaptic plasticity and cognition 
via RGS12 by positively modulating N-VGCC. The enhanced CA3-CA1 synaptic strength then 
conferred neuroprotection. 

Recently there have been a series exciting studies demonstrating the power of neuromodulation in 
reducing Alzheimer pathology with either 40Hz light or sound, targeting brain low gamma 
oscillation. Thus, this study is very timely. Although some mechanims have been elucidated in 
earlier reports, the involvement of RGS12 acting on N-VGCC from this paper is clearly very novel. 
Besides high quality data centering on synaptic plasticity as the core, this study also employs 
multidisciplinary approaches, which is the main strength. The progression of presentation was 
especially logical. The authors have shown it for the first time that a noninvasive therapy with 
40hz LED stimulation could not only mitigate CA1 neuronal death but also improves synaptic 

plasticity and the recovery of cognition. Overall, this is a very well written manuscript with clear, 
strong and mostly consistent and well-presented data that support their claim. The following 
comments and suggestions are for the authors to consider. 

1. 2VO is essentially a model of global cerebral ischemia, which occurs as a result of cardiac arrest, 
not stroke. There is great distinction in pharmacology as well as pathophysiology between the two. 
In order to avoid confusion, I suggest using “cerebral ischemia” instead of “stroke” in describing 
your results. 
2. Global ischemia also induces cell death in other brain regions including the cortex and lateral 
striatum. Within the hippocampus, somatostatin (+) neurons are said to be more vulnerable than 
the CA1 neurons to global ischemic insult. It would be useful to characterize your model by 
showing areas of cell death and determine whether other regions also benefited from the 40Hz 
light therapy. If so, the mechanism of protection might extends beyond CA3-CA1 synaptic 
plasticity. 
3. Along the same vein, it would be interesting to determine whether light therapy improves blood 
flow in visual cortex and the hippocampus. More sensitive equipment like laser speckle imaging 
might be required to detect changes. If possible, [14C] iodoantipyrine for postmortem analysis 
would work too. Also, particularly in the 7d and 14d time points, vessel density in the 
hippocampus should be determined in the 2VO with and without 40Hz light therapy. 
4. Water maze acquisition should include pathlength data. 
5. It’s unclear why the proteomics was conducted in 7d time point while the majority of the data 
were based on the 3d time point. From the perspective of neuroprotection, the protective 
mechanism should be activated very early on in order to prevent the delayed neuronal death in the 
CA1. 
6. The authors once emphasized that the 2VO didn’t affect CA3, a needed feature for their 
experiment. However, in figure S6, it appeared the spine density in CA3 began to deteriorate after 
d3 in the 2VO group. Can the authors elaborate what effect this might have on the synaptic 
plasticity? 
7. Did blocking RGS12 also reduce neuroprotection in the CA1 in the light treated 2VO group? 
8. Figure S7, the authors should list the top 5 or 10 up- and down-regulated proteins. Simply 
describing the numbers is hard to get a sense about the extent of changes upon each treatment. 

Jialing Liu 

Reviewer #2 (Remarks to the Author): 

The major claims of this paper are that 40 Hz light stimulation induces gamma rhythms in the 
hippocampus, improves hippocampal-dependent memory, improves synaptic plasticity, and is 
neuroprotective in a mouse model of stroke. The paper includes a variety of different physiological, 
behavioral, and anatomical methods, but many of the tests were not done rigorously. The 
conclusions of the paper are not convincingly supported by the results presented. The hypotheses 
and rationale for performing many of the experiments are also often unclear. Specific comments 
are detailed below: 

Major Points: 
1. The authors do not convincingly demonstrate that the 40 Hz LED stimulation generates 40 Hz 
oscillations in the hippocampus. Specific points related to this general point are listed below. 
a) In Figure 1C, it appears as though 1/f noise differs between normal light and 40 Hz LED groups. 
Also, the 40 Hz LED stimulation is associated with a sharp peak at 40 Hz, which is not physiological 
and is instead likely to be a stimulation artifact. 
b) The recordings shown in Figure 1B are too low resolution to see anything. 
c) Why are there 2 peaks in Figure 1B (below)? Could these be theta harmonics? What was 
observed at lower frequencies? 
d) Hippocampal rhythms are affected by animals’ behavior (e.g., movement speed). There is no 

demonstration that behavior was the same across groups. For example, it is possible that the 
effects were observed because the light stimulation made the mice run faster. 
e) There are no peaks observed in Figure 1E, just broadband power. It is unclear what is actually 
being measured here when power is taken from the 30-50 Hz band. Were any differences 
observed at lower frequencies? 
f) The animals’ movement speed likely differed during novel object exploration, but the authors did 
not appear to control for differences in movement speed. 
2. Another major problem with the paper is that the authors conclude that 40 Hz stimulation 
produces the effects that they observe, but, at least for most of the figures, there were no controls 
performed using other types of sensory stimulation. So, it is unclear whether any type of sensory 
stimulation, not just 40 Hz light flickering, would produce similar effects as those reported in 
Figures 4-7. 
3. Does this light stimulation produce temperature differences that could affect recovery from 
stroke? One could imagine that those LEDs could produce some heat. 
4. There is no interaction effect reported to support the authors’ claim that slow gamma is the 
hippocampal rhythm that is selectively affected in 2VO mice. 
5. Figure 3B-C does not seem to control for potential differences in running speeds across groups. 
A similar point applies to Figure 3E. Could this again be due to differences in movement speed? 
6. Regarding statistical analyses, why was a repeated measures analysis not performed for the LTP 
measurements? 
7. The authors make statements that are not supported up by the results, even if the results were 
convincingly supported (e.g., “Enhancing CA1 low gamma power using 40 Hz light alleviated 2VO-
induced cognitive deficits through the strengthening of CA3-CA1 synapses”). 
8. The explanation for the different types of light stimulation is unclear. Were the different 
stimulation patterns (e.g., 10 Hz, 30 Hz, etc.) delivered in different mice? 
9. It is unclear exactly how the novel object exploration fit in. Were all mice subjected to this 
paradigm? Or only those mice implanted with electrodes? On which days exactly did this occur? 

Minor Points: 
1. The rationale for looking at NMDA-R-independent LTP is unclear, considering that the authors 
test memory in the Morris water, and this type of memory has been shown to require NMDA 
receptors (a Richard Morris Nature paper from the 1980’s). 
2. The rationale for the open field test was unclear. Why would the 2VO manipulation be expected 
to affect anxiety or behavior on this test? 

Reviewer #3 (Remarks to the Author): 

This is an interesting study which aims to determine if entrainment of hippocampal gamma could 
alleviate stroke-induced cognitive dysfunction. The authors present the case that post-stroke 
motor and cognitive dysfunctions are associated with the perturbation of specific brain oscillation 
frequencies and, importantly, such oscillations have the potential to be non-invasively and 
externally modulated. This is a comprehensive study which has the potential to provide novel and 
interesting data to the field. However, some methodological concerns need addressing; 
The authors have performed two-vessel occlusion of the common carotid arteries which produces a 
selective damage of the CA1 pyramidal neurones – the authors need to be clear what (disease) 
they are modelling – 2VO offers a global and transient ischemia that more clinically represents 
cardiac arrest rather than the regional focal ischemia seen following cerebral stroke which is 
achieved via other experimental methods. I think it is rather an over extrapolation to suggest that 
results obtained from this study may therefore have clinical relevance as a therapeutic for stroke 
prior to such work being conducted in a regionally focal model of stroke. 
The justification of the inclusion of sham animals is unclear given the statistical comparison is 2VO 
vs 2VO+LED. 

It’s unclear why only 5 animals were used to determine ischemia/reperfusion-induced changes in 
rCBF? Were the same 5 animals used to determine the changes in MABP? 
In vivo experiments should be reported according to the Arrive guidelines and therefore include 
details of; total animal used, inclusion/exclusion criteria, how animal numbers were determined, if 
any, and for what reasons, animals were excluded. In some instances (e.g. Morris water maze) n 
values (4) appear low – how do the authors know the appropriate number of animals were used? 
Presentation of results is a little confusing as does not match order of experimental detail provided 
in methods. For example, I would have expected reporting of rCBF and MABP data first. Also it 
would be useful for the authors to show confirmation of CA1-selective damage following the 2-VO. 
The authors need to show that the two x 2VO experimental groups received the same amount of 
damage (i.e. change in rCBF) and that the only difference between the groups was the presence, 
or absence, of LED.