Author Response

Reviewer #1 (Public Review):

This manuscript presents the first evidence for a plastic enhancement in the response of pial cortical arterioles to external stimulation. Specifically, they show (p8; Figure 3A-C) that repeated application of a visual stimulus at 0.25 Hz, at the upper edge of the vasomotor response, leads to a greater change in the diameter of pial arterioles at that frequency. This adds to the earlier, referenced work of Mateo et al (2017) that showed locking - or entrainment of pial arteriole vasomotion - by stimuli at different (0.0 to 0.3 Hz) frequencies.

We thank the reviewer for positively identifying the value of our manuscript.

The manuscript has a major flaw. Much as there is plasticity that leads to an increase in the amplitude of vasomotion at the drive frequency, the authors need to show reversibility. This could possibly be accomplished by driving the visual system at a different frequency, say 0.15 Hz, and observing if the 0.25 Hz response is then diminished. The authors could then test if their observation is repeatable by again driving at 0.25 Hz. Unless I missed the presentation on this point, there is no evidence for reversibility.

The reviewer has raised a very important point of view. In our experiments, the visually induced vasomotion (or visual stimulus-triggered vasomotion) was always entrained by repeated trials of the 0.25 Hz temporal frequency stimuli. When the visual stimulation stops, the vasomotion frequency lock to 0.25 Hz quickly dissipates. After saturated training with this stimulus, the parameters of the visual stimulus were switched, for example to 0.15 Hz. The animal quickly adapted to this new stimulus paradigm and the vasomotion was frequency-locked to 0.15 Hz. The adaptation to this new paradigm occurred well within 5 minutes. In Fig. 5, various paradigms were randomly tested. In some of the trials, 0.25 Hz stimulus was tested after 0.15 Hz. The vasomotion also quickly adapted back to the 0.25 Hz. We agree with the reviewer that this reversibility could have been explicitly documented in the manuscript.

Drew, P. J., A. Y. Shih, J. D. Driscoll, P. M. Knutsen, D. Davalos, P. Blinder, K. Akassoglou, P. S. Tsai, and D. Kleinfeld. 2010. 'Chronic optical access through a polished and reinforced thinned skull', Nature Methods, 7: 981-84.

Morii, S., A. C. Ngai, and H. R. Winn. 1986. 'Reactivity of rat pial arterioles and venules to adenosine and carbon dioxide: With detailed description of the closed cranial window technique in rats', Journal of Cerebral Blood Flow & Metabolism, 6: 34-41.

Reviewer #2 (Public Review):

Sasaki et al. investigated methods to entrain vasomotion in awake wild-type mice across multiple regions of the brain using a horizontally oscillating visual pattern which induces an optokinetic response (HOKR) eye movement. They found that spontaneous vasomotion could be detected in individual vessels of their wild-type mice through either a thinned cranial window or intact skull preparation using a widefield macro-zoom microscope. They showed that low-resolution autofluorescence signals coming from the brain parenchyma could be used to capture vasomotion activity using a macro-zoom microscope or optical fibre, as this signal correlates well with the intensity profile of fluorescently-labelled single vessels. They show that vasomotion can also be entrained across the cortical surface using an oscillating visual stimulus with a range of parameters (with varying temporal frequencies, amplitudes, or spatial cycles), and that the amplitude spectrum of the detected vasomotion frequency increases with repeated training sessions. The authors include some control experiments to rule out fluorescence fluctuations being due to artifacts of eye movement or screen luminance and attempt to demonstrate some functional benefit of vasomotion entraining as HOKR performance improves after repeat training. These data add in an interesting way to the current knowledge base on vasomotion, as the authors demonstrate the ability to entrain vasomotion across multiple brain areas and show some functional significance to vasomotion with regards to information processing as HOKR task performance correlates well with vascular oscillation amplitudes.

We thank the reviewer for summarizing the value of our study and recognizing its significance.

The aims of the paper are mostly well supported by the data, but some streamlining of the data presentation would improve overall clarity. The third aim to establish the functional significance of vasomotion in relation to plasticity in information processing could be better supported by the inclusion of some additional control experiments.

We thank the reviewer for recognizing our vast amount of data supporting our findings. We agree that better data presentation could have improved the clarity of the manuscript.

Specifically:

The clarity and comprehensibility of the paper could be significantly enhanced by incorporating additional details in both the introduction and discussion sections. In the introduction, a succinct definition of the frequency range of vasomotion should be provided, as well as a better description of the horizontal optokinetic response (i.e. as they have in the results section in the first paragraph below the 'Entrainment of vasomotion with visual stimuli presentation' sub-heading). The discussion would benefit from the inclusion of a clear summary of the results presented at the start, and the inclusion of stronger justification (i.e. more citations) with regards to the speculation about vasomotion and neuronal plasticity (e.g. paragraph 5 includes no citations).

We agree that a better description of vasomotion and horizontal optokinetic response could have been provided in the introduction. As the reviewer suggests, the discussion could also have started with the following summary of the results.

“We show that visually induced vasomotion can be frequency-locked to the visual stimulus and can be entrained with repeated trials. The initial drive for the vasomotion, or the sensory-evoked hyperemia, must be coming from the neuronal activity in the visual system. The vasomotion is likely triggered by activation of the neurovascular interaction (Kayser, 2004; van Veluw et al., 2020). Surprisingly, the entrained vasomotion was observed not only in the visual cortex but also widely throughout the surface of the brain and deep in the cerebellar flocculus. The global entrainment could be realized through separate mechanisms from the local neurovascular coupling. What is also unknown is where the plasticity occurs. The neuronal visual response in the primary visual cortex could potentially decrease with repeated visual stimulation presentation as the adaptive movement of the eye should decrease the retinal slip. With repeated training sessions, a more static projection of the presented image will likely be shown to the retina. The neurovascular coupling could be enhanced with increased responsiveness of the vascules and vascular-to-vascular coupling could also be potentiated.”

The novel methods for detecting vasomotion using low-resolution imaging techniques are discussed across the first four figures, but this gets a little bit confusing to follow as the authors jump back and forth between the different imaging and analysis techniques they have employed to capture vasomotion. The data presentation could be better streamlined - for instance by presenting only the methods most relevant for the functional dataset (in Figures 5-7), with the additional information regarding the various controls to establish the use of autofluorescence intensity imaging as a valid method for capturing vasomotion reduced to fewer figure panels, or moved to supplementary figures so as to not detract from the main novel findings contributed in this study.

We apologize for the confusing presentation of the data. Many of the initial figures were technical; however, we feel that following these steps was necessary to logically conclude that shadow imaging of the autofluorescence could be used as an indicator of vasomotion. We do agree with the reviewer that going back and forth between different techniques can be confusing. We could have added separate supplementary figures to introduce the various methods used upfront before going into the findings.

The authors heavily rely on representative traces from individual vessels to illustrate their findings, particularly evident in Figures 1-4. While these traces offer a valuable visualization, augmenting their approach by presenting individual data points across the entire dataset, encompassing all animals and vessels, would significantly enhance the robustness of their claims. For instance, in Figures 1 and 2, where average basal and dilated traces are depicted for a representative vessel, supplementing these with graphs showcasing peak values across all measured vessels would enable the authors to convey a more holistic representation of their data. Or in Figure 3, where the amplitude spectrum is presented for individual Texas red fluorescence intensity changes in V1 across novice, trained, and expert mice, incorporating a summary graph featuring the amplitude spectrum value at 0.25Hz for each individual trace (across animals/imaging sessions), followed by statistical analysis, would fortify the strength of their assertions. Moreover, providing explicit details on sample sizes for each individual figure panel (where not a representative trace), including the number of animals or vessels/imaging sessions, would contribute to transparency and aid readers in assessing the generalisability of the findings.

We agree with the reviewer that summarization of the data across a number of vessels/imaging sessions would lead to more generalization of the findings. However, contrary to what the reviewer described, we did summarize the vessel diameter expansion events across multiple vessel observations in Fig. 1F, G. The vasomotion parameters were not summarized for observation in intact skull shown in Fig. 2. However, this figure was intended just to show that vessel boundary cannot be well defined in intact skull imaging and Texas Red intensity or autofluorescence intensity fluctuation would give a better indication of vessel diameter fluctuation. In Fig. 3G, the peak ratio of 0.25 Hz was calculated for individual animals at Novice, Trained, and Expert levels and summarized for n = 5 animals. Statistical analysis was also done. The variability between imaging sessions within individual animals was not analyzed; thus, this could have been indicated.

In the experiments where mice are classed as "novice", "trained" or "expert", the inclusion of the specific range of the number of training sessions for each category would improve replicability.

We agree with the reviewer that classification on the level of training should have been explicitly indicated. Mice experiencing the first visual training session were defined as “Novice”. The mice that have experienced 3 training sessions are the “Trained” mice and the performance of the “Trained” mice during the 4th training session was evaluated. Mice that experienced 8 to 11 rounds of visual training sessions are the “Expert” mice.

The authors don't state whether mice were habituated to the imaging set-up prior to the first data collection, as head-fixation and restraint can be stress-inducing for animals, especially upon first exposure, which could impact their neurovascular coupling responses differentially in "novice" versus "trained" imaging sessions (e.g. see Han et al., 2020, DOI: https://doi.org/10.1523/JNEUROSCI.1553-20.2020). The stress associated with a tail vein injection prior to imaging could also partially explain why mice didn't learn very well if Texas Red was injected before the training session. If no habituation was conducted in these experiments, the study would benefit from the inclusion of some control experiments where "novice" responses were compared between habituated and non-habituated animals.

We agree with the reviewer that stress could well affect spontaneous vasomotion as well as visually induced vasomotion (or visual stimulus-triggered vasomotion). As the reviewer suggested, we could have compared the habituated and non-habituated mice to the initial visually induced vasomotion response. In addition, whether the experimentally induced increase in stress would interfere with the vasomotion or not could also be studied. With the Texas Red experiments, we observed that tail-vein injection stress appeared to interfere with the HOKR learning process. In the experiments presented in Fig. 3, Texas Red was injected before session 1. Vasomotion entrainment likely progressed with sessions 2 and 3 training. Before session 4, Texas Red was injected again to visualize the vasomotion. The vasomotion was clearly observed in session 4, indicating that the stress induced by tail-vein injection could not interfere with the generation of visually induced vasomotion.

The experiments regarding the brain-wide vasomotion entrainment across the cortical surface would benefit from some additional information about how brain regions were identified (e.g. particularly how V1 and V2 were distinguished given how close together they are).

The brain regions were identified by referring to the Mouse Brain Atlas. As the skull was intact, the location of bregma, lambda, and midline was clearly visible. We agree with the reviewer that strict separation of V1 and V2 could be difficult if we rely on the brain atlas alone. However, what we wanted to emphasize was that there was no specific localization of the vasomotion entrainment effect.

Whilst the authors show that HOKR task performance and vasomotion amplitude are increased with repeated training to provide some support to their aim of investigating the functional significance of vasomotion with regards to information processing plasticity, the inclusion of some additional control experiments would provide stronger evidence to address this aim. For instance, if vasomotion signalling is blocked or reduced (e.g. using optogenetics or in an AD mouse model where arteriole amyloid load restricts vasomotion capacity), does flocculus-dependent task performance (e.g. HOKR eye movements) still improve with repeated exposure to the external stimulus.

We agree that experimental intervention to vasomotion is ideal to test the functional significance of vasomotion. As pharmacological intervention lacks specificity, we are currently exploring the optogenetic approach. We have never thought of using the AD mouse as a model of restricted vasomotion by amyloid, and we agree this would be an interesting model to study. However, the AD mouse model would also have deficits other than the restricted vasomotion. On the other hand, we could test whether the repeated presentation of slowly oscillating visual stimuli can have beneficial effects in improving the cognitive abilities of AD model mice.

Reviewer #3 (Public Review):

Summary:

Here the authors show global synchronization of cerebral blood flow (CBF) induced by oscillating visual stimuli in the mouse brain. The study validates the use of endogenous autofluorescence to quantify the vessel "shadow" to assess the magnitude of frequency-locked cerebral blood flow changes. This approach enables straightforward estimation of artery diameter fluctuations in wild-type mice, employing either low magnification wide-field microscopy or deep-brain fibre photometry. For the visual stimuli, awake mice were exposed to vertically oscillating stripes at a low temporal frequency (0.25 Hz), resulting in oscillatory changes in artery diameter synchronized to the visual stimulation frequency. This phenomenon occurred not only in the primary visual cortex but also across a broad cortical and cerebellar surface. The induced CBF changes adapted to various stimulation parameters, and interestingly, repeated trials led to plastic entrainment. The authors control for different artefacts that may have confounded the measurements such as light contamination and eye movements but found no influence of these variables. The study also tested horizontally oscillating visual stimuli, which induce the horizontal optokinetic response (HOKR). The amplitude of eye movement, known to increase with repeated training sessions, showed a strong correlation with CBF entrainment magnitude in the cerebellar flocculus. The authors suggest that parallel plasticity in CBF and neuronal circuits is occurring. Overall, the study proposes that entrained "vasomotion" contributes to meeting the increased energy demand associated with coordinated neuronal activity and subsequent neuronal circuit reorganization.

We thank the reviewer for providing a thorough summarization of our manuscript.

Strengths:

The paper describes a simple and useful method for tracking vasomotion in awake mice through an intact skull.

The work controls for artefacts in their primary measurements.

There are some interesting observations, including the nearly brain-wide synchronization of cerebral blood flow oscillations to visual stimuli and that this process only occurs after mice are trained in a visual task.

This topic is interesting to many in the CBF, functional imaging, and dementia fields.

We thank the reviewer for positively recognizing the strength of the paper.

Weaknesses:

I have concerns with the main concepts put forward, regarding whether the authors are actually studying vasomotion as they state, as opposed to functional hyperemia which is sensory-induced changes in blood flow, which is what they are actually doing. I recommend several additional experiments/analyses for them to explore. This is mostly further characterizing their effect which will benefit the interpretations.

We recognized that the terminology used in our paper was not explicitly explained. Traditionally, “vasomotion” is defined as the dilation and constriction of the blood vessels that occurs spontaneously at low frequencies in the 0.1 Hz range without any apparent external stimuli. Sensory-induced changes in the blood flow are usually called “hyperemia”. However, in our paper, we used the term, vasomotion, literally, to indicate both forms of “vascular” “motion”. Therefore, the traditional vasomotion was called “spontaneous vasomotion” and the hyperemia induced with slow oscillating visual stimuli was called “visually induced vasomotion”.

Using our newly devised methods, we show the presence of “spontaneous vasomotion”. However, this spontaneous vasomotion was often fragmented and did not last long at a specific frequency. With visual stimuli that slowly oscillated at temporal frequencies close to the frequency of spontaneous vasomotion, oscillating hyperemia, or “visually induced vasomotion” was observed.

Neuronal calcium imaging would also benefit the study and improve the interpretations.

In our paper, we mainly studied the visually induced vasomotion (or visual stimulus-triggered vasomotion). Therefore, visual stimulation must first activate the neurons and, through neurovascular coupling, the initial drive for vasomotion is likely triggered. However, visually induced vasomotion is not observed in novice animals. Therefore, the visually induced vasomotion is not a simple sensory reaction of the vascular in response to neuronal activity in the primary visual cortex. We also do not know how the synchronized vasomotion can spread throughout the whole brain. Where the plasticity for vasomotion entrainment occurs is also unknown. To identify the extent of the neuronal contribution to the vasomotion triggering, whole brain synchronization, and vasomotion entrainment, simultaneous neuronal calcium imaging would be ideal. However, due to the fact that fluorescent Ca2+ indicators expressed in neurons would also be distorted by the “shadow” effect from the vasomotion, exquisite imaging techniques would be required.

The plastic effects in vasomotion synchronization that occur with training are interesting but they could use an additional control for stress. Is this really a plastic effect, or is it caused by progressively decreasing stress as trials and progress? I recommend a habituation control experiment.

As also pointed out by reviewer #2, we agree that, whether stress would affect visually induced vasomotion or not could be studied. Studying the visually induced vasomotion in mice well-habituated to the experimental apparatus would give an idea of whether stress could truly be a profounding factor affecting vasomotion. On the other hand, whether acutely induced stress can interfere with the already entrained vasomotion could also be studied. In the experiments presented in Fig. 3, Texas Red was injected via the tail vein, which would be quite stressful for the mouse. However, in the trained mouse, visually induced vasomotion could be observed regardless of the stress. It is likely that stress can interfere with the acquisition of vasomotion entrainment, but the already acquired entrainment will not be canceled with acute stress induced by tail-vein injection. We agree that further relationship between stress and vasomotion and plasticity related to vasomotion entrainment could be investigated.

Appraisal

I think the authors have an interesting effect that requires further characterization and controls. Their interpretations are likely sound and additional experiments will continue to support the main hypothesis. If brain-wide synchrony of blood flow can be trained and entrained by external stimuli, this may have interesting therapeutic potential to help clear out toxic proteins from the brain as seen in several neurodegenerative diseases.

We thank the reviewer for the positive evaluation of our manuscript. Strong entrainment of visually induced vasomotion was observed with a simple presentation of slowly oscillating visual stimuli for several days. This is a totally non-invasive method to train the vasomotion capacity. As the reviewer recognizes, potential benefits for the treatment of dementia and neurodegenerative diseases could be evaluated with further studies.

Figures and data

Spontaneous vasomotion detection

Autofluorescence signals could be used for vasomotion detection through the intact skull

Vasomotion entrainment with the oscillating visual stimulus

Autofluorescence signals could be used to quantify the vasomotion entrainment

Vasomotion entrainment over a wide range of visual stimulation parameters

Fluorescence fluctuations are not the results of eye movement artifact

A concomitant increase in the HOKR performance and vasomotion entrainment