Elife 93721 v1

RESEARCH ARTICLE
Plastic vasomotion entrainment

Daichi Sasaki, Ken Imai, Yoko Ikoma, Ko Matsui*
Super-network Brain Physiology, Graduate School of Life Sciences, Tohoku University,

Sendai, Japan
Abstract The presence of global synchronization of vasomotion induced by oscillating visual

stimuli was identified in the mouse brain. Endogenous autofluorescence was used and the vessel
‘shadow’ was quantified to evaluate the magnitude of the frequency-locked vasomotion. This
method allows vasomotion to be easily quantified in non-transgenic wild-type mice using either the
wide-field macro-zoom microscopy or the deep-brain fiber photometry methods. Vertical stripes
horizontally oscillating at a low temporal frequency (0.25 Hz) were presented to the awake mouse,
and oscillatory vasomotion locked to the temporal frequency of the visual stimulation was induced
not only in the primary visual cortex but across a wide surface area of the cortex and the cerebellum.
The visually induced vasomotion adapted to a wide range of stimulation parameters. Repeated trials
of the visual stimulus presentations resulted in the plastic entrainment of vasomotion. Horizontally
oscillating visual stimulus is known to induce horizontal optokinetic response (HOKR). The amplitude
of the eye movement is known to increase with repeated training sessions, and the flocculus region
of the cerebellum is known to be essential for this learning to occur. Here, we show a strong correla-
tion between the average HOKR performance gain and the vasomotion entrainment magnitude in
the cerebellar flocculus. Therefore, the plasticity of vasomotion and neuronal circuits appeared to
occur in parallel. Efficient energy delivery by the entrained vasomotion may contribute to meeting
*For correspondence: the energy demand for increased coordinated neuronal activity and the subsequent neuronal circuit
matsui@med.tohoku.ac.jp reorganization.
Competing interest: The authors
declare that no competing
interests exist. eLife assessment
Funding: See page 22 This article presents important results indicating a plastic enhancement in the vasomotion response
of pial cortical arterioles to external stimulation in awake mice using a wide range of external visual
Sent for Review
stimulation paradigms. The evidence for this interesting effect, with broad potential applications, is
20 November 2023
Preprint posted solid. These results are relevant for scientists and clinicians interested in the regulation of blood flow
21 November 2023 in the brain.
Reviewed preprint posted
07 February 2024
Reviewed preprint revised
27 March 2024 Introduction
Version of Record published Energy consumption in the brain is considered to be super-efficient compared to that of modern
17 April 2024 computers (Attwell and Laughlin, 2001; Howarth et al., 2012; Sokoloff, 1960). Revealing the prin-
ciple underlying the relationship between energy delivery, metabolism, and information processing
Reviewing Editor: Brice
Bathellier, CNRS, France would lead to our understanding of the unique feature of the biological brain that evolved to opti-
mally utilize the limited energy supply. The neuronal circuit requires an adequate energy supply for
‍ ‍Copyright Sasaki et al. This
the proper functioning of information encoding and inducing plastic changes. Energy (i.e., oxygen
article is distributed under the
and glucose) is delivered to the brain via the blood vessels. The on-demand vascular motion appears
terms of the Creative Commons
Attribution License, which
to allow localized energy delivery to specific brain regions or global regulation of the efficiency of
permits unrestricted use and energy delivery.
redistribution provided that the Vasodilation and vasoconstriction spontaneously occurring at seconds time scale (~0.1 Hz) have
original author and source are been reported (Davis et al., 2008; Fujii et al., 1990; Hundley et al., 1988; Jones, 1853). Such spon-
credited. taneous ‘vasomotion’ without any apparent external stimuli was first described in the vein of the bat
Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721 1 of 26

Research article Neuroscience
wing (Jones, 1853) and was subsequently observed mainly in the arteries and arterioles of numerous
species (Fujii et al., 1990; Hundley et al., 1988). In mice, the temporal pattern of such spontaneous
vasomotion is two orders of magnitude different from the frequency of heartbeat (~10 Hz; Ho et al.,
2011; Kramer et al., 1993). Therefore, vasomotion should be considered distinct from pulsation
due to heartbeat. The slow vasomotion is likely beneficial for the efficient delivery of blood in small
vessels as has been indicated by mathematical modeling (Meyer et al., 2002). Vasomotion has also
been suggested to facilitate the clearance of unwanted substances such as Aβ (Aldea et al., 2019;
van Veluw et al., 2020).
Although vasomotion can spontaneously occur on its own without the presentation of any apparent
external stimuli, it can also be affected by neurovascular coupling mechanisms (Rivadulla et al., 2011;
van Veluw et al., 2020; Villringer and Dirnagl, 1995). Neuronal firing triggers the release of vasodi-
lator signals from neurons and/or astrocytes (Zhu et al., 2022). In response to these signals, the blood
vessels dilate through the relaxation of the smooth muscle cells (SMCs) and the pericytes (Davis et al.,
2008; Peppiatt et al., 2006). Due to these signals, the frequency, amplitude, or phase of an already
ongoing spontaneous vasomotion can be affected. Whether the blood vessel is spontaneously oscil-
lating or static, it can react to the external presentation of sensory stimuli. The vasodilation response
to the neural activity caused by the sensory stimulation is termed ‘functional hyperemia’ (Nippert
et al., 2018; Stickland et al., 2019; Zhu et al., 2022). Functional hyperemia is triggered by the
neurovascular coupling mechanisms. The functional MRI signals largely reflect signals created by the
changes in the blood flow (Glover, 2011; Ogawa et al., 1990).
Here, both spontaneous vasomotion and functional hyperemia induced by horizontally oscillating
visual stimuli at temporal frequencies similar to the naturally occurring spontaneous vasomotion were
studied. In this article, the term ‘vasomotion’ is used to describe both forms of vascular motion. Spon-
taneous vasomotion and visually induced vasomotion were examined on the surface of the cerebral
cortex through intact crania using a macro-zoom microscope or studied with an optical fiber inserted
deep in the cerebellum using fiber photometry methods in vivo mice.
We show the global entrainment of the visually induced vasomotion throughout the surface of the
brain in mice with repeated visual stimulation. Functional hyperemia confined to a sensory-specific
region has been previously reported (van Veluw et al., 2020). Our unique observation was that
the visually induced vasomotion developed with repeated training and became perfectly frequency-
locked to the presented stimuli. Such plastic entrainment occurred globally throughout the whole
cortex, and it was not limited to the visual cortex.
The horizontally oscillating visual pattern that was used in this study is known to induce optokinetic
response (HOKR). Repeated exposure to such a pattern results in an increased amplitude of the HOKR
eye movement (Ito, 1982; Kanaya et al., 2023; Waespe and Henn, 1977). Neuronal circuit plasticity
required for the HOKR learning takes place in the flocculus region of the cerebellum. Visually induced
vasomotion dynamics at the flocculus, the deep brain region, were studied using the fiber photometry
method. A concomitant increase in the HOKR performance and the magnitude of the frequency-
locked vasomotion was observed. These results suggest the plastic change of vasomotion dynamics
may support and interact with the neuronal circuit plasticity.
Results
Presence of spontaneous vasomotion
Blood vessels in the brain can change their diameter through the actions of the SMCs and the peri-
cytes (Davis et al., 2008; Peppiatt et al., 2006). Here, we observed spontaneous vasomotion in vivo
in unanesthetized head-restrained awake mice. We sought to understand how vascular motion is
regulated by sensory stimuli, how visually induced vasomotion can be entrained, and the functional
significance of vasomotion plasticity in information processing in the brain.
Anesthesia or the creation of an invasive cranial window could potentially perturb naturally occur-
ring vasomotion. Therefore, we studied the presence of spontaneous vasomotion in the brain of
awake wild-type mice through the skull using a macro-zoom microscope. To visualize the blood
vessels, a fluorescence dye, TexasRed dextran, was administrated through the tail vein. Blood vessels
on the surface of the brain could be visualized through an intact skull when the skull is coated with
a transparent resin; however, the images were inevitably blurred. Therefore, in initial experiments,

A TexRed B
500 µm 20 µm
C Single line slice

D Basal Dilated
intensity (a.u.)
2 2 2 1.87(a.u.)
1.65(a.u.)
1.65(a.u.)
1.65(a.u.
.
TexRed
1 1 1
0 0 37.8 µm 0 41.7
1 7 µm
1.
41.7
50 µm
EVessel diameter F
TexRed intensity
Peak amp (%)
50
5%
TexRed intensity R2 = 0.845
0
0 10 20 30
Peak amp (%)
10% Vessel diameter
G 10 sec
1
relative freq.
Cumulative
0.5
0
0 30 60 0 10 20 30 0 3 6
interval (sec) Peak amp (%) Width (50%) (sec)
Figure 1. Spontaneous vasomotion detection. (A) A fluorescence image of a mouse brain through the thinned-
skull cranial window using a single-photon wide-field macro-zoom microscope. TexasRed was intravenously
injected and the blood vessels on the brain surface (between bregma and lambda) could be readily resolved.
(B) Magnified image of a single blood vessel. TexasRed fluorescence intensity profile was calculated at the cross-
section indicated by the blue line. (C) An example of a single-line intensity profile. Raw traces (gray bold line) were
filtered to derive the boundaries between the vessels and parenchyma. (D) TexasRed intensity profiles averaged
for multiple lines. The profile during the basal, undilated phase (solid line shown on the left, the same profile
shown as a dotted line on the right) and the dilated phase (solid line on the right, dotted line on the left). Peak
amplitudes of the intensity profiles (red cross) and full width at 10% maximum (horizontal lines). (E) An example of
a time-series data of the calculated vessel diameter and TexasRed intensity. (F) Correlation of the peak amplitude
of the calculated vessel diameter with the TexasRed peak intensity (n = 214 peaks). (G) Characteristics of the vessel
dilation events across multiple vessels were summarized. Inter-event interval (left), the peak amplitude of the vessel
diameter (middle), and the half-width of the duration of individual events (right) are shown for individual sessions
collected from multiple vessels (gray lines, n = 9 sessions from five vessels of three animals; duration of each
session was from 1.5 to 11 min). Averaged cumulative histograms are shown as blue lines.
the thinned-skull cranial window (Drew et al., 2010; Morii et al., 1986) was created (Figure 1A).
Spontaneous vasodilation/vasoconstriction oscillations were observed. To quantify the spontaneous
vasomotion dynamics, we attempted to measure the vessel diameter; however, even through the
thinned-skull cranial window, the exact boundary between the vessel and the brain parenchyma was
difficult to determine. Therefore, the TexasRed fluorescence intensity profile perpendicular to the
axis of blood flow in the vessel was measured, and the full width at 10% maximum of the TexasRed
fluorescence was taken as an indication of vessel diameter (Figure 1B and C; 45.7 ± 9.6 [S.D.] µm, n
= 5 vessels observations; see validation of the use of full width at 10% maximum for vessel diameter
estimation in the ‘Materials and methods’ section). The diameter of the selected vessels suggests that
mostly the pial arteries or the arterioles were observed for the stereomicroscope studies.
Spontaneous vessel diameter dynamics could be revealed; however, the quantification was
pixelated due to the resolution limit of the microscope (Figure 1E, top). Therefore, the peak TexasRed
fluorescence within the blood vessels was also quantified. The vessel diameter and the peak TexasRed

fluorescence correlated well; that is, a larger vessel diameter resulted in a higher peak fluorescence
(Figure 1F). Higher resolution of the vasomotion dynamics could be examined with the peak fluores-
cence measurement method (Figure 1E, bottom). Spontaneous vasodilation events were observed
with the near-instantaneous rise and an apparent exponential decay. The cumulative frequency of
the inter-event interval, the peak amplitude of the dilated vessel diameter, and the half-width of the
duration of individual events were summarized (Figure 1G). The vasodilation events occurred rela-
tively periodically (15.8 ± 4.8 [S.D.] s), were relatively fixed in amplitude (10.4 ± 3.7 [S.D.]%), and the
half-width of duration was also relatively constant (1.6 ± 0.4 [S.D.] s; n = 9 sessions from five vessels
observations).
It is possible that even the creation of the thinned skull may perturb the naturally occurring vaso-
motion as heat or vibration created by the drilling was reported to influence the surface of brain
tissue (Augustinaite and Kuhn, 2020). Thus, the blood vessels were observed through the intact skull
coated with a transparent resin. The blood vessels could be surprisingly well resolved with this method
without the use of a two-photon microscope; however, compared to the thin skull observation, the
boundary between the vessel and the brain parenchyma was more vague (Figure 2A–C). TexasRed
fluorescence intensity profile perpendicular to the vessel was often shallower than that observed
with the thinned skull; however, the full width at 10% maximum of the profile and the peak intensity
appeared to be correlated in the intact skull as well (Figure 2D). As the intensity profile was shallow
with intact skull imaging, the full width at 10% maximum of the profile appeared to be less reliable as
an indicator of vessel diameter and measurements of the TexasRed intensity fluctuations would better
represent the vessel volume changes stemming from vasomotion.
During these macro-zoom microscopy recordings, we noticed that an intense autofluorescence
from the brain tissue could be detected with ~430 nm excitation and ~540 nm emission. Most of
the autofluorescence appeared to be coming from the brain parenchyma. The inside of the blood
vessels was mostly devoid of this autofluorescence. Therefore, with the simultaneous TexasRed and
autofluorescence imaging, the blood vessels and the brain parenchyma could be separately visu-
alized (Figure 2E and F). Dynamic fluorescence intensity changes of the TexasRed and autofluo-
rescence were simultaneously observed (Figure 2G). The two fluorescence traces nearly completely
mirrored each other (Figure 2H and I). Therefore, the inverted autofluorescence dynamics would also
reflect the vasomotion dynamics. In later experiments, 400-µm-diameter optical fibers will be used
to measure the vasomotion dynamics in the deep brain areas. To simulate the situation using images
from the brain surface, a 500-µm-diameter circular region of interest (ROI) was drawn and the average
TexasRed fluorescence dynamics were plotted (Figure 2J and K). As apparent from the spectrogram
of the trace, several cycles of oscillatory fluctuations at low frequencies (0.1–0.5 Hz) were sponta-
neously observed. The frequency of the oscillatory fluctuation within a cluster was not strictly fixed
at a constant value. The occurrence of clusters was also not periodic, and the duration of a cluster
also varied. The presence of similar low-frequency vascular oscillations has been reported in previous
studies (Davis et al., 2008; Jones, 1853), but in those studies, the cycle of fluctuation appeared to
be constant and the periodic cycles continued without much interruption. It is possible that sponta-
neous vasomotion detected in our system through the intact skull in awake in vivo mice was less peri-
odic because spontaneous behaviors such as whisking, twitching, or struggling could cause functional
hyperemia resulting in the interruption of apparent periodic oscillations.
Entrainment of vasomotion with visual stimuli presentation

Horizontally oscillating visual stimuli are known to induce involuntary eye movement and the line of
sight follows the stimuli in head-fixed mice. This response to the stimuli is termed horizontal optoki-
netic response (HOKR). Repeated presentation of the stimuli also induces a gradual increase in the
amplitude of the eye movement and the small flocculus region of the cerebellum has been shown to
be responsible for this learning (Aziz et al., 2014; Ito, 1982). Whether visually induced vasomotion
(or functional hyperemia) occurs in response to this HOKR-inducing visual stimuli in the cortex was
examined. Immediately after TexasRed was tail vein injected, using the whole brain surface imaging
with the macro-zoom microscope, TexasRed fluorescence fluctuation in response to the HOKR stimuli
was studied (Figure 3A and B).
Horizontally oscillating vertical black and white stripes were presented to the head-fixed mouse
with a temporal frequency of 0.25 Hz and with a maximal visual angle (amplitude) of 17° for 15 min

A TexRed B
500 µm 20 µm
C Single line slice D Basal Dilated
Intensity (a.u.)
2.32(a.u.)
2 2 1.76(a.u.) 2
TexRed
1 1 1
0 0 43.4 µm 0 51.4 µm
50 µm
E G TexRed Intensity
Intensity
s
5%
AutoFluo Intensity
3%
20 µm 1 min
TexRed
F H I
AI (%)
5
TexRed
10% TI (%)
-5 10
inverted AutoFluo 10 sec
AutoFluo 5%
R2 = 0.859
20 µm AutoFluo 3%
-10
J K TexRe
Te
TexRed
Red iin
R n 500 µ
µmm diameter ROI
R I
RO
2%
Freqency (Hz)
0.3
0.5
200 µm 0 0.15
5 min
Figure 2. Autofluorescence signals could be used for vasomotion detection through the intact skull. (A) A
fluorescence image of a mouse brain through the intact skull (between bregma and lambda). Although with less
resolution, individual blood vessels containing TexasRed could be visualized. (B) Magnified image of a single blood
vessel. (C) A single-line intensity profile. (D) Averaged TexasRed intensity profile at the basal (left) and the dilated
(right) states. The intensity profiles were shallower compared to the profiles analyzed with thinned-skull images.
Therefore, rather than the vessel diameter estimated by the full width at 10% maximum of the intensity profile, the
TexasRed intensity would likely better reflect the volume changes of the blood vessels. (E) A merged image of the
TexasRed signal in the blood vessel (magenta) and the autofluorescence signal likely coming exclusively from the
brain parenchyma (green). (F) Spatial intensity profile along the cross-section of TexasRed and autofluorescence
signals in (E). (G) The temporal transitions of the TexasRed and the autofluorescence intensity signals at a region
of interest (ROI) encompassing the vessel are shown in (E). The two signals mostly mirrored each other, suggesting
that vessel dilation appears as the TexasRed intensity increase and the autofluorescence intensity decrease. (H) An
enlarged segment of the traces is shown in (G). The inverted autofluorescence trace (light green) resembles the
TexasRed intensity trace. (I) Autofluorescence intensity (AI) plotted against the TexasRed intensity (TI) for the
duration of the traces shown in (G) (n = 3440 data points). (J) An image of the mouse brain through the intact skull.
500 diameter µm circular ROI is shown (dotted yellow line). This diameter corresponds closely to the diameter
(400 µm) of the optical fiber used in fiber photometry experiments. (K) Spontaneous TexasRed fluctuation (top) was
detected through the intact skull within the ROI shown in (J). The spectrum below shows that the low temporal
frequency vasomotion does not continuously occur and short phases of vascular oscillation occur with frequency
not locked to a narrow specific range.
per session (Figure 3A). In response to the first visual training session, fixed frequency oscillation
of the TexasRed intensity at the primary visual cortex (V1) was not observed. TexasRed signals in
these ‘Novice’ mice, fluctuated with non-fixed low frequencies which consisted mainly of <0.3 Hz
(Figure 3C, left). After a couple of more training sessions and just before the fourth training session,

A B
V1
TexRed 1 mm
C Visual stimulus (0.25 Hz)
Novice Trained Expert 10 sec

TexRed @V1
1%
spectrum (%)
0.4 0.4 0.4

10 sec
Amplitude
0.2 0.2 0.2
0 0 0
0 0.5 1 0 0.5 1 0 0.5 1
Freqency (Hz) Freqency (Hz) Freqency (Hz)
D Expert
Pre Visual stim.
stim. Post Visual sti
stim.
m.
TexasRed
@V1 1%
1%
0.4
Freq.
(Hz)
0.5 0.5 0.5

0 0 0 0.1
1 min 1 min 5 min
E
0.4 Visual sti
stim.
m. 0.4 Visual stim.
stim. 0.4 Visual sti
stim.
m.
Amplitude spectrum
@0.25 Hz (%)
0.2 0.2 0.2
0 0 0
0 5 10 15 0 5 10 15 0 5 10 15
Time from Visual stim.
start (min)
F Novice Trained Expert GExpert scaled
7 10
S1
S1 V1
V1
2
V2
Cb
Cb
0 2
H 2 mm
I TexasRed @each-ROI
in whole brain surface-ROI
** V1
Peak ratio of 0.25 Hz
*
10
V2
5 S1
0 Cb
Novice Trained Expert 0.5%
4 sec
Figure 3. Vasomotion entrainment with the oscillating visual stimulus. (A) Fluorescence images were taken from
the whole brain surface through the intact skull using the macro-zoom microscope in awake mice. Horizontally
oscillating (at 0.25 Hz temporal frequency) vertical stripes are shown as the visual stimuli on the two perpendicular
monitors placed in front of the mouse. (B) Representative image of the near-whole brain surface after intravenous
injection of TexasRed. The location of region of interest (ROI) in V1 is shown with the yellow dotted line.
(C) TexasRed signals at V1-ROI during visual stimulation are shown. After repeated sessions, the vasomotion
gradually became entrained to the frequency of the visual stimulus. At the first training session (Novice, left), no
Figure 3 continued on next page

Figure 3 continued
specific peak was observed in the amplitude spectrum (bottom) of the TexasRed signals (middle). After several
trainings (Trained, at fourth training session, middle), a 0.25 Hz peak (arrowhead) in the amplitude spectrum
appeared. After saturated training (Expert, at 8th–11th training session, right), the 0.25 Hz oscillation of the
TexasRed signal was apparent in the raw traces (middle). (D) An example data from an Expert mouse. Immediately
after the onset of a visual stimulation session, the 0.25 Hz band appeared in the TexasRed trace (left). The 0.25 Hz
band dissipated immediately upon the cessation of the visual stimulation (middle). The 0.25 Hz band lasted
throughout the 15 minute session in this particular mouse, albeit the amplitude became smaller toward the end
of the session (right). (E) The amplitude values at 0.25 Hz in the spectrogram were plotted against time for three
different animals. The left graph is from the same Expert animal and the same session as shown in (D). The two
other graphs (middle, right) are from different sessions. The amplitudes at 0.25 Hz during the visual stimulation
were mostly higher than those before the stimulation (horizontal gray line); however, the amplitude did not stay
constant in most cases. The vasomotion appears to come in and out of the frequency lock to the visual stimulus.
(F) The peak amplitude at 0.25 Hz over the mean amplitude in the 0.1–1 Hz range (PR0.25) was calculated for each
pixel in the whole brain during the visual stimulation for Novice, Trained, and Expert mice. PR0.25 increase with
repeated sessions was not restricted to the visual cortex but a near-uniform increase was observed throughout
the whole brain surface. The color bar indicates PR0.25. (G) The color scale for the Expert example shown in (F) is
saturated. Therefore, a different color scale (right) was applied for the same data. (H) The averaged PR0.25 in
the whole brain surface ROI gradually increased as the training progressed (n = 5 animals). Novice vs. Trained,
p=0.039, *p<0.05; Novice vs. Expert, p=0.0030, **p<0.005; paired t-test with Holm correction for multiple test.
Data from individual animals are shown in gray, and the averaged data across all animals are shown in red. Data are
presented as mean ± SEM, as error bars. (I) TexasRed signals in ROIs at V1, V2, S1, and cerebellum (Cb). Signals in
different locations fluctuated with similar frequency with similar phases.
TexasRed was administered again. In these ‘Trained’ mice, the TexasRed signal in the cortex fluctuated
periodically. Fourier transform of the TexasRed signal at V1 during the 15 min training session revealed
a clear sharp peak in the amplitude spectrum at 0.25 Hz, which was not observed in the Novice mice
(Figure 3C, middle). This 0.25 Hz is the temporal frequency of the HOKR oscillatory visual stimuli.
We also noticed that, although the amplitudes were often much smaller, second and third harmonic
signals were sometimes observed. The second harmonic signal could be explained if visually induced
vasomotion reacts to both directions of oscillating stimuli. The origin of the third harmonics was unde-
termined. These harmonic signals were not always observed, and the magnitude of these signals was
variable compared to the robust frequency-locked signal.
With further repeated training sessions (8–11 training sessions), the TexasRed signal fluctuation
further becomes more strongly tuned to the frequency of the visual stimuli (‘Expert’, Figure 3C, right).
In this Expert mouse, the 0.25 Hz fluctuation in the TexasRed signal becomes visible even in the raw
traces. Using this Expert mouse, the time course of the vasomotion frequency locked to the visual
stimulus was examined (Figure 3D). Before the visual stimulus, the amplitude at 0.25 Hz was negli-
gible; however, as soon as the visual stimulus started, a sharp rise in 0.25 Hz frequency amplitude
increased. The amplitude gradually declined but stayed significant until the cessation of the visual
stimulus and the vasomotion amplitude locked to the visual stimulus frequency quickly dissipated
(Figure 3D and E, left panel). This characteristic of an instant onset and a steady increase was not
always shared in all animals. In many cases, the frequency locked to the 0.25 Hz came in and out
throughout the visual stimulation session; however, mostly throughout the session, the vasomotion
amplitude at 0.25 Hz frequency was above the baseline measured before the visual stimulation (exam-
ples in Figure 3E, middle and right panel).
Visual stimulus affecting vasomotion or hyperemia in the V1 was more or less expected; however,
does it affect other regions of the cortex? To examine this, data from the near-whole brain surface
observation with a macro-zoom microscope was studied. As the TexasRed fluorescence was low,
acquired images were Gaussian filtered in the spatial XY dimension to reduce the pixelated noise
at the expense of spatial resolution reduction without losing much of the temporal resolution. Then,
the TexasRed signal fluctuation during the 15 min session was examined for every pixel imaged. The
peak of the amplitude of the TexasRed signal at 0.25 Hz temporal frequency was calculated and was
divided with the mean amplitude at 0.1–1 Hz range (peak ratio of 0.25 Hz; PR0.25). This normalization
process was necessary because each region had a difference in the baseline fluctuation level of the
fluorescence signal. Color-coded map of the PR0.25 was mostly uniform throughout the cortex and
gradually increased from Novice, Trained, to Expert (Figure 3F). Averaged PR0.25 in the whole brain

surface ROI gradually increased as the training progressed (Figure 3H). The increase in the PR0.25
was so great in the Expert that the color map was mostly saturated in Figure 3F, right panel. There-
fore, the color code was scaled down in Figure 3G. The location of V1, secondary visual cortex (V2),
primary sensory cortex (S1), and the cerebellum (Cb) was roughly identified based on the coordinates
of the mouse brain atlas by referring to the bregma and lambda locations. PR0.25 in V1 and V2 was
high but not particularly high compared to S1 or Cb or, in fact, anywhere else that could be observed.
An example of the actual TexasRed signal fluctuation trace in various ROI is shown in Figure 3I. The
0.25 Hz signal fluctuations were apparent in all ROI and, in addition, the phase of the signal fluctuation
was also nearly synchronized (n = 5 animals; data not shown). These data show that frequency and
phase-locked vasomotion are not restricted to the visual-related area, and enhancement and entrain-
ment of vasomotion are observed widely throughout the surface of the brain.
The use of autofluorescence signal for examination of vasomotion

The small cerebellar flocculus region deep and away from the cranial surface is responsible for the
cerebellar-dependent HOKR motor learning. To examine the vasomotion in such a deep brain region,
optical fiber was implanted into the cerebellum (Figure 4C). The eye movement amplitude normally
increases in response to repeated sessions of HOKR visual stimulation training. However, we found
that, if TexasRed had to be injected before every training session, the mouse did not learn very well.
It is possible that the stress induced by the tail vein injection interfered with the learning process.
However, as demonstrated below, an already acquired HOKR learning with multiple training sessions
does not get lost with the stressful injection. An alternative method to measure the vessel volume
dynamics without the use of TexasRed fluorescence in the blood vessels was required to examine the
development of visually induced vasomotion during the training sessions. To accomplish this, the use
of fluorescence in the brain parenchyma was attempted.
As shown in Figure 4A, the blood vessels are devoid of the fluorescence from the YFP expressed
in astrocytes, and thus the blood vessels appear as dark regions in the images. Therefore, dilation
or constriction would appear as a decrease and increase in the fluorescence signal of the YFP. Such
concept of ‘shadow’ imaging of the dynamics of brain blood volume has been demonstrated using
the fiber photometry method previously (Ikoma et al., 2023a; Ikoma et al., 2023b). Here, we first
used a transgenic mouse, Mlc1-tTA::tetO-YCnano50, which expresses FRET-based CFP and YFP conju-
gated sensor protein specifically in astrocytes, including Bergmann glial cells (Kanemaru et al., 2014;
Tanaka et al., 2012). Instead of exciting the CFP with ~430 nm, YFP was directly excited by deliv-
ering ~505 nm light through the implanted optical fiber in the cerebellum, and the YFP fluorescence
(dYFP) was detected using the same optical fiber. The excitation filter used strictly restricted the
exposed light from exciting the CFP (see ‘Materials and methods’ for details). The dYFP fluorescence
would not be affected by the Ca2+ and would only be affected by the local brain blood volume and
possibly the cytosolic pH as YFP protein is sensitive to changes in the pH. It is known that YFP fluores-
cence can be quenched by acidification. Both TexasRed and dYFP signals simultaneously monitored
with fiber photometry fluctuated with the 0.25 Hz temporal frequency directly matching the visual
stimulus, but mirrored in the direction of change (Figure 4D). The TexasRed signal oscillations were
locked to the temporal frequency of the visual stimulus. This shows that, not only in the cortex but also
deep in the cerebellum, vasomotion can be induced with visual stimulation. Cross-correlation of the
TexasRed and dYFP signals shows a negative peak with ~0 s lag time (Δt; Figure 4E and F). The lag
time (Δt) between TexasRed and dYFP was calculated and the phase shift was determined by dividing
Δt by the presented visual stimulation oscillation cycle time (5 or 4 s, depending on the experiment),
which was then multiplied by 360° and was added 180°. The average phase shift of dYFP relative to
TexasRed was 184.4° ± 17.6° (S.D.) (Figure 4G; n = 13 segments with 5 min durations each), indi-
cating that these signals were nearly completely inverted. This shows that dYFP signals can be used
for assessing the vasomotion dynamics. The cytosolic pH may change also; however, even if there are
effects of the cytosolic pH fluctuations on the fluorescence amplitude, pH changes appear to be in
phase with the oscillatory vasomotion.
Unfortunately, robust HOKR learning could not be reliably induced in this line of transgenic mice for
unknown reasons. Therefore, C57BL/6NJcl wild-type mice were used for the experiments described
in the rest of the manuscript. As introduced in Figure 2E, autofluorescence excited with 430 nm light
could be detected in the wild-type mice. Using the images taken with the macro-zoom microscope

A TexRed dYFP
200 µm 200 µm
B C
Optical fiber
Flocculus
D TexRed
1%
1%
dYFP 5 sec
E TexRed F Corr. coef.

0.5 X-Corr. G
0.5% TexRed vs dYFP
0 184.4 deg
0.5%
-0.5 Δt = 0 sec
1 sec 0 180 360
dYFP -10
Lag time (sec)
0 10
Phase shift (deg)
H TexRed I J
0.4 TexRed vs AutoFluo
0.2% 246.9 deg
0
0.1%
1 sec -0.4 ec 0
Δt = 0.8 sec 180 360
AutoFluo -10 0 10 Phase shift (deg)
K L Novice Trained
Novice Trained TexRed
TexRed
spectrum (%) spectrum (%)
0.4 0.4
TexRed
Amplitude
0.2 0.2
0.3% 0 0
0 0.5 1 0 0.5 1
AutoFluo 0.2 AutoFluo
AutoFluo 0.2
Amplitude
0 2%
0.2% 0.1 0.1
1 sec 1 sec 0 0
0 0.5 1 0 0.5 1
M Freqency (Hz) Freqency (Hz)

PR0.25_AutoFluo
R2 = 0.769
Peak ratio of 0.25 Hz (PR0.25) = 10
Peak amplitude @ 0.25 Hz

Mean amplitude in 0.1 - 1.0 Hz range 5
0
0 10 20
PR0.25_TexasRed
Figure 4. Autofluorescence signals could be used to quantify the vasomotion entrainment. (A) Images of the
TexasRed fluorescence in the blood vessels and the direct YFP fluorescence expressed in astrocytes of a transgenic
mouse. The blood vessels appear as dark ‘shadows’ in the dYFP fluorescence image. (B) An optical fiber was
implanted in the cerebellum close to the flocculus and another optical fiber (highlighted in green) from the
fiber photometry apparatus was connected to this implanted fiber. Horizontally oscillating visual stimulation was
presented on the monitors. A hot mirror reflected the IR image of the eye and the eye location was monitored
using a camera. (C) Location of the optical fiber implantation relative to the cerebellar flocculus. (D) Simultaneously
recording of the TexasRed and the dYFP signals from the optical fiber implanted in the flocculus during the
horizontal optokinetic response (HOKR) visual stimulation session. The dYFP mirrored the TexasRed signal
fluctuations. Fluorescence signal data were filtered at 150–350 mHz. (E) 20 5 s segments of the TexasRed and
Figure 4 continued on next page

Figure 4 continued
the dYFP signal traces were extracted and averaged. The two traces nearly completely mirrored each other. The
temporal frequency of the visual stimulation presented to the transgenic mouse was 0.20 Hz. (F) Cross-correlation
between the two traces had a negative peak with a lag time of Δt of 0 s. (G) The phase shift between TexasRed and
dYFP signals was calculated for n = 13 segments with 5 min durations each. (H) In a separate set of experiments,
the TexasRed signal and the endogenous autofluorescence signal were simultaneously recorded in wild-type mice.
20 4 s segments were extracted and averaged. The traces were not completely inverted and a certain amount
of phase shift was observed. The temporal frequency of the visual stimulation presented to the wild-type mouse
was 0.25 Hz. (I) Cross-correlation of TexasRed and Autofluorescence signals. Δt of 0.8 s was observed in this
particular example. (J) Phase shifts in TexasRed and autofluorescence signals were calculated for n = 28 segments.
(K) Averaged data of 35 traces of the extracted 4 s segments of the TexasRed and the autofluorescence signals
during the first training (Novice) and the fourth training (Trained). Data was filtered with 150–350 mHz. An increase
in the amplitudes was observed from both signals. (L) Amplitude spectrums for TexasRed and autofluorescence
during the 5 min of the first training (Novice) and that of the fourth training (Trained). Signal data were high-
passed filtered at 50 mHz. (M) PR0.25 of the TexasRed signals and the autofluorescence signals in the 5 min
during the visual stimulation were plotted against each other. A clear correlation was observed indicating that the
autofluorescence signal can be used to estimate the magnitude of the vasomotion frequency-locked to the visual
stimulus.
and limiting the ROI in a small region surrounding the blood vessels, mirrored traces of the TexasRed
and autofluorescence were observed (Figure 2H). However, when the signals collected using the
400 µm diameter fiber photometry were analyzed, TexasRed fluorescence and autofluorescence did
not mirror completely and a significant shift in the phase was observed (Figure 4H–J; 246.9° ± 49.9°
[S.D.], n = 28 segments). The endogenous substance in the brain parenchyma that produces autoflu-
orescence was not identified but it could be flavin or other related substances (Croce and Bottiroli,
2015; Huang et al., 2002; Islam et al., 2013). Production and degradation of flavin and other metab-
olites may be induced by the fluctuation in the blood vessel diameter with a fixed delay time. The
phase shift in the autofluorescence could be due to the additive effect of ‘shadow’ imaging of the
vessel and to the concentration fluctuation of the autofluorescent metabolite.
The TexasRed fluorescence and the autofluorescence signals are shown for the initial sessions
(Novice, Figure 4K) and after several training sessions (Trained). The amplitude of the 0.25 Hz temporal
frequency component of both the TexasRed and the autofluorescence signals increased with training
(Figure 4K and L). PR0.25 of autofluorescence was plotted against that of the TexasRed for multiple
training sessions and a strong correlation between these two parameters was found (Figure 4M).
Therefore, although the autofluorescence dynamics result from the fluctuations reflecting the ‘shadow’
of vasomotion and an unknown fluctuation source, the PR0.25 apparently reflects the amplitude of the
vasomotion locked to the visual stimulation cycle. In the remainder of the article, the autofluorescence
fluctuations were mainly examined to understand the dynamics of the visually induced vasomotion.
Vasomotion followed the visual stimulation dynamics over a wide

parameter range
Optimal visual stimulation properties for inducing frequency-locked vasomotion were searched. The
visual stimulation used in all other sections of this study was the following. The temporal frequency
of horizontal oscillation was 0.25 Hz; the spatial amplitude of the horizontal oscillation was 17° in
the visual angle of the mouse; the horizontal spatial cycle of the vertical stripes was 6.4° in the visual
angle. First, the temporal frequency was changed from 0.25 Hz to 0.15 Hz or 0.50 Hz while keeping
other parameters fixed. Interestingly, the autofluorescence fluctuations followed the changes in the
temporal frequency, and a clear sharp amplitude at the frequency locked to the presented visual
stimulation was evident in the amplitude spectrum in all conditions (Figure 5A). The peak ratio of the
autofluorescence was examined for temporal frequency ranging from 0.15 to 0.50 Hz and normalized
to that of 0.25 Hz (PR0.25; Figure 5D, left panel). No significant difference was found, showing that
vasomotion can lock to any temporal frequency within the range examined. It should be noted that
the mice were first trained with 0.25 Hz temporal frequency stimuli, and subsequently tested with
multiple temporal frequency stimuli in pseudo-random order and the original 0.25 Hz was tested
again sometime during the sequence. The visually induced vasomotion quickly adapted to any new
frequency tested and the reversibility of the adaptation was confirmed.

A Temporal freq (Amplitude : 17 deg, Spatial cycle : 6.4 deg)

0.15 Hz 0.25 Hz 0.5 Hz
Visua Stim.
Visual Sti
10 deg
0.5%
AutoFluo 5 sec
0.25 Hz
0.15 0.15 Hz
0.15 0.15 0.15
spectrum (%)
Amplitude
0.50
0.50 Hz
0.1 0.1 0.1
0.05 0.05 0.05
0 0 0
0 0.5 1 0 0.5 1 0 0.5 1
Freqency (Hz) Freqency (Hz) Freqency (Hz)
B Amplitude (Temporal freq : 0.25 Hz, Spatial cycle : 6.4 deg)
4 deg 25 deg 0.25 5 Hz
4 deg
AS (%)
Visual Stim. 0.1
0.25 Hz 10 deg
0
0 0.5 1
AutoFlu
l o
AutoFluo
AS (%)
0.1 25 deg
0.5% 0
5 sec 0 0.5 1
Freqency (Hz)
C Spatial cycle (Temporal freq : 0.25 Hz, Amplitude : 17 deg)
3 deg 18 deg 0.255 Hz
3 deg
AS (%)
0.1
0
Visual Stim. 0 0.5 1
AS (%)
0.1 18 deg
0.25
25 Hz
0.25 Hz
Auto
utoF
utoFluo
l o
lu
AutoFluo 10 deg
0
0 0.5 1
0.5% Freqency (Hz)
D 5 sec
Relative Peak Ratio
1.5 1.5 1.5

(norm. to original)
1 1 1
0.5 0.5 0.5
0 0 0
0.2 0.3 0.4 0.5 4 8.5 13 17 21 25 3 6.4 9 12 18
0.15 0.25
Temporal freq (Hz) Amplitude (deg) Spatial cycle (deg)
Figure 5. Vasomotion entrainment over a wide range of visual stimulation parameters. (A–C) Autofluorescence
signals at the flocculus in response to visual stimulation with various parameters. In the original condition, the
temporal freq (the temporal frequency of horizontal oscillation) was 0.25 Hz, the amplitude (the spatial amplitude
of the horizontal oscillation) was 17° in the visual angle of the mouse, and the spatial cycle (the horizontal spatial
cycle of the vertical stripes) was 6.4°. All the fluorescence signal data were filtered with 50–1000 mHz. (A) The
temporal frequency of visual oscillation was varied. The location of the peaks in the autofluorescence signal
amplitude spectrogram exactly matched the frequency of the visual stimulus in almost all cases (arrowhead).
(B) The amplitude of the visual oscillation was varied. (C) The spatial cycle of the stripe patterns was varied. (D) The
relative peak ratio of the autofluorescence signal at the temporal frequency matching that of the presented visual
stimulus was calculated. This relative peak ratio was normalized to that of the original condition. Varying the
temporal freq, the amplitude, or the spatial cycle almost had no effect on the relative peak ratio. This suggests that
vasomotion can tune in to any visual stimulation within the parameter range that we tested. Significant differences
were shown only when the spatial cycle was varied (spatial cycle, 6.4° [original] vs. 9°, p=0.0028, *p<0.0125; 6.4°
[original] vs. 18°, p=0.015, *p<0.017; one-sample t-test with Holm correction for multiple tests). Individual mice’s
data (as an average from the three sessions of the 5 min test) are shown in pale green, and the average data from
the examined mice (n = 5 mice) are shown in dark green.
Similarly, the effect of changing the spatial amplitude of the horizontal oscillations was examined,
while keeping other parameters fixed. A large amplitude would require the mouse to move its eyes
largely within their eye sockets. The temporal frequency-locked vasomotion was apparent in the low
4° amplitude as well as in the high 25° amplitude (Figure 5B). The relative peak ratio normalized
to that of 17° amplitude showed that the vasomotion was substantially induced in response to any

amplitude between 4° and 25° (Figure 5D, middle panel). Lastly, the horizontal spatial cycle of the
vertical stripes was varied (Figure 5C). Again, the vasomotion could follow the visual stimuli irrespec-
tive of the fineness of the presented stripes (Figure 5D, right panel). The combination of parameters
of the visual stimuli used in the rest of the article was well within the adequate range that would
induce maximal visually induced vasomotion.
Vasomotion is not the consequence of eye movements

We have shown above that the bulk dynamics of vasomotion under the optical fiber could be quan-
tified with autofluorescence intensity fluctuations using fiber photometry. HOKR visual stimulation
induces both eye movement and frequency-locked vasomotion. Here, we examined whether the eye
movement itself is the cause of vasomotion.
First, the right eye trace in response to the horizontally oscillating visual stimulus was examined
(Figure 6A). The eye tracked the visual stimulation well and there was virtually no phase shift in the
eye trace relative to the visual stimulation cycle (Figure 6B). The TexasRed signal fluctuation in the
flocculus in the right hemisphere was oscillatory with the visual stimulation; however, the phase of
the TexasRed signal was either 0° or 180° relative to the visual stimulation, depending on the animal
(Figure 6C). This indicates that in the 0° phase-shifted animals, the maximum vasodilation and vaso-
constriction occur when the right eye is at the maximal nasal and temporal locations, respectively.
Conversely, in the 180° phase-shift animals, it is the other way around. In either mouse, the maximum
vasodilation and vasoconstriction occur when the speed of the eye movement reaches zero. The
reason why the phase of the vasomotion becomes fixed to 0° or 180°, depending on the animal, is
unresolved.
As the vasomotion apparently followed the visual stimuli unbelievably well, we suspected several
artifacts affecting the fluorescence recording. First, we suspected that the visual stimulation itself is
penetrating the back of the eye, through the brain, and to the flocculus of the cerebellum. Thus, the
photo intensity detected with the optical fiber could be coming directly from the oscillatory visual
stimulus. To address this possibility, the mouse was anesthetized by isoflurane (Figure 6D). Under
anesthesia, the eye fails to follow the screen. The vasomotion was detected with the ‘shadow’ imaging
method by studying the dYFP fluctuations. No oscillatory signals were detected in the dYFP. Next, in
the TexasRed injected mouse, the HOKR visual stimulation was presented, which induced a nice eye
movement following the visual stimulus (Figure 6E). However, in this case, we did not send the exci-
tation light through the optical fiber but kept the PMT active. If the visual stimulus on the presented
screen is somehow reaching the optical fiber, then it should be detected in this condition, but it was
not.
Finally, we investigated the possibility that the fluorescence signal fluctuation is due to the motion
artifact. The physical eye movement could produce a movement in the brain tissue and the optical
fiber location relative to the blood vessels could be moved, leading to the fluctuation of the fluores-
cence signals. When the mouse is presented with visual stimulation, in the trained mouse, the eye
usually follows the visual stimulus completely. However, even during a single session, the frequency-
locked TexasRed signal was not always apparent. In an example shown in Figure 6F, the eye trace
was completely in sync with the visual stimulation either in the left panel segment or in the right.
However, TexasRed signal fluctuation with an amplitude spectrogram of 0.25 Hz was not apparent in
the left panel segment but was significant in the right panel segment. This observation argues against
the possibility that the detected fluorescence fluctuation is the direct result of eye movement. The
amplitude of the oscillatory eye movement was plotted against the PR0.25 of the TexasRed during a
single visual session (Figure 6G). The cycle of visual stimulation producing either a large or small eye
movement did not always result in a large or small amplitude fluctuation of the vasomotion, respec-
tively, and no correlation was found.
Possibility of the contribution of vasomotion entrainment to the HOKR

performance
Repeated HOKR training usually results in a larger amplitude of the eye oscillations, which reaches
close to the actual visual presentation amplitude. We have shown above that the frequency-locked
vasomotion in the cortex also got entrained with repeated training sessions (Figure 3). It has been
shown that HOKR performance increase relies on the neuronal and glial activity in the cerebellar

A Nasal B Eye trace phase

Visual Stim. Temporal Visual Stim.
Anim.
#1
10 deg
Eye trace #2
#3
#4
#5
3 deg
-90 0 90 180 270 360
TexRed
TexRe
Redd
C TexRed phase (deg)
Visual Stim.
1%
AutoFluo
AutoFluo
F Anim.
#1
#2
#3
0.2% #4
4 sec #5
D F -90 0 90 180 270 360

(deg)
Visual Stim.
Under isoflurane
Visua Stim.
Visual S m.
Sti m Eye trace 10 deg
3 deg 2.0
Freq. [Hz]
10 deg 0.5 0.25 Hz 0.25 Hz
Eye trace
0 0
0.1
dYFP
1 deg TexRed
1% 0.25
Freq. [Hz]
2% 0.5 0.5
0.25 Hz
5 sec
0 0
E 10 sec 0.1
G 8
PR0.25_TexRed
No excitation
6
Visual Stim.
17 deg 4 R2 = 0.120
Eye trace
4 deg 2
PMT detection 0.002 V 0
0V 0 3 4 5
TexRed 5 sec Eye degree [deg]
Figure 6. Fluorescence fluctuations are not the results of eye movement artifact. (A) An example of the visual
stimulation time course, the eye trace, the TexasRed signal, and the autofluorescence signal fluctuations are
shown. The data was filtered with 50–500 mHz. (B) The phase of the eye trace relative to the visual stimulus. The
visual stimulus was presented with a 4 s cycle (0.25 Hz), thus, the eye trace data was segmented with 4 s durations
during the first 5 min of each training. The segmented data were aligned, averaged, and fitted with a sine curve.
The phase difference between the visual stimulus and the fit to the averaged eye trace was calculated. The mean
of the phase in each animal across multiple sessions is shown as a vertical black bar. The average phase lag was 2.3
± 1.0° in five animals, indicating that the eye trace synchronize nearly completely with the visual stimulus. (C) The
phase of the TexasRed signal fluctuations relative to the visual stimulus. The average TexasRed signal fluctuation
with a 4 s cycle was calculated from segments extracted from several sessions, each with 1–6 min in duration. The
phase lag of the TexasRed signal relative to the visual stimulus was clustered either at 0° or 180°. This shows that
the local brain blood volume in the right flocculus becomes the largest when the right eye is either in the most
nasal position or the temporal position. (D) Under anesthesia with isoflurane, the eye movement was not induced.
The dYFP signal from the YCnano50 expressed in astrocytes also did not oscillate. (E) With no excitation light sent
to the optical fiber, the PMT detected no signal (0 V). The presented visual stimulus did not reach the fiber optics
to directly induce oscillatory signals. (F) The amplitude of the eye movement was normally nearly constant during
a single session; however, slightly strong (left) and weak (right) eye movements were observed. The TexasRed
signal fluctuation at the frequency locked to the visual stimulus was not constant during the session and PR0.25
of the TexasRed signal could be seen in some (right) but not the other (left) segments of the session. (G) PR0.25
of TexasRed signal was plotted against the oscillatory eye movement amplitude in 40 s segments within a single
visual stimulation session (15 min). The first segment was excluded (n = 21 segments total). No correlation was
found between these two factors.
flocculus (Kanaya et al., 2023). We next evaluated whether the plasticity of the vasomotion dynamics
in the flocculus correlates with the HOKR performance increase.
HOKR-spaced training paradigm was used. Briefly, 15 min training sessions were repeated at 1 hr
intervals four times on day 1 (i.e., the training day; Figure 7A). Averaged amplitudes of the eye move-
ment during the first and last 3 min of each training session were calculated. The calculated averaged

A Day2 Day5
1st Train 2nd 3rd 4th
1 hour 2 min 2 min

15 min
B 1st Train 4th Train Day 2

Visual Stim.
10 deg
Eye trace
5 deg
AutoFluo
0.5%
10 sec
C 1st Train 4th Train Day 2 Day 5
Eye trace 5 deg
0.3%
AutoFluo 1 sec
D E
Relative Gain @Day 5 PR0.25_AutoFluo

8
Relative Gain
1.5
6
HOKR
4
2
1.0
0
1st 2nd 3rd 4th Day2 Day5 1st 2nd 3rd 4th Day2 Day5
F G
1.5
Relative Gain
1.5
(norm. to 4th-ave.)
HOKR
R2 = 0.801
R2 = 0.556
1.0
1.0
0 0
0 2 4 6 8 0 4 8 12 16
PR0.25_AutoFluo PR0.25_AutoFluo @4th-fin
Figure 7. A concomitant increase in the horizontal optokinetic response (HOKR) performance and vasomotion
entrainment. (A) Schematic schedule of the HOKR spaced training paradigm. The 15 min training sessions were
repeated at 1 hr intervals for four times on day 1 (the training day). The average amplitudes of the eye movement
during the first and last 3 min of each training session were analyzed (during the period indicated by the blue box
in the schematics). The 2 min tests measuring long-term memory were done on days 2 and 5. (B) Representative
traces of the visual stimulus, the eye trace, and the autofluorescence during the first and the fourth training on
day 1 and the test done on day 2. The amplitude of the eye movement and the vasomotion gradually increased
in amplitude with repeated sessions. (C) The eye traces and the autofluorescence traces were segmented in
4 s durations, corresponding to the visual stimulation frequency of 0.25 Hz, and aligned and averaged. (D) The
amplitude of the eye movement relative to that of the start of the first training session on day 1 was refined as the
HOKR relative gain. Average HOKR relative gain across n = 15 animals is plotted (mean ± SEM). (E) PR0.25 of the
autofluorescence signals was averaged across n = 15 animals and plotted against sessions. (F) Averaged HOKR
relative gain was plotted against the averaged PR0.25 of autofluorescence signals. Error bars indicated S.D. (n =
15 animals). (G) HOKR relative gain on day 5 normalized to that on the fourth training on day 1 was plotted against
the PR0.25 of autofluorescence signals at the end of the fourth training on day 1 (n = 15 animals).
eye amplitudes were normalized to the initial amplitude measured at the first 3 min of the first training
session. This value was termed the ‘relative gain’. A brief 2 min test was done on day 2 and 5 to eval-
uate the long-term changes in the performance. Along with the HOKR eye movement measurements,
autofluorescence signals were measured with the optical fiber implanted close to the flocculus. The
amplitude of the autofluorescence signal at a temporal frequency locked to the visual stimulation
frequency (0.25 Hz) was analyzed. As above, this amplitude was divided by the mean amplitude in the
0.1–1 Hz range to derive the PR0.25. As the autofluorescence fluctuations were often noisy, the auto-
fluorescence signals during the first and last 5 min of each training session were used for the PR0.25
calculation. The long-term memory tests done on days 2 and 5 were only 2 min in duration, thus, the
full 2 min were used for the PR0.25 calculations.

As described previously, HOKR performance increased with repeated training sessions during day
1 (Figure 7B-D). With the current experimental conditions, the average HOKR performance tested on
days 2 and 5 was nearly the same as the performance on the last session of day 1; however, long-term
changes in the performance were sometimes apparent in individual cases (Figure 7C). The PR0.25 of
the autofluorescence signal changes with the spaced training protocol basically followed the plastic
changes of the eye movement (Figure 7E). PR0.25 tended to increase with repeated training sessions.
The HOKR relative gain was calculated for each test (i.e., 10 tests total; the early and the late time
tests of each of the four sessions on day 1, and tests on days 2 and 5), and the HOKR relative gain was
averaged across all animals examined (n = 15 mice). Similarly, the PR0.25 in autofluorescence fluctua-
tions were averaged across animals in 10 tests. The average HOKR relative gains were plotted against
the average PR0.25 values and a strong correlation was found (Figure 7F; each data point represents
an average score of n = 15 mice and 10 data points are shown for the 10 tests). In addition, it should
be pointed out that a large variance was found both in the HOKR relative gain and the PR0.25 of the
autofluorescence fluctuations across animals (X and Y error bars indicate S.D. in Figure 7F). Therefore,
in individual animal recordings, the strong correlation between the HOKR relative gain and the PR0.25
of the autofluorescence fluctuations may not always be apparent. It is possible that the plasticity
governing HOKR and vasomotion increases could be induced by a parallel process.
Correlations between HOKR and vasomotion across individual animals were searched. HOKR
relative gain on day 5 normalized to that for the fourth training on day 1 was plotted against the
PR0.25 of the autofluorescence fluctuations during the last 5 min of the fourth training session on
day 1 (Figure 7G). Interestingly, a moderate but significant correlation between these parameters
was found (n = 15 mice). This may suggest that the level of visually induced vasomotion contributes
to the consolidation of HOKR performance in later days. These results imply a complex and intimate
relationship between vasomotion and neuronal circuit plasticity, learning, and memory.
Discussion
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 visually induced vasomotion is
likely triggered by activation of the neurovascular interaction (Kayser et al., 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 entrain-
ment 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. Repeated visual stimulus may induce synaptic
plasticity not in the visual system but at the neuronal projections from the retina to a central nucleus
that controls a brain-wide vasomotion, such as in the autonomic nerve system (Korte et al., 2023).
Instead, the neurovascular coupling could be enhanced with increased responsiveness of the vascules
and vascular-to-vascular coupling could also be potentiated.
We show that the fluorescence signal from the brain parenchyma follows the vasomotion because
the blood vessels largely lack fluorescence signals within the filter band that we observe. This is
described as ‘shadow imaging’. What was rather puzzling was that autofluorescence signals were
phase-shifted in time. This suggests that these autofluorescence signals have an anti-phase ‘shadow
imaging’ component and another component that is phase-shifted in time. Glucose and oxygen are
likely to be abundantly delivered during the vasodilation phase compared to the vasoconstriction
phase of vasomotion. These molecules will trigger cell metabolism and endogenous fluorescent
molecules such as NADH, NADPH, and FAD may increase or decrease with a certain delay. These
molecules are required for biochemical reactions to occur. Therefore, the concentration fluctuation
of these metabolites could lag in time to the changes in the blood flow. It is also expected that these
metabolites may fluctuate according to the neuronal activity that triggers visually induced vasomotion
or functional hyperemia.
The stress in head restraint mice could well affect spontaneous vasomotion as well as visually
induced vasomotion. Whether the experimentally induced increase in stress would interfere with the

vasomotion or not could also be studied. With the TexasRed experiments, we observed that tail-vein
injection stress appeared to interfere with the HOKR learning process. In the experiments presented
in Figure 3, TexasRed was injected before session 1. Vasomotion entrainment likely progressed with
session 2 and 3 training. Before session 4, TexasRed was injected again to visualize the vasomotion.
The oscillatory 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.
It is not possible to distinguish whether the visually induced oscillatory vasomotion observed in
this study was driven by the mechanisms of spontaneous vasomotion or functional hyperemia. Both of
them may be involved in the visually induced vasomotion. The spontaneous vasomotion requires the
coordinated activity of the vascular SMCs. Oscillatory release of calcium ions from the sarcoplasmic
reticulum results in oscillations in the membrane potential of SMCs. Via the gap junctions between
SMCs, synchronized oscillations are created (Haddock et al., 2006; Haddock and Hill, 2002; Hill
et al., 1999; Peng et al., 2001; Schuster et al., 2001). Blood flow in the central nervous system was
assumed to be regulated primarily by the SMCs in precapillary arterioles; however, capillaries lacking
smooth muscle have been shown to be controlled by pericytes (Peppiatt et al., 2006). The sponta-
neous vasomotion observed in our study could be the result of constriction and dilation of arterioles or
capillaries controlled by SMCs or pericytes, respectively. Astrocytes also constitute the neurovascular
unit; however, whether astrocytic calcium is involved in controlling local cerebral blood flow is in ques-
tion (Ozawa et al., 2023). It is still possible that astrocytes could regulate vasomotor amplitudes via
stretch-mediated feedback (Bazargani and Attwell, 2016; Haidey et al., 2021). Spontaneous vaso-
motion has been suggested to be driven by the periodic neural activity in the resting state (Martuzzi
et al., 2009; Mateo et al., 2017). Neuronal projections from the neuronal nuclei projecting to the
whole brain may be controlling and synchronizing vasomotion via neurovascular coupling (Korte
et al., 2023). It is also possible that ultra-slow vasomotion could be driven not only by the neuronal
rhythms but also by the oscillatory systemic changes, such as blood pressure and carbon dioxide in
the blood (Panerai et al., 1998; Sassaroli et al., 2012; Wise et al., 2004).
Functional hyperemia has been shown to be induced by visual stimulation (Heeger et al., 2000;
Kayser et al., 2004; Niessing et al., 2005; Rivadulla et al., 2011; Shaw et al., 2021; Stickland
et al., 2019). Visual stimulation evokes neuronal action potential firing and through the neurovascular
coupling mechanisms, vasomotion is affected. If a pulsatile visual stimulus is periodically presented,
that would result in vasomotion fluctuation with the same frequency as the presented visual stimulus
(Kayser et al., 2004; van Veluw et al., 2020). Previously, such visual stimulus-evoked vasomotion was
reported to be restricted to the visual cortex (van Veluw et al., 2020). However, in our study, we show
that vasomotion entrainment occurs with repeated HOKR visual stimulation, and the coordinated
vasomotion, which is frequency-locked to the visual stimulus, was observed not only in the visual
cortex but also widely throughout the surface of the cortex and the cerebellum, and also deep in the
flocculus region of the cerebellum. It is possible that vasomotion entrainment is induced in other areas
as well, including the limbic system and the brain stem.
Vasomotion could be beneficial for regulating the blood flow and the vascular surface area, which
would affect the supply of nutrients (e.g., glucose) and oxygen in the brain. Mathematical modeling
has shown that effective vascular resistance decreases with vasomotion (Meyer et al., 2002). Low-
frequency vasomotion has been shown to be beneficial for tissue oxygenation, especially in regions
where perfusion is restricted (Davis et al., 2008; Nilsson and Aalkjaer, 2003). As astrocyte processes
surround the blood vessels, the coordinated activity of astrocytes is presumed to be important for the
efficient delivery of the energy source to neurons for information coding and plasticity. Furthermore, it
was recently reported that vasomotion could also drive paravascular clearance or intramural periarte-
rial drainage (IPAD) (van Veluw et al., 2020). Initially, arterial pulsation from the heart was considered
to be the driving force for the IPAD; however, simulation modeling suggested that vasomotion was
required for the efficient IPAD (Aldea et al., 2019).
40 Hz sensory stimulation has been proposed to be potentially beneficial for the treatment of
Alzheimer’s disease (AD) (Adaikkan et al., 2019; Iaccarino et al., 2016; Martorell et al., 2019).
However, it was recently claimed that the 40 Hz flickering light does not entrain gamma oscillations
and suppress amyloid beta (Aβ) accumulation in the brains of the AD model mouse (Soula et al.,
2023). In another study, it was shown that flashing visual stimuli could increase the amplitude of vaso-
motion, which resulted in an increase in the paravascular clearance rates in the visual cortex of awake

mice (van Veluw et al., 2020). In mice with cerebral amyloid angiopathy, impaired paravascular clear-
ance and Aβ accumulation occur (Charidimou et al., 2017; Peng et al., 2016). It was also suggested
that low-frequency arteriolar oscillations could drive the drainage of soluble waste products (van
Veluw et al., 2020).
In our study, we show that the HOKR-driving oscillatory visual stimulus, which is much slower
(0.25 Hz) than the 40 Hz, is effective in producing a brain-wide oscillatory vasomotion. In addition,
repeated presentation with the spaced training protocol was effective in driving the plastic vaso-
motion entrainment. Paravascular clearance and drainage of waste products, such as Aβ, may be
enhanced with vasomotion entrainment. We show that synaptic plasticity change related to cerebellar
motor learning is enhanced in parallel with the vasomotion entrainment. It is possible that vasomotion
enhancement may increase the meta-plasticity and provide a basis for efficient neuronal plasticity to
occur. In the cerebellum, neuron-to-astrocyte (or Bergmann glia) (Matsui et al., 2005; Matsui and
Jahr, 2004; Matsui and Jahr, 2003) and astrocyte-to-neuron (Beppu et al., 2021; Beppu et al., 2014;
Sasaki et al., 2012) signaling pathways have been identified and such neuron-astrocyte interaction
has been shown to be essential for the online HOKR learning to occur (Kanaya et al., 2023). Late
offline HOKR learning was dependent in part on the phagocytosis activity of astrocytes (Morizawa
et al., 2022). It is possible that the energy required for the long-term plastic changes in the brain is
supported by the cooperative actions of the astrocytes and the entrained vasomotion. Aβ has been
shown to constrict capillaries and the resulting slight hypoxia and neuronal damage may actually be
the cause of dementia in AD (Nortley et al., 2019). The slow sensory stimulation may possibly be
effective for AD treatment; however, evidence of enhanced Aβ drainage and cognitive decline resis-
tance with such sensory stimulus using AD model mice would be required in the future to support
such a claim.
Materials and methods

Animals
All experiments were performed in accordance with the recommendations of the Regulations for
Animal Experiments and Related Activities at Tohoku University (2019LsA-017-10). Efforts were made
to minimize animal suffering and reduce the number of animals used. Mice were housed individually
for more than 7 d under standard laboratory conditions with food and water ad libitum.
C57BL/6NJcl male mice (2–4 months old) were used in this study in order to reduce the variance
of HOKR performance among animals (Aziz et al., 2014). Mlc1-tTA::tetO-YCnano50 double transgenic
mice were also used, which express the tetracycline transactivator (tTA) under the astrocyte-specific
Mlc1 promoter (Tanaka et al., 2012). The expressed tTA activates the tetO promoter and the FRET-
based Ca2+ sensor YCnano50 is expressed specifically in astrocytes, including the cerebellar Bergmann
glial cells (Kanemaru et al., 2014). Astrocyte Ca2+ dynamics associated with vasomotion were of
potential interest to study; however, HOKR learning could not be reliably induced in Mlc1-tTA::tetO-
YCnano50 transgenic mice for unknown reasons. Thus, we concentrated on the vasomotion dynamics
resolved by the autofluorescence detection for most of the article. Only the YFP part of the CFP-YFP
conjugated protein of YCnano50 was used to assess the ‘shadow’ imaging principle for detecting vaso-
motion dynamics.
Wide-field macro-zoom microscope

For surgical preparation, mice were anesthetized under ~5% isofluorane for induction and 0.5–
2.0% isoflurane mixed with the air for maintenance. The scalp was cut open and the periosteum
was gently removed to expose the crania. A stainless steel chamber frame (CF-10; Narishige,
Tokyo, Japan) was used as a holding jig to place the head in place under the microscope. The
chamber frame was attached to the edges of the exposed crania of the mouse to avoid obstruc-
tion of the view of the cortex and the cerebellum using a synthetic resin (Super bond C&B; Sun
Medical Company, Shiga, Japan) mixed with charcoal particles. In the thinned skull imaging (Drew
et al., 2010), the skill was gently thinned with a drill with precautions not to penetrate the skull.
In both thinned skull and intact skull imaging (Morii et al., 1986), the crania were covered with
a UV-curing resin (Jelly Nail; IML Inc, Tokyo, Japan) to prevent desiccation of the bone. The resin
greatly improved the transparency of the skull, and it could protect the field of view for over weeks.

The resin and the short UV exposure are commonly applied to human fingernails without health
concerns. Even after the short UV exposure for curing resin, fluorescence proteins in the mouse
brain were not photobleached. A tube made of black aluminum was attached to the top of the
mouse’s head so as not to obstruct the view of the microscope. All gaps between the head and
this custom-made tube were sealed with the Jelly Nail resin with charcoal particles to prevent light
from the video monitor used for HOKR visual stimulus from leaking in. The mice were allowed to
recover from the surgery for at least 3 d.
Prior to setting the mouse under the microscope, the mouse was intravenously injected via the
tail vein with TexasRed dextran (70,000 MW, Invitrogen; prepared 25 mg/kg in saline at 0.1 ml).
Immediately after the injection, the mouse was mounted on a chamber holder (MAG-1 and MAG-A;
Narishige) using the chamber frame fixed to its skull. The mouse’s body was loosely restrained with a
plastic cylinder. The head-fixed mouse was set under a wide-field macro-zoom microscope (MVX10;
Olympus, Tokyo, Japan) equipped with an image splitting optics (W-VIEW GEMINI A12801-01; Hama-
matsu) and a digital CMOS camera (ORCA-Flash4.0 V3; Hamamatsu Photonics, Shizuoka, Japan). An
inverted cone made of a black opaque sheet was attached to the objective lens to prevent the outside
light from leaking in and prevent the fluorescence excitation light from the microscope from reaching
the mouse’s vision and startling the mouse. The inverted cone was capped by the tube attached to
the mice’s head, thus, the light insulation was complete.
The excitation light to the microscope was provided by an LED-based light source (Niji; Blue Box
Optics, Blackwood, UK). For excitation of the TexasRed, excitation of the autofluorescence, and direct
excitation of the YFP (dYFP) of the YCnano50, were accomplished with 560 nm LED light source with a
bandpass filter of ET580/25x (Chroma, VT), 440 nm LED light source with a bandpass filter ET430/24x
(Chroma), and 490 nm LED light source with a bandpass filter ET505/20x (Chroma), respectively. A
multi-band beamsplitter (69008bs; Chroma) was used to send multiple excitation wavelength lights to
the mouse while allowing the emission lights of the TexasRed, the autofluorescence, and the dYFP to
pass through. When necessary, the emission light of the TexasRed and that of the autofluorescence or
the dYFP were split using a dichroic mirror (T590lpxr, Chroma). The wavelength of the emission lights
was restricted using a custom bandpass filter (passband: 473 ± 12 nm and 625 ± 15 nm, Chroma), a
bandpass filter ET540/40m (Chroma), or a bandpass filter ET540/30 m (Chroma) for the detection of
TexasRed, autofluorescence, or dYFP, respectively.
The frame exposure time of the camera was 89 ms and frames were recorded at 5 Hz sampling
frequency. The magnification of the microscope was ×0.8 or ×6.3. All settings of the camera were
controlled by the HCImageLive software (Hamamatsu). The power of excitation light at the wave-
lengths of 560 nm and 440 nm delivered from Niji were 1.7 mW and 3.2 mW with ×0.8 magnification,
respectively. The power of excitation light with the wavelength of 560 nm, 440 nm, and 490 nm
with ×6.3 magnification were 8 mW, 13 mW, and 4 mW, respectively. In all of the recording configura-
tions, each excitation light was sequentially delivered in 91 ms pulses with 109 ms intervals.
Fiber photometry
Prior to surgical implantation of the optical fiber, the mouse was anesthetized with three types of
mixed anesthesia, consisting of 0.75 mg/kg of medetomidine hydrochloride (Domitol; Nippon Zenyaku
Kogyo Co., Ltd, Fukushima, Japan), 4 mg/kg of midazolam (Midazolam, Sandoz Inc, Japan), and 5 mg/
kg of butorphanol tartrate (Vetorphale; Meiji Seika Pharma Co., Ltd, Tokyo, Japan). A screw (6 mm
head diameter and 10 mm length) was attached to the cranial bone with its head down between
bregma and lambda using a synthetic resin (Super bond C&B; Sun Medical Company). This screw was
used as a holding jig to keep the mouse’s head fixed toward the visual stimulus presenting monitor.
A glass optical fiber (core diameter 400 µm, 0.39 NA; FT400UMT; Thorlabs, NJ) was implanted in
the cerebellum close to the flocculus (coordinates X = 2.0 mm, Y = –5.65 mm, Z = 2.9 mm, α = 10°).
This optical fiber was fixed using a synthetic resin (Super bond C&B) mixed with charcoal particles
to prevent external lights from penetrating the resin. For recovery from three types of mixed anes-
thesia, 0.75 mg/kg of atipamezole hydrochloride (Antisedan; Zenyaku Kogyo Co., Ltd, Tokyo, Japan)
was administered. The mice were allowed to recover from surgery for at least 5 d. When necessary,
the mice were intravenously injected via the tail vein with TexasRed dextran prior to recording. Each
mouse was head-restrained by tightening the screw on the head of the mouse to the custom-made
apparatus and its body was loosely restrained in an upward-sloping plastic cylinder.

A custom-made optical apparatus for fiber photometry was used (Lucir, Tsukuba, Japan). Exci-
tation lights were delivered from the SPECTRA X Light Engine (Lumencor, OR). An optical fiber from
the fiber photometry apparatus was connected to the optical fiber implanted in the mouse with a
ferrule-to-ferule connection using a split sleeve. Excitation light was sent to this optical fiber and the
power of excitation light with the wavelength of 575 nm, 510 nm, and 440 nm were 3 mW, 11 µW, and
2.2 mW, respectively, at the tip of the implanted optical fiber. The emitted light came back through
the optical fiber, reached the fiber photometry apparatus, and was detected using the two photomul-
tiplier tubes (PMTs) (PMTH-S1-CR316-02; Zolix Instruments Co., Ltd, China) with current-to-voltage
amplifiers (HVC1800; Zolix). The electronic signals from the PMTs were digitized with Micro1401-3
plus ADC12 top box (CED, Cambridge, UK) and recorded using the Spike2 software (CED) at a 5 kHz
sampling frequency. In all of the recording configurations, each excitation light was delivered in 20 ms
pulses with 180 ms intervals. The signals were averaged within the 20 ms pulse, resulting in an effec-
tive sampling frequency of 5 Hz.
TexasRed and dYFP were excited with the wavelength of 575 nm and 510 nm, respectively, and
the excitation wavelengths were restricted with a multi-bandpass filter 69008x (Chroma). Specifically,
the light for directly exciting YFP was delivered via the relevant band of 503 nm/19.5 nm (FWHM) of
the multi-bandpass filter 69008x. With this filter, negligible light with a wavelength below 490 nm is
passed through. The excitation wavelength of CFP is below 490 nm, and thus excitation of CFP can
be assumed to be negligible with the light used for directly exciting YFP. A multi-band beamsplitter
(ZT445/514/594rpc; Chroma) was used to send multiple excitation lights to the mice and pass through
the emission lights of TexasRed or dYFP. The emission lights of TexasRed and dYFP were split by a
dichroic mirror, FF605-Di02 (Semrock, IL). The wavelength of the emission lights was restricted with a
bandpass filter ET640/30x (Chroma) for TexasRed and with ET539/21x (Chroma) for dYFP detection.
For excitation of TexasRed and autofluorescence, lights with the wavelength of 575 nm and 440 nm
restricted by a multi-bandpass filter 69008x (Chroma) were used. A multi-band beamsplitter 69008bs
(Chroma) was used to send multiple excitation lights to the mice and pass through the emission lights
of TexasRed and autofluorescence. The emission lights of TexasRed and autofluorescence were split
by a dichroic mirror FF605-Di02 (Semrock). The wavelength of the emission lights was restricted with
a bandpass filter ET645/75m (Chroma) for TexasRed and with ET537/29m (Chroma) for autofluores-
cence detection.
For excitation of only the autofluorescence, light with the wavelength of 440 nm restricted with a
bandpass filter ET436/20x (Chroma) was used. A dichroic mirror T495lpxr (Chroma) was used to send
the excitation light to the mice and pass through the emission light of the autofluorescence. The
wavelength of the emission light was directly restricted with a bandpass filter FF01-550/88 (Semrock)
for autofluorescence detection.
Visual stimulus presentation

For visual stimulation, sinusoidal oscillations of vertical stripes displayed on two PC monitors arranged
orthogonally were presented to the animal. The visual stimuli were created and controlled with the
ImageJ/Fiji software (National Institute of Health, MD) or the custom program written in MATLAB
(MathWorks, Natick, MA), for use with the macro-zoom microscope or the fiber photometry, respec-
tively. In experiments other than the ones shown in Figure 5, the amplitude of the oscillations was
17° in the visual angle of the mouse, the temporal frequency of the oscillations was 0.25 Hz, and the
spatial cycle of the displayed stripe pattern was 6.4°.
Analysis of the eye movement for the HOKR experiments

In fiber photometry experiments, the eye movements were simultaneously recorded. The method
of recording and analyzing eye movement was previously described in detail (Kanaya et al., 2023).
During the HOKR experiments, the mouse was exposed to white noise sounds, which helped keep the
mouse alert. An angled hot mirror, which reflects only the IR light, was placed between the mouse’s
right eye and the monitor, thus the visual field of the mouse was unobstructed. The monitored eye (the
right eye) was illuminated with two infrared (IR) LEDs. The corneal reflections of the IR LEDs were used
as the reference point. The reflected IR image of the eye was captured using a CCD camera (DN3V-
30BU; Shodensha Inc, Osaka, Japan) with the IR-cut filter removed.

The captured video taken at 30 fps was analyzed using DeepLabCut (Mathis et al., 2018), and the
center of the pupil was tracked (see Kanaya et al., 2023 for details). The angle of the eye direction
was calculated based on the pupil and camera position using a previously introduced method (Saka-
tani and Isa, 2004). When the difference in the center eye position between two consecutive frames
(1/30 s) was more than 1° in visual angle, it was assumed that a saccadic eye movement had occurred.
These incidents of saccadic movements were removed. Drifts in the eye movement were eliminated
by a bandpass filter with a 100–500 mHz frequency. The amplitudes of the eye were calculated by
the difference between the averaged maximum peak and minimum peak. HOKR relative gain was
calculated by normalizing the amplitude of the eye movement at the start of the first training session.
Vessel diameter and vasomotion analysis with macro-zoom microscope

images
Images acquired with the macro-zoom microscope were analyzed with ImageJ/Fiji and AxoGraph
(Axograph Scientific, New South Wales, Australia) software. The blood vessels focused and studied
were likely to be arteries or arterioles found on the surface of the brain which were filled with TexasRed
fluorophore via intravenous injection. The blood vessel in focus was rotated so that the flow of the
blood would be aligned with the vertical axis. The stack of images taken as time series was cropped
with the blood vessel in the center of the image, and this stack was named the ‘original stack’. The
background intensity was measured within the ROI on the left and right of the vessel and an average
value was calculated for each image of the stack. A new stack with the same XY pixel dimensions as
the ‘(1) original stack’ was created and each image of the stack had a single uniform value of the back-
ground intensity calculated above. This background stack was subtracted from the ‘(1) original stack’
to create the ‘(2) background subtracted stack’. Due to the dilution of the TexasRed fluorophore and
the photobleaching effect, the fluorescence in the blood vessels tends to fade in time. Thus, a normal-
ization procedure to counter the fading effect was developed. First, using the above ‘(2) background
subtracted stack’, an ROI was set well within the blood vessels and a time-series data of the average
intensity within the ROI was calculated. This data was strongly low-pass Gaussian filtered at 5 mHz,
and the transient fluctuations of intensity due to vasomotion were totally eliminated, leaving only
the baseline decline of intensity due to the fading effect was left. Again, a new stack with the same
XY pixel dimensions as the ‘(1) original stack’ was created and each image of the stack had a single
uniform value of the baseline intensity calculated above. The ‘(2) background subtracted stack’ was
divided by this baseline stack to create the ‘(3) baseline normalized stack’. With this procedure, the
images are normalized to the basal TexasRed intensity in the blood vessels. TexasRed intensity differs
among vessels and trials, thus this procedure is effective in the normalization of multiple experiments.
The ‘(3) baseline normalized stack’ was resliced with a line vertical to the vessel using the ImageJ/Fiji
software and the temporal change of the TexasRed profile along this line was created. Multiple lines
vertical to the vessel were averaged, allowing a better signal-to-noise ratio. This data was low-pass
Gaussian filtered (~0.05 pixel) in each slice. The left side and the right side of the vessel often had
different background intensity levels. Therefore, these cross-sectional profiles of the blood vessels
were processed with a flat-sloping baseline function in AxoGraph to eliminate this horizontal slope.
The cross-sectional profile of an undilated vessel in the basal phase was referred and 10% of the
maximum value in the profile was calculated. The full width of the cross-section profile at this threshold
value was defined as the vessel diameter. Since a confocal or two-photon microscope was not used,
an ideal optical section image could not be obtained. Therefore, the fluorescence intensity of the line
profile across the vessel would be expected to increase toward the center of the vessel as the thick-
ness of the vessel increases. One would not expect to see a square line profile. Therefore, full width at
half-maximum value of the line profile would likely be an underestimate of the actual vessel diameter.
Approximately 10% is the minimum intensity that we could distinguish from the background intensity
fluctuations. Full width at 10% maximum should be considered just an index of the actual diameter,
and the ‘true’ diameter of the vessels could not be determined. In most of the current study, we dealt
only with the change of the vessel diameter relative to the basal diameter. The calculated vessel diam-
eter at each time point was normalized to the vessel diameter in the undilated phase.
The vasodilation events were detected using the event detection function in AxoGraph. Thresholds
were determined arbitrarily as the amplitudes of the vasodilation varied across the vessels. Inter-event
interval and peak amplitudes were calculated. Unit dilation time was defined as the full-width time at

the half-maximum of the amplitude of the vasodilation. Inter-event interval, peak amplitude, and unit
dilation time were transformed into probability density function and the cumulative relative frequency
was calculated.
Frequency analysis
Essentially, the same procedure was used for analyzing the temporal frequency profile of the fluores-
cence signal data recorded from either the macro-zoom microscopy or the fiber photometry. First, the
fluorescence fade was corrected and the baseline value of the fluorescence signals was normalized.
Using MATLAB, the original fluorescence signal data taken with a sampling frequency of 5 Hz were
divided by their own low-pass filtered data (the passband was 50 mHz and the stopband was 10 mHz).
The result was subtracted by 1 and multiplied by 100 to transform the data into a percentage display.
5 min or 15 min of data taken during visual stimulus presentation were processed by fast Fourier trans-
form (FFT). The amplitude spectrum and spectrogram were created by executing FFT or short-term
Fourier transform (STFT), respectively, by custom programs written in MATLAB. For the spectrogram;
the window size was 120 s, 60 s, or 40 s, and the step size was 10 s, 4 s, or 2 s.
The magnitude of the fluorescence signal frequency locked to the presented temporal frequency
of the visual stimulus was expressed as the peak ratio of 0.25 Hz (PR0.25). For deriving the PR0.25,
first, the peak amplitude of the fluorescence signal at 0.25 Hz was calculated. Next, the mean ampli-
tude of the signal in the 0.1–1 Hz range was calculated. PR0.25 is the signal amplitude at 0.25 Hz
divided by the mean at 0.1–1 Hz.
The whole brain surface map of the PR0.25 magnitude presented in Figure 3F was created using
the following methods. The raw stacked image data taken during the visual stimulus presentation
were first processed by using a Gaussian filter with σ = 165 µm in the x and y dimensions. The time-
series data of each pixel were normalized using the method described above. The time-series data
of each pixel were then processed using FFT, the PR0.25 during the 15 min of visual stimulation was
calculated, and the magnitude of the PR0.25 was color-coded and mapped.
Phase analysis
The phase shifts between the periodic oscillatory signals of the TexasRed and the dYFP signals, or
between the TexasRed and the autofluorescence signals, were evaluated using the cross-correlation
analysis. The lag time of the negative peak around 0 s in the cross-correlation was defined as the Δt.
The temporal cycle of the horizontally oscillating visual stimuli was 4 s. A negative peak was usually
found within the ±4 s range in the cross-correlation graph in almost all sessions. Data with no negative
peak within this range were excluded from further analysis.
The phase shifts between the visual stimuli and the eye movement were calculated using the
following method. The eye movement data during the first 5 min of each training was divided into
segments of 4 s in duration, resulting in a series of 75 traces. These traces were averaged and fitted
with a sinusoidal curve. The phase of the eye movement was calculated using this fitted curve to
the averaged eye movement trace. The phase shifts between the visual stimuli and the TexasRed
signal were analyzed similarly. However, as shown in Figure 6F, although the eye movement reliably
followed the visual stimulation throughout the session, vasomotion apparently came in and out of the
frequency locked to the visual stimulation. Therefore, PR0.25 was calculated for the TexasRed traces
and only when the PR0.25 was higher than 3 that segment of the TexasRed trace was extracted. Using
these selected segments of the TexasRed signals, the phase shift between the visual stimulation and
the TexasRed signal was calculated.
Statistical analysis
All statistical tests were done with Origin Pro 8.6 software (OriginLab, MA). Data are presented as
means ± SEM, except in Figure 7F. The number of samples is indicated in the figure legend. Statistical
analyses were performed with the paired t-test (two-sided), one-sample t-test, and Holm correction
for multiple tests.
Acknowledgements
We are thankful to all members of the Ko Matsui laboratory for their invaluable assistance at every stage
of the experiments. This work was supported by JSPS Fellowships 22KJ0262 (to DS), Grant-in-Aid for

Early-Career Scientists 22K15218 (to YI), Grant-in-Aid for Transformative Research Areas (A) 'Glial
Decoding': 20H05896 (to KM), 'Biology of Behavior Change': 23H04659 (to KM), Grant-in-Aid for
Scientific Research on Innovative Areas 'Brain Information Dynamics': 18H05110, 20H05046 (to KM),
Grant-in-Aid for Scientific Research (B): 19H03338, 22H02713 (to KM), Research Foundation for Opto-
Science and Technology (to KM), Takeda Science Foundation (to KM), the NOVARTIS Foundation for
the Promotion of Science (to KM), and the Uehara Memorial Foundation (to KM).
Additional information
Funding
Funder Grant reference number Author
Japan Society for the 22KJ0262 Daichi Sasaki

Promotion of Science
Japan Society for the 22K15218 Yoko Ikoma

Japan Society for the 20H05896 Ko Matsui






Research Foundation Ko Matsui

for Opto-Science and
Technology
Takeda Science Ko Matsui

Foundation
NOVARTIS Foundation Ko Matsui
Uehara Memorial Ko Matsui

Foundation
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Daichi Sasaki, Conceptualization, Data curation, Formal analysis, Funding acquisition, Visualization,
Methodology, Writing - original draft, Writing - review and editing; Ken Imai, Formal analysis, Vali-
dation, Methodology, Writing - review and editing; Yoko Ikoma, Supervision, Funding acquisition,
Validation, Methodology, Writing - review and editing; Ko Matsui, Conceptualization, Resources,
Supervision, Funding acquisition, Validation, Methodology, Writing - original draft, Project administra-
tion, Writing - review and editing
Author ORCIDs
Ko Matsui ‍ ‍http://orcid.org/0000-0003-1068-9705
Ethics
This study was performed in strict accordance with the recommendations in the Regulations for
Animal Experiments and Related Activities at Tohoku University. All of the animals were handled
according to approved institutional animal care and use committee protocols of the Tohoku University

(2019LsA-017-10). All surgery was performed under anesthesia, and every effort was made to mini-
mize suffering and to reduce the number of animals used.
Peer review material

Reviewer #2 (Public Review): https://doi.org/10.7554/eLife.93721.3.sa1
Reviewer #3 (Public Review): https://doi.org/10.7554/eLife.93721.3.sa2
Author response https://doi.org/10.7554/eLife.93721.3.sa3
Additional files
Supplementary files
• MDAR checklist
Data availability
The datasets required to replicate the results and reproduce the figures in the manuscript are freely
available in the project's Dryad Digital Repository (https://doi.org/10.5061/dryad.15dv41p4f).
The following dataset was generated:
Author(s) Year Dataset title Dataset URL Database and Identifier
Sasaki D, Imai K, 2024 Dataset for "Plastic https://doi.org/ Dryad Digital Repository,
Ikoma Y, Matsui K vasomotion entrainment" 10.5061/dryad. 10.5061/dryad.15dv41p4f
in eLife 15dv41p4f
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RESEARCH ARTICLE

Plastic vasomotion entrainment

Super-­network Brain Physiology, Graduate School of Life Sciences, Tohoku University,

Abstract The presence of global synchronization of vasomotion induced by oscillating visual

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  1 of 26

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  2 of 26

C Single line slice

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  3 of 26

Entrainment of vasomotion with visual stimuli presentation

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C Single line slice D Basal Dilated

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  5 of 26

Novice Trained Expert 10 sec

0.4 0.4 0.4

0.2 0.2 0.2

0.5 0.5 0.5

0.2 0.2 0.2

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The use of autofluorescence signal for examination of vasomotion

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  8 of 26

E TexRed F Corr. coef.

M Freqency (Hz) Freqency (Hz)

Peak amplitude @ 0.25 Hz

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  9 of 26

Vasomotion followed the visual stimulation dynamics over a wide

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  10 of 26

A Temporal freq (Amplitude : 17 deg, Spatial cycle : 6.4 deg)

1.5 1.5 1.5

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  11 of 26

Vasomotion is not the consequence of eye movements

Possibility of the contribution of vasomotion entrainment to the HOKR

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  12 of 26

A Nasal B Eye trace phase

D F -90 0 90 180 270 360

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  13 of 26

1 hour 2 min 2 min

B 1st Train 4th Train Day 2

Relative Gain @Day 5 PR0.25_AutoFluo

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  14 of 26

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Materials and methods

Wide-field macro-zoom microscope

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Visual stimulus presentation

Analysis of the eye movement for the HOKR experiments

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  19 of 26

Vessel diameter and vasomotion analysis with macro-zoom microscope

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Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721  21 of 26

Japan Society for the 22KJ0262 Daichi Sasaki

Japan Society for the 22K15218 Yoko Ikoma

Japan Society for the 20H05896 Ko Matsui

Japan Society for the 23H04659 Ko Matsui

Japan Society for the 18H05110 Ko Matsui

Japan Society for the 20H05046 Ko Matsui

Japan Society for the 19H03338 Ko Matsui

Super-network Brain Physiology, Graduate School of Life Sciences, Tohoku University,

Sasaki et al. eLife 2024;13:RP93721. DOI: https://doi.org/10.7554/eLife.93721 1 of 26

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