Original Article

Experimental cortical stroke inducesaberrant increase of sharp-waveassociated ripples in the hippocampus
and disrupts cortico-hippocampal
communication

Journal of Cerebral Blood Flow &Metabolism
2020, Vol. 40(9) 1778–1796! Author(s) 2019
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DOI: 10.1177/0271678X19877889
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Ji-Wei He1,2,*, Gratianne Rabiller1,2,*, Yasuo Nishijima1,2,3,
Yosuke Akamatsu1,2,3, Karam Khateeb4,
Azadeh Yazdan-Shahmorad4,5 and Jialing Liu1,2

Abstract
The functional consequences of ischemic stroke in the remote brain regions are not well characterized. The current
study sought to determine changes in hippocampal oscillatory activity that may underlie the cognitive impairment
observed following distal middle cerebral artery occlusion (dMCAO) without causing hippocampal structural damage.
Local field potentials were recorded from the dorsal hippocampus and cortex in urethane-anesthetized rats withmultichannel silicon probes during dMCAO and reperfusion, or mild ischemia induced by bilateral common carotid
artery occlusion (CCAO). Bilateral change of brain state was evidenced by reduced theta/delta amplitude ratio andshortened high theta duration following acute dMCAO but not CCAO. An aberrant increase in the occurrence of sharpwave-associated ripples (150–250 Hz), crucial for memory consolidation, was only detected after dMCAO reperfusion,
coinciding with an increased occurrence of high-frequency discharges (250–450 Hz). dMCAO also significantly affected
the modulation of gamma amplitude in the cortex coupled to hippocampal theta phase, although both hippocampal thetaand gamma power were temporarily decreased during dMCAO. Our results suggest that MCAO may disrupt the balance
between excitatory and inhibitory circuits in the hippocampus and alter the function of cortico-hippocampal network,
providing a novel insight in how cortical stroke affects function in remote brain regions.

Keywords
Local field potential, phase amplitude coupling, modulation index, connectivity, distal middle cerebral artery occlusion,common carotid artery occlusion
Received 8 April 2019; Revised 22 July 2019; Accepted 25 July 2019

Introduction
Stroke aﬄicts 800,000 people per year and has become
the leading cause of adult disability in the US. Apartfrom sensory and motor impairment, post-stroke cognitive impairment in various domains from attention
deficits to memory problems is not uncommon.1–3
The hippocampus is involved in memory function,
but direct ischemic lesions strategic to the hippocampus
or parahippocampal regions are rarely observed in
middle cerebral artery (MCA) stroke.4–6 Consistent
with clinical observation, cognitive impairment can bedetected in animal models of stroke in the absence of1

Department of Neurological Surgery, UCSF, San Francisco, CA, USA
Department of Neurological Surgery, SFVAMC, San Francisco, CA, USA
3
Department of Neurosurgery, Tohoku University Graduate School of
Medicine, Sendai, Japan
4
Departments of Bioengineering and Electrical and Computer
Engineering, University of Washington, Seattle, WA, USA
5
Center for Integrative Neuroscience and Department of Physiology,
University of California, San Francisco, CA, USA
2*These authors contributed equally to the work.
Corresponding author:
Jialing Liu, Department of Neurological Surgery (112C), University ofCalifornia at San Francisco and Department of Veterans Affairs Medical
Center, 1700 Owens Street, San Francisco, CA 94158, USA.
Email: jialing.liu@ucsf.edu

He et al.
hippocampal injury.7,8 However, the neural changes in
the hippocampus that may underlie the functional
impairment induced by cortical stroke are not wellunderstood.
Stroke leads to down regulation in the GABAergictransmission,9,10 changes in connectivity among functional networks between the cortex and hippocampus11,12 as well as a host of changes in brain
oscillations serving to communicate between brain networks,13,14 all of which can potentially aﬀect brain
regions remote to stroke. For example in the hippocampus, the firing of sharp-wave-associated ripples (SWRs),
a high-frequency activity in the pyramidal layer15 that
requires temporally precise local interactions between
excitatory and inhibitory neurons in the CA1,16–18 canpotentially be disrupted by MCAO that alters GABA
receptor expression.9 The selective elimination of
SWRs during post-learning sleep resulted in impairmentof memory,19,20 suggesting that the dysregulation of
SWRs may interfere with memory consolidation andtransferring memory from the hippocampus to cortex.
A second potential target of MCAO can be the theta
rhythm, a nearly sinusoidal oscillation at 4–7 Hz originated from the hippocampus, not only correlates withmemory performance but also is involved in memory
consolidation during REM sleep.21,22 Conversely, thehippocampal non-theta state, also known as the slow
wave state (SWS), is associated with immobility or
SWS sleep.23,24 Manipulating the hippocampal theta
rhythm alters cognition, further suggesting a causal
role of the hippocampal theta in cognition.25,26 The
third likely target, cortical gamma oscillations, including
low- (30–60 Hz) and high gamma oscillations (60–150 Hz), are known to play a role in sensory stimulation,27,28 declarative memory,29 attentional selection,30
cognition,31 and assist in encoding and retrieval ofmemory traces in the hippocampal formation.32–34
Ample evidence suggests that theta oscillations provide
a temporal reference for the exchange of information
among diﬀerent brain networks, whereas fastergamma-frequency oscillations nested within theta
cycles underlie local information processing in thecortex.35,36 Thus, hippocampal theta-gamma coupling,in which gamma oscillations are modulated by theta
oscillations, is stronger during memory replay.
Conversely, poor hippocampal coupling between thetaand gamma could predict poor memory performance.37,38 Likewise, reduced modulation of cortical
gamma amplitude during hippocampal theta phase
was detected in a mouse model of Alzheimer’s Disease
with cognitive deficit,39 and in schizophrenic patientsassociated with working memory dysfunction.40
The perturbation of brain oscillations occurs in
response to the reduction of cerebral blood flow(CBF) and energy deprivation, resulting in decreased

1779
amplitude in high-frequency rhythms in electroencephalography (EEG) and increased power in slower frequency rhythms.13,41 Yet little is known about the
dynamics of hippocampal oscillations in the course ofbrain ischemia and how they respond to critical dip of
CBF that causes irreversible neuronal damage in thecortex, leading to the disruption of connectivity
between cortex and the other brain networks including
the hippocampal formation.11 Changes in the inhibitory and excitatory neurotransmitter systems havebeen well documented in the motor cortex after
stroke42,43; however, little is known regarding how
they change other remote brain regions such as the
hippocampus and their eﬀects on neuronal activity.
The current study sought to determine how distal
MCA stroke aﬀected cortical and hippocampal oscillatory activity. Our results indicate that ischemic strokein the cortex profoundly aﬀects multiple hippocampal
oscillations, among which the dysregulation of SWRs
and the altered theta-gamma modulation may contribute to cognitive impairment post stroke.

Methods and materials
Animals and housing
All experiments were conducted in accordance with the
Guide for Care and Use of Laboratory Animals(National Research Council. 2011. Guide for the Careand Use of Laboratory Animals: Eighth Edition.
Washington, DC), reported in compliance with theAnimals in Research: Reporting In Vivo Experiments(ARRIVE) guidelines, and were approved by the San
Francisco VA Medical Center Institutional Animal
Care and Use Committee (IACUC). A total of 24
adult male Sprague-Dawley rats approximately 2.5
months of age weighing 250 g were purchased fromCharles River Laboratories (Wilmington, MA) andhoused in institutional standard cages (two rats percage) on a 12-h light/12-h dark cycle with ad libitum
access to food and water before the experimental procedures. The identity of the test subject was blinded toinvestigators who performed the stroke surgery andelectrophysiology recording and who analyzed thedata.

Experimental stroke
Stroke was induced unilaterally in rats by the distal
middle cerebral artery occlusion (dMCAO) method in
combination with supplemental proximal artery occlusion of the bilateral common carotid arteries (CCAO)
under isoflurane (1.5%)/O2(30%)/N2O(68.5%) anesthesia as described previously,44 producing cortical
infarct restricted to the somatosensory (SS) cortex in1780
the absence of direct hippocampal injury.8 In brief, a
1.5 mm diameter burr hole 1 mm rostral to the anterior
junction of the zygoma and temporalis bone was madewith a dental drill. The dura mater was carefully
pierced with a 30-gauge needle. The main trunk of theleft MCA was ligated permanently above the rhinal
fissure with a 10–0 suture, and the bilateral common
carotid arteries (CCA) were occluded temporarily for
60 min with 4–0 sutures. The sutures were then removed
to restore blood flow, and the cervical incision
was closed. Core temperature was maintained at
37 ! 0.5" C with a heating blanket and rectal thermistor
servo-loop throughout the procedure. Due to the wellknown eﬀect of reduced CBF on brain EEG rhythms,
an additional cohort of rats with temporary bilateral
CCAO was included to diﬀerentiate the eﬀect of temporary CBF reduction from permanent cortical infarct
on brain oscillations.

RecordingElectrophysiological recordings were performed using
multisite extracellular silicon probes (NeuroNexus
Technologies) under urethane anesthesia for 2 h(Sigma, 15 mg/kg i.p.) as described.8,45 Following craniotomy, two electrodes (A1x32-6mm-50-177 into theipsilateral side and A1x16-5mm-100-703 into thecontralateral side, respectively) were slowly inserted
into the brain after the dura mater was resected totarget the dorsal hippocampus at (AP: #3.3 mm;
ML: ! 2 mm) via a stereotaxic frame (David Kopf
Instruments, Tujunga, CA, USA). The electrode andrecording sites in the brain are shown in Figure 1(a)to (c). Real-time data display and an audio aid wereused to facilitate the identification of proper recording
location while advancing electrodes until characteristic
signals from stratum pyramidale and stratum radiatum
were detected and recorded. A 2-h multi-channel recording from bilateral sensorimotor cortex and dorsal
hippocampus was collected from each rat. Data werestored at a sampling rate of 32 kHz after band-pass filtering (0.1–9 kHz) with an input range of ! 3 mV
(Digital Lynx SX, Neuralynx, USA). Approximately
10% rats died during recording, and the data were
excluded from analysis.

Tissue preparation and infarct assessment
One to 1.5 h after dMCAO reperfusion, rats were perfused transcardially with 4% PFA in 0.1 M phosphatebuﬀer, pH 7.4. The brains were collected, post fixed
overnight in 4% PFA and placed in 30% sucrose solution for 24 h. Brains were cut coronally in 40 um-thicksections and stored at 4" C. Serial coronal sections were
stained using the hematoxylin and eosin (H&E)

Journal of Cerebral Blood Flow & Metabolism 40(9)method. Infarct volume was measured by subtracting
the volume of intact tissue in the ipsilateral side fromthat in the contralateral side using Stereoinvestigator
software (Microbrighfield, VA).46

AnalysisPrior to data processing, the precise locations of the
pyramidal and radiatum layers were confirmed manually by using oﬀ-line analysis software (Neuroscope,GNU) with respect to unit activity, sharp waves andSWRs. Prior to quantification, all recordings were initially band-pass filtered at 0.1–9 K Hz with an input
range of ! 3 mV (Digital Lynx SX, Neuralynx, USA)and down-sampled to 1250 Hz (Matlab, MathWorks,
USA). Occasionally, data conversion failed in thisstep in one or more channels due to low-quality recordings, resulting in missing data in aﬀected channels. Only
rats with complete down-sampled data for all channels
and with all-time points were included in the analysis.
SWRs and high-frequency discharges (HFDs) eventswere analyzed from CA1 pyramidal layer of the hippocampus. For the analysis of hippocampal recordings
and theta state selection, data from the stratum lacunosum moleculare (slm) layer were selected over otherlayers for the main analyses based on the high signal-tonoise ratio in slm (Figure 1(d) to (i)).47,48
Local field potentials (LFPs) were band-pass filtered
for the following frequency bands: theta (4–7 Hz), delta(0.1–3 Hz), low gamma (LoG: 30–60 Hz) and highgamma (HiG: 60–150 Hz). Regarding brain state detection, two states of high theta/delta (HTD) and low
theta/delta (LTD) were defined by first calculating
Hilbert amplitudes for both frequency bands inslm.49–51 The amplitude envelopes were then smoothedwith a Gaussian kernel (s ¼ 1 s, 8 s window) and theratio of hippocampal theta to delta band amplitude(T/D) was calculated. The result was then convolved
with a smoothing Gaussian kernel (s ¼ 10 s, 80 s
window), after which a manually set threshold ratio
was used to capture sustained HTD and LTD periods.
Periods of SWRs and HFDs were detected from thesmoothed LFPs following Hilbert transformation (150–250 Hz for SWRs; 250–450 Hz for HFDs) of recordingsin the CA1 pyramidal layer. SWRs and HFDs were
defined as periods in which the smoothed envelope
exceeded a threshold amplitude six times greater thanthe baseline standard deviation for at least 15 ms.
SWRs and HFDs were analyzed only during LTD(non-theta state or SWS) period per previous
method.52 The average occurrence of events was examined between baseline, occlusion and reperfusion periods. Occurrence of events was derived by dividing thenumbers of events by the duration of LTD period (inseconds).

He et al.

1781

Figure 1. The amplitude of theta oscillation peaks at the stratum lacunosum moleculare. (a) Schematic representation of the A1x165mm-100-703 Neuronexus probe. (b) An example of the H&E-stained brain section showing the probe location with the corresponding channels spanning the brain layers. (c) Local field potential traces of a 2-s multichannel recording spanning from the cortex(top two traces) to the hippocampus (bottom traces) using a 16-channel NeuroNexus probe in a urethane-anesthetized rat without
vessel occlusion. Anatomical structure corresponding to each channel is labeled as cortex (ctx), stratum oriens (so), stratum
pyramidale (sp- trace labeled in red), stratum radiatum (sr) and stratum lacunosum moleculare (slm). In the frequency domain, theta(d: max between 3 and 7 Hz) and low gamma-band (e: LoG; median between 20 and 50 Hz) amplitudes for each channel werecomputed. White (L: left) and black (R: right) bars: simultaneous bilateral recording within the same animal (N ¼ 12). Mean spectrograms of the pyramidal layer channels (red), one cortical channel (dotted black), and one molecular layer channel (solid black) are
shown for theta (f) and low gamma (g). Typically, the slm channels chosen for analysis are located 400–600 mm below the pyramidal
layer. (i–h), 5000 ms representative traces of multichannel recording during Low-theta/delta (h) state and high-theta/delta state (i).
Green channels label the cortical layers, red channel the pyramidal layer of the CA1 and purple channel the stratum lacunosum
moleculare layer. Scale bar: 250 ms and 1 mv.

1782
To determine phase-amplitude coupling between
hippocampal theta and cortical gamma oscillations,the modulation index (MI) was calculated using thealgorithm described previously.53 Briefly, either theslm layer or CA1 pyramidal layer were used to analyze
the hippocampal theta phase coupling with the cortical
gamma amplitude during HTD periods. In particular,
band-pass filters for low gamma (LoG) and highgamma (HiG) were set at 30–60 Hz and 60–150 Hz,
respectively. A Hilbert transform was applied toobtain the amplitude time series of the cortical LFPs
and the phase time series of the hippocampal LFPs. To
determine the MI, the phase time series were grouped
into 18 bins, wherein the mean amplitude for eachphase bin was calculated and standardized over thesum of all mean amplitudes.

Statistical analyses
Data are expressed as mean !SD. For data with anormal distribution, as tested with Kolmogorov–
Smirnoﬀ tests (GraphPad Prism, v5.00), one-way
ANOVA was performed to assess the main eﬀect ofacute dMCAO or CCAO on each variable. Post hoc
Bonferroni’s multiple comparisons were used to characterize the temporal profile. Student’s t test was used
to assess the lateralization eﬀect. For non-normal distributed data sets, nonparametric tests were applied;e.g. Friedman test for multiple comparisons of paired
samples, and Kruskal–Wallis test for multiple comparisons of independent samples followed by a post hoc
Dunn’s Multiple Comparison test. P values less than
0.05 were considered as significant.

Results
Cortical ischemic stroke increases the occurrence ofhippocampal SWRs and HFDs
To determine whether cortical stroke aﬀects the synchrony of the hippocampal circuit by altering the interplay between excitation and inhibition, we recorded
neural activity as LFPs from diﬀerent depths startingat deep cortical layers down to the hippocampus in rats
under urethane anesthesia and analyzed the occurrenceof SWRs. Consistent with previous findings, SWRs
were detected in the pyramidal layer and accompaniedby sharp waves in the stratum radiatum (Figure 2(a)and (b)). There were few ripple events taking place
during baseline due to the predominance of theta
brain state in this period. In contrast, there was a significant increase in the rate of ripple occurrence during
ischemic reperfusion of dMCAO compared to baselinein both the ipsilateral and contralateral CA1 (Figure2(c)). Unlike dMCAO, mild ischemia via temporary

Journal of Cerebral Blood Flow & Metabolism 40(9)CCAO did not alter SWRs occurrence even during
the periods when the duration of SWSs was increased.
Besides SWRs, the occurrence of HFDs in the range of
250–450 Hz was also increased during ischemic reperfusion following dMCAO but not CCAO (Figure 2(d)),
resulting in the increased proportion of HFDs in thepathological range compared to the physiological rangeof ripples (Figure 2(e)).
Under urethane anesthesia, dMCAO produced anischemic lesion mainly in the somatosensory cortex(Figure 2(f)), a location similar to that seen under isoflurane anesthesia.8,44 Infarct volume determined at 1–
1.5 h after reperfusion was 24.85 ! 7.50 mm3, significantly smaller than the lesion size observed at five toseven weeks after dMCAO in our previous studies.44

Cerebral ischemia alters brain states in the
hippocampus
Because an increased duration of the delta brain state isoften associated with adverse neurological function54,55
and T/D amplitude ratio is regarded as a potential biomarker of post-stroke cognitive outcome,56 we thus
examined the eﬀect of cerebral ischemia on brain
states. Since LFP data recorded in the slm layer displayed the most robust signal-to-noise ratio in theta
amplitude compared to those recorded in the stratum pyramidal layer or cortical layers (Figure 1(d) to(g)), this layer was selected as the main source ofthe hippocampal data for theta-related studies.
Despite the greatest changes in LFP power occurring
immediately following the occlusion of MCA or CCAs,
statistical comparisons were made only during thesecond half of the occlusion period due to incomplete
recording among rats that underwent dMCAO
procedures.
We found that during baseline recording, the hippocampus is dominated by the HTD state (Figure 3(a)).
However, HTD duration was reduced following CCAO
in the bilateral hippocampi and following dMCAO in
the ipsilateral hippocampus. CCAO also increased thefrequency of altering states between HTD and LTD byshortening the duration of each state, as demonstrated
by a temporary but significant increase in the numberof discontinuing HTD periods during CCAO in contralateral hemisphere (Figure 3(b)). Although T/D amplitude ratio tended to reduce after mild cerebral ischemia
by CCAO, a significant and greater reduction of T/Damplitude ratio was found only after dMCAO and persisted throughout ischemic reperfusion (Figure 3(c)).
The prolonged decrease in the hippocampal T/D amplitude ratio following dMCAO is reminiscent of thedelta predominance, i.e. a typical feature in non-theta
state, frequently observed in human EEG recording
near infarct shortly after stroke onset,57 suggestingFigure 2. Cortical stroke increases the occurrence of sharp-wave-associated ripples and high-frequency discharges in the hippocampus. (a) Selective traces of local field potential (LFP) during baseline (left) and dMCAO reperfusion (right) period recorded in theipsilateral CA1 pyramidal layer (red) and the stratum radiatum layer 200 mm below (blue). Each sharp wave event in the radiatum (light
blue arrows) was associated with high-frequency oscillations, also known as the sharp-wave-associated ripples (SWRs) (orange
arrows) in the CA1 pyramidal layer. Increased occurrence of SWRs with longer duration is often observed during reperfusion ofdMCAO compared to those during baseline. (b) Frequency spectrograms of the LFP recording from CA1 pyramidal layer during
dMCAO display the presence of physiological SWRs in the frequency range of 150–250 Hz (left) and the high-frequency discharges in
the range of 250–450 Hz embedded in the ripple discharges (right), corresponding to traces in the left and right panel in A,
respectively. An increase in the occurrence of SWRs (150–250 Hz) (c) and HFDs (250–450 Hz) (d) was detected in the bilateral CA1
pyramidal layers during MCA reperfusion compared to baseline in the bar graphs, respectively (ipsilateral: open bar panel; contralateral: solid bar panel). (e) A relative increase in HFDs to SWRs was observed in the contralateral CA1 during occlusion andreperfusion of dMCAO. In contrast, CCAO did not increase either SWRs or HFDs occurrence during reperfusion but only transiently
increased the numbers of HFDs relative to SWRs in the contralateral CA1 pyramidal layer during the first 30 min of occlusion.
*p < 0.05, **p < 0.01, ***p < 0.001 (for dMCAO); §p < 0.05 (for CCAO) for multiple comparisons between baseline and other levels.
dMCAO: N ¼ 10; CCAO: N ¼ 7. (f) Hematoxylin and eosin (H&E)-stained coronal brain sections of a stroked rat showing the infarct
area (black arrows). The volume of the infarct is 24.85 ! 7.50 mm3 or 5.36 ! 0.99% of the ipsilateral hemisphere (N ¼ 4). Tissue wascollected 1–1.5 h after reperfusion. (g) Higher magnification of the ipsilateral infarct area and contralateral homotopic area in thecortex. The red arrow shows the pnykotic nucleus of an apoptotic cell. Scale bars are 100 um for magnification % 10 and 20 um for
magnification % 40.

1784

Journal of Cerebral Blood Flow & Metabolism 40(9)Figure 2. Continued.

that ischemic stroke might potentially aﬀect neuralactivity of brain regions beyond the cortex.

Cortical ischemic stroke temporarily impacts
hippocampal theta and gamma oscillations
Because a wealth of literature suggests that the reduction of cerebral blood flow results in the decrease orloss in EEG amplitude and frequency,13,14,57 we next
determined whether brain ischemia aﬀected oscillationsin the hippocampus that is remote from the ischemic
core. We found that the delta power was only transiently increased for less than 5 min in the hippocampal
slm layer following an episode of mild ischemia via
bilateral CCAO; interestingly, it normalized quickly
likely due to the robust collateral flow compensation
via the circle of Willis.58 Similar changes in delta
power were detected in the contralateral hippocampus(Figure 4).
Specifically during the HTD phase, a temporary
reduction of theta and gamma power was detectedbilaterally in the hippocampal slm during dMCAO
period (Figure 4(c) and (d)). Theta and gamma power
was most aﬀected during the initial period of occlusion,
while gradually restored during ischemic reperfusion(Figure 4). However, despite the gradual recovery ofbilateral hippocampal oscillations towards baseline
level, the power of delta, theta and gamma exhibited
great fluctuations during ischemic reperfusion ofdMCAO, suggesting a lasting eﬀect of stroke on brain
oscillations (Figure 4(a) and (b)).
A similar reduction of theta and gamma power was
also observed in the contralateral CA1 field of the

hippocampus only after dMCAO (Figure 5). CCAO
induced a transient increase in delta power in ipsilateral
CA1 during the first 30 min of occlusion period, further
suggesting that hippocampal blood flow was only
impacted for a very brief period of time during
ischemia.

dMCAO alters cortical gamma amplitude coupled tothe hippocampal theta phase
In light of the significance of cross-frequency coupling
between oscillations in network communication andinformation exchange, we investigated the dynamics
of theta–gamma interactions during acute ischemia insimultaneous recordings from the hippocampus andcortex. We found that two distinct gamma bands inthe cortex displayed dynamic and diﬀerential phaseamplitude modulation. There was an increase in themodulation index between cortical high gamma bandand hippocampal theta (4–7 Hz) (MIHiG-theta) or low
frequency oscillations (0–4 Hz) (MIHiG-Lowfreq) during
MCA occlusion and reperfusion period in the ipsilateral CA1 layer during HTD phase (Figure 6(a), (c) and(e)). Increased coupling of cortical low gamma and hippocampal low frequency oscillations (MILoG-Lowfreq)
was observed during MCA occlusion in bilateral CA1(Figure 6(a), (c) and (e)). Interestingly, the main highgamma activity appeared to be coupled to the phase ofdelta wave oscillation during MCA occlusion, whichfurther shifted towards lower frequencies (<1.5 Hz)
during reperfusion. Increased MIHiG in theta and low
frequencies phases was also detected in the ipsilateralslm layer during dMCAO occlusion periodHe et al.

1785

Figure 3. Cerebral ischemia reduces high theta state duration and decreases theta/delta ratio. The total duration of high theta/delta
brain states was summed for each 30-min period in the ipsilateral- (upper panel with open bars) and contralateral (lower panel withsolid bars) hippocampus slm layers for rats subjected to CCAO (blue) or dMCAO (red). (a) A temporary decrease in the HTD
duration was detected during both dMCAO and CCAO occlusion period compared to baseline. (b) A significant increase in thetransition between HTD and LTD, expressed as the number of HTD states, was observed during the first 30 min of CCAO occlusionin the contralateral hippocampus. (c) T/D ratio was decreased in the bilateral slm during dMCAO but not CCAO occlusion andpersisted during ischemic reperfusion compared to baseline period. *p < 0.05, **p < 0.01, ***p < 0.001 (for dMCAO); §p < 0.05
(for CCAO) for multiple comparisons between baseline and other levels. dMCAO: N ¼ 10; CCAO: N ¼ 7.

(Figure 7(a), (c) and (e)). A slight reduction in MILoGLowFreq was found in the contralateral slm layer during
CCAO occlusion (Figure 7(b) and (f)).
The main eﬀects of dMCAO or CCAO on brain
oscillations over time periods during acute ischemia
are summarized in Supplementary Table 1. Persistent
reduction of theta-gamma modulation is consistent
with the reduction of T/D ratio during the same
period of time. Because LTD occurrence was increased
during the occlusion period, the analysis was also
extended to LTD. However, there was no significant
change in the above parameters compared to baseline
duringLTDperiodofdMCAO
animals(Supplementary Table 2). Neither stroke nor mild
ischemia induced significant changes in delta or
gamma oscillations in the intact ipsilateral cortex
distal to ischemic lesion, or in the homotopic contralateral side (Supplementary Figure 1). The main eﬀects ofdMCAO or CCAO over time periods on delta andgamma power are summarized in Supplementary
Table 3.

Discussion
While the eﬀect of reduced CBF on scalp EEG has been
well documented in humans, the specific oscillatory
activity changes in deeper brain regions remote fromthe stroke lesion site still remain poorly understood.
With extracellular multi-site electrophysiology recordings conducted under urethane anesthesia, we detected
temporary decreases in the power of theta and gamma
oscillations following dMCAO but not CCAO, whichwere likely attributable to a quick recovery of CBF in
the latter due to robust collateral compensation. A
reduction in the high theta brain state was observed
after both dMCAO and CCAO, although a significant
and persistent reduction of T/D amplitude ratio was
only found after dMCAO. Furthermore, dMCAO but

1786

Journal of Cerebral Blood Flow & Metabolism 40(9)Figure 4. Cortical stroke induces temporary reduction in the power of theta and gamma oscillation in the hippocampal slm.
(a and b), representative 2-h time series for the parameters as indicated in rats subjected to temporary cortical stroke (dMCAO)
or mild ischemia (CCAO) for ipsilateral (a) and contralateral (b) recordings in the slm. Data are expressed in logarithm scale. Blacklines and gray shades represent mean and SD, respectively. (c and d), Quantification of temporal changes in hippocampal oscillatory
activity in ipsilateral (upper open bar panels) (c) and contralateral hippocampus (lower solid bar panels) (d). The continuous data foreach variable were grouped into four time periods as baseline (0–30 min), 1st occlusion (30–60 min), 2nd occlusion (60–90 min) andreperfusion (90–120 min). One-way analysis of variance (ANOVA) for the time periods was used to determine changes induced by
CCAO (blue) or dMCAO. Data for the 1st occlusion period in the dMCAO group were only partially plotted or excluded for statistics
due to a procedural delay. A follow-up post hoc analysis was performed to reveal a detailed temporal profile for each variable ifnecessary. Theta and low gamma (LoG) power was reduced in the bilateral stratum lacunosum moleculare during dMCAO occlusioncompared to baseline, while high gamma (HiG) power was reduced contralaterally. Unlike dMCAO, mild ischemia via CCAO did not
induce significant changes in theta or gamma oscillations compared to baseline. The time series plots show greater dynamic changes ofpower in all frequencies during dMCAO reperfusion not revealed by the averages of power during the time period *p < 0.05,**p < 0.01, ***p < 0.001. dMCAO: N ¼ 10; CCAO: N ¼ 7.

He et al.

1787

Figure 4. Continued.

not CCAO induced an increased occurrence of the
SWRs and HFDs during ischemic reperfusion period,
suggesting that cortical stroke aﬀected the interplay
between excitatory and inhibitory circuit in the hippocampus. The more pronounced changes in hippocampal oscillations following the dMCAO likely reflect
permanent neuronal loss in the cortex that resulted in
the disruption of brain connectivity between the cortex
and hippocampus or other limbic regions, as evidenced
by the change in phase-amplitude coupling between
hippocampal theta and cortical gamma oscillations.

Imaging-based connectivity data show that stroke inhumans leads to network disturbance not only in thevicinity of the brain lesion but also between remotecortical areas in bilateral hemispheres.59,60 Brainregions in the entorhinal cortex, perirhinal cortex andthe temporal lobe are reciprocally connected with thedMCAO lesioned regions in the rats, suggesting thatexperimental stroke may alter the input from thedamaged regions to the lateral entorhinal cortex, and/or by changing the synaptic activity among the remaining intact regions including the hippocampus.

1788

Journal of Cerebral Blood Flow & Metabolism 40(9)Figure 5. Stroke reduces theta and gamma power in the contralateral CA1 pyramidal layer. Comparison of temporal changes inhippocampal oscillatory power in the ipsilateral (A: upper open bar panel) and contralateral hemisphere (B: lower solid bar panel).
Multiple comparisons between baseline and the other time periods revealed that dMCAO reduced theta, low- and high-gamma powerin the contralateral hemisphere during occlusion compared to baseline period, while CCAO transiently increased delta power during
the first 30-min period of occlusion in the ipsilateral CA1 pyramidal layer. *p < 0.05 (for dMCAO); §p < 0.05 (for CCAO): p < 0.05.
dMCAO: N ¼ 10; CCAO: N ¼ 7.

He et al.

1789

Figure 6. Cortical gamma power is modulated by hippocampal low frequency phase in the CA1 following cortical stroke.
Comparison of temporal modulation of cortical gamma amplitude by theta phase (4–7 Hz) (a–d) and low frequency (0–4 Hz) phase(e–f) in the CA1 of the ipsilateral (open bar panels) and contralateral hemisphere (solid bar panels) revealed an increase of the thetagamma and low frequency wave-gamma modulation index in the ipsilateral hemisphere during acute dMCAO. Cross frequency
coupling was confirmed by the phase-amplitude comodulogram computed for LFP signals from baseline (left), during occlusion(one to two middle panels) and after reperfusion (right) in the ipsilateral and contralateral hemisphere following dMCAO (a) andCCAO (b) within the grids as indicated. The CA1 theta phase and low-frequency wave phase modulation of the cortical layer gamma
oscillations was increased for both low and high gamma bands, respectively. The increase of MIHiG-Theta and MiHiG-Low Freq also
extended to the reperfusion phase of dMCAO (c, e). CCAO did not lead to significant changes of MI of either gamma bands. *p < 0.05,**p < 0.01, ***p < 0.001 (for dMCAO) for multiple comparisons between baseline and other levels. dMCAO: N ¼ 10; CCAO: N ¼ 7.

1790

Journal of Cerebral Blood Flow & Metabolism 40(9)Figure 7. Cortical gamma power is modulated by hippocampal low frequency phase in the slm following cortical stroke. Comparisonof temporal modulation of cortical gamma amplitude by low frequencies oscillations phase in the slm layer of the ipsilateral andcontralateral hemisphere revealed an increase of the theta-high gamma modulation index (a–d) and an increase of the coupling
between high gamma and lower frequencies (0–4 Hz) (e–f) in the ipsilateral hemisphere during acute dMCAO. The phase-amplitude
comodulogram (a–b) confirmed the coupling between theta or low frequencies and high oscillations observed in the bar graphrepresentations (c–f) in the ipsilateral hemisphere during MCA occlusion. In contrast, little change in theta-gamma modulation or
lower frequencies coupling was observed following CCAO except for a brief decrease of MiHiG-Theta (c) and MiLoG-Low Freq (e) during
the CCA occlusion in the slm layer of the ipsilateral side. *p < 0.05, **p < 0.01, (for dMCAO); §p < 0.05 (for CCAO) for multiple
comparisons between baseline and other levels. dMCAO: N ¼ 10; CCAO: N ¼ 7.

He et al.
Our recent connectome analysis at the mesoscale confirmed that dMCAO reduced the reciprocal connections of the remaining intact brain regions involved inspatial information processing,11 which could lead toaltered connectivity and functional activation among
these regions. Some of the perturbation of brain connectivity reported after stroke could be attributed toaltered excitotary and inhibitory drives. For example,
GABAaR downregulation is observed after stroke bothpre- and post-synaptically in ischemic area,61 while invitro blockade of GABAaR increases the excitability of
infarcted slices after ischemia.62 In addition, GABAaR
and GABAbR are downregulated after hippocampal
ischemia in guinea pig63 and after focal ischemia in
mice,9 respectively. Because a long-term disruption ofcortico-hippocampal networks is necessary for cognitive decline, one limitation of the current study is thelack of brain oscillation data during chronic stroke. A
thorough investigation in relevant electrophysiologicalsignature in rats with chronic stroke is underway64 andwould be insightful to understand the long-term adaptation of brain recovering from acute ischemic brain
injury.
Because the generation and regulation of ripple
firing appears to depend on the precise interplay
between excitatory pyramidal neurons and inhibitory
interneurons,65–67 the aberrant ripple occurrence
observed in our stroke model is likely due to alteredexcitatory and inhibitory networks in the hippocampus caused by the ischemia-induced deaﬀerentation.
Consistent with this notion, augmented ripple oscillations were found in mutant mice with reduced AMPA
receptor in PV(þ) interneurons.16 However, the neuronal populations participating in the regulation ofphysiological ripple oscillations and their phenotypes
are complex, requiring orchestrated actions of synchronous activation of excitatory pyramidal cells andcertain classes of inhibitory GABAergic interneurons.68–70 Focal pharmacological blockade of
GABAA receptors abolished ripples,18 while an antagonist of a GABAAR5 enhanced the power and duration of the SWRs.62,67 Apart from the acutedisturbance of excitatory and inhibitory circuits asobserved in the current stroke study, a number ofmouse models of neurodegeneration with altered hippocampal inhibitory drive also displayed changes in
the SWR. For example, diminished ripple occurrence
rate was found in aged mice with human
Apolipoprotein E4 knocked in71 that led to the lossof hilar GABAergic interneurons,72 while rTg4510
transgenic mice, a model of tauopathy and dementia,
had reduced inhibitory control of hippocampal network and smaller amplitude-SWRs.73
Similar to our observation, mice undergoing unilateral photothrombosis in the hippocampus also

1791
displayed bilateral HFDs.51 Unlike the dMCAO
stroke that did not produce hippocampal injury, unilateral hippocampal photothrombotic stroke induced
extensive hippocampal infarction and elicited an immediate DC voltage shift and reduced gamma activity
ipsilaterally. However, some HFDs recorded in the hippocampal photothrombotic model in the range of 250–
450 Hz and reminiscent of epileptiform dischages werealso detected in our dMCAO model. Consistent with
the notion that aberrant or pathological ripples are
implicated in neurological diseases and functional deficits,74 high amplitude SWRs were reported in epilepsy
patients.75
It is well established that CBF reduction induces
EEG abnormality due to energy deprivation-inducedchanges in the membrane potential of neurons.13,14
Progressive loss of fast frequency rhythms and increasein low frequency ones such as the delta wave is often
observed with increasing degree of brain ischemia in theipsilateral cortical regions. Gamma oscillations in the
hippocampus are particularly sensitive to decreases in
pO2.76 The impairment in functional activation andaberrant hippocampal oscillations could also be
caused by below-infarct-threshold level of CBF reduction that goes far beyond the ischemic core, as supported by a recent study showing that the disruptionof neural activity in the barrel cortex surrounding
photothrombotic microinfarcts appeared to be inversely correlated with the distance from the infarctborder, resulting in a greater volume of neural deficits compared to the volume of infarct core by
12 folds.77
Hippocampal theta rhythm has been implicated in
the coding of information and coordination of cellular
processes related to memory.21,78–80 Apart from cognitive processes, the frequency and power of the hippocampal theta vary with behavior and were reportedlyaﬀected by movement speed,81,82 visual novelty83 andnasal respiration.84 Increased hippocampal theta amplitude was detected even under urethane anesthesia following the treatment of cognition-enhancing drugs.85
However, the change in hippocampal theta power recorded under anesthesia following brain ischemia
detected in our study is unlikely to be related to changes
in behavior as observed in the awake state. Instead, it ismore likely that the brain is transitioning into theenergy saving mode of slower oscillations after ischemia
due to reduced oxygen metabolism, coinciding with aconcurrent reduction in the amplitude of gamma oscillations. Stroke-induced reduction in the amplitudes ofmultiple frequency bands appears to persist until atleast one month after stroke (unpublished observation),
implicating a long-lasting eﬀect of ischemic injury on
the disruption of brain activity. Similar to ischemic
stroke, traumatic brain injury that produced cognitive

1792
impairment was also associated with reduced hippocampal theta amplitude and frequency.86
Interaction between oscillations in diﬀerent frequencies can occur via phase-amplitude coupling, resulting
in synchronization of amplitude envelope of fasterrhythms within the phase of slower rhythms. Phaseamplitude coupling is universally observed in several
brain regions including the hippocampus, basal ganglia
and neocortex of many species.53 Recently, the theta
phase modulation of gamma amplitude during place
cell firing has received great attention, and has beencausally linked to spatial navigation and cognitive performances in rodents.87–89 Unexpectedly, we found thatthe coupling between cortical gamma power and hippocampal theta as well as low frequency oscillations
was increased during dMCAO occlusion and persisted
after reperfusion. Interestingly, increased modulationof low gamma power was reported in the near deathstate of Wistar rats, although by alpha- (10–15 Hz) andtheta oscillations (5–10 Hz),90 providing a frame work
to explain the lucid consciousness experienced during
near death. On the other hand, the increased MI
detected in our stroke model may reflect an attempt
to compensate for the loss of connectivity between thecortex and hippocampus during ischemia, although thestate of cognitive processing or consciousness of thesestroke rats remains unknown. Consistent with previous
data, the slow wave phase that modulates the gamma
power in our study is likely to be related to anesthesia.90 In addition, another recent study found that anear-theta and respiration-entrained rhythm (>1 Hz)
was coupled to the 80–120 Hz gamma band in several
brain regions of freely moving mice,91 suggesting that
gamma can be modulated by a variety of slow oscillations relevant to the physiological states during crossfrequency coupling.
The main impediment curbing the translation of ourfindings stems from the recording condition, since general anesthetics are known to reduce spike activity.92
Nonetheless, urethane is considered a good choice ofanesthetic for physiological studies due to its modest
eﬀect on neuronal activity, e.g. it causes a small potentiation of GABA and glycine inhibition as well as a small
reduction in glutamate excitation in the brain comparedto other more selective anesthetics.93 Moreover, urethane anesthesia not only preserves all the brain rhythms
investigated in our study, but also induces spontaneousand rhythmic brain state alternations mimicking thenatural sleep in mice and rats.92,94 In fact, most of
our earlier knowledge in cell type-specific brain oscillations in the hippocampus came from studies using
urethane anesthesia.65 Due to its minimal eﬀects on
autonomic and cardiovascular systems and longlasting anesthetic eﬀect,95 it oﬀers a stable and reproducible platform for our experimental paradigm.

Journal of Cerebral Blood Flow & Metabolism 40(9)Furthermore, it is well established that hippocampal
theta rhythm recorded under urethane96 is comparable
to awake recording.97 A recent optogenetic study also
confirms that septal cholinergic input generates hippocampal theta in both awake and anesthetized conditions,98 further aﬃrming that urethane anesthesia isappropriate for studying network activity involving
the hippocampus. Lastly, urethane anesthesia has
been successfully used in the stroke community to
study patterns of intracortical LFPs as a diagnostic
for epileptiform discharge,99 as well as other oscillatorychanges,13,14 and it led to a similar infarct size as halothane anesthesia.100 Despite the eﬀect of anesthesia on
neuronal activity, many electrophysiological changes
detected in our study are specific to MCA stroke,
since they are not present during baseline nor following
CCAO that were also recorded under the same condition of anesthesia.
Apart from stroke diagnosis and monitoring the eﬃcacy of thrombolysis, a number of electrophysiologicalcorrelates have also been used to predict post stroke
disability and recovery.13 For example, an aberrant
interhemispheric asynchrony measured by EEG is a
useful predictor for post-stroke disability that outperforms conventional methods of focusing on delta
oscillations,101–103 although the overreliance on interhemispheric balancing as indices for recovery has beenchallenged.104 However, electrophysiological correlates
for cognitive recovery are not well established. For
example, the reliability of P300 wave, a large EEG positivity that peaked around 300 ms after the stimulus
appeared, in assessing post stroke cognitive processes
such as attention and memory has not been
proven,105,106 due to wide influence on P300 by biological and environmental factors.107 It is also suggested that the P300 may not be suitable for all
stroke subtypes, especially MCA stroke, nor forchronic stroke. To the contrary, the aberrant changeof hippocampal oscillations in the current study may
be a useful biomarker for post stroke cognitive function
if properly validated in awake rats performing behavioral tasks during chronic stroke, as some variables
have been proposed as the potential neural substrates
of cognitive impairment in brain disease.108,109
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of thisarticle: This work was supported by NIH grant R01
NS102886 (JL), VA merit award I01BX003335, Research
Career Scientist award IK6BX004600 (JL), the Eunice
Kennedy Shiver National Institute of Child Health &Human Development of the National Institutes of Healthunder Award Number K12HD073945 (AY), and the Center
for Neurotechnology (CNT, a National ScienceHe et al.
Foundation Engineering Research Center under Grant EEC1028725) (AY).

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.

Authors’ contributions
Ji-Wei He: performed the recording and analysis, and contributed to manuscript writing.
Gratianne Rabiller: performed the recording and analysis,
and contributed to manuscript writing.
Yasuo Nishijima: performed the dMCAO CCAO surgery.
Yosuke Akamatsu: performed the dMCAO CCAO surgery.
Karam Khateeb: performed a part of the data analysis.
Azadeh Yazdan-Shahmorad: performed some data analysis
and contributed to the overall manuscript editing.
Jialing Liu: provided the overal idea, design, logistics andwriting of the manuscript.

Supplemental material
Supplemental material for this paper can be found at thejournal website: http://journals.sagepub.com/home/jcb

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