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Dihydromyricetin improves social isolation anxiety induced synaptic loss and astrogliosis in mice hippocampus

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Dihydromyricetin improves social isolation anxiety induced synaptic loss and astrogliosis in mice hippocampus

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Dihydromyricetin Improves Social Isolation Anxiety Induced
Synaptic Loss and Astrogliosis in Mice Hippocampus

by

Chen Xue

A Thesis Presented to the
FACULTY OF THE USC School of Pharmacy
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
Molecular Pharmacology and Toxicology

August 2021

Copyright 2021 Chen Xue
ii
Acknowledgements
First and foremost, I am extremely grateful to my mentor, Prof. Jing Liang, for her
strong support and encouragement throughout my study and research. I wouldn’t go this
far without her help and patience. I also want to express appreciation to my other
committee members, Prof. Daryl L. Davies, and Prof. Martine Culty, for reviewing my
thesis and giving out advice and adjustments. Besides, I thank Dr. Richard W. Olsen
(UCLA School of Medicine) for useful discussions, and Dr. Xuesi M. Shao (UCLA School
of Medicine) for the biostatistics assay. This work was supported by the National Institute
of Health grants AA017991 (to JL), AA022448 (to DLD), and Carefree Biotechnology
Foundation.
Despite the hard time I had in the past two years like everyone else because of
COVID, I am glad that I finally made it. I sincerely thank Dr. Joshua Silva (a previous Ph.D.
student) and Alzahra (a Ph.D. candidate) in our lab for their mentoring and helping. I
sincerely thank Dr. Xiao-Nan Zhang for biochemical consultant and discussion. Apart
from them, I truly appreciate the support and assistance from all my partners and lab
mates, Jimmy, Ray, Angel, Saki, Lucy, Carson, and Max. I will always cherish the time
we spent together in the past two years. Finally, I am willing to say thanks to my beloved
parents for their understanding and selfless support.

iii

TABLE OF CONTENTS
Acknowledgements...........................................................................................................ii
List of Figures...................................................................................................................iv
Abbreviations………………………………………………………….……………...……….... v
Abstract............................................................................................................................vi
Introduction.......................................................................................................................1
Chapter1: Materials and methods .....................................................................................8
Animal housing and social isolation model.............................................................8
Drug preparation and administration......................................................................8
Behavioral test........................................................................................................9
Immunohistochemistry fluorescent staining..........................................................11
Image acquisition and analysis.............................................................................13
Chapter2: Result ............................................................................................................14
Mice declined cognition/memory and elevated anxiety level caused by social
isolation can be reversed by DHM treatment
Social isolation anxiety influenced GFAP-positive hippocampal astrocyte density
can be adjusted by DHM
Social isolation induced astrocyte morphological changes can be modified by DHM
Social isolation anxiety induced synaptic failure restored by DHM
Chapter3: Discussion......................................................................................................24
Bibliography....................................................................................................................27

iv
List of Figures
Figure 1. DHM ameliorates reduced the frequency and amplitude of mIPSCs after social
isolation or in AD animal model…………………………………………………………………3
Figure 2. DHM reduces 4-week social isolation-induced anxiety and improves
cognition/memory……………………………………………………………………...………16
Figure 3. DHM decreases 4-week social isolation-induced higher astrocyte density…...18
Figure 4. GFAP-positive astrocyte morphological analysis………………………………...20
Figure 5. Images of Synapse-astrocyte tripartite after 4-week social isolation…………...22

v
Abbreviations
AD Alzheimer’s Disease PFA Paraformaldehyde
DHM Dihydromyricetin VGAT Vesicular GABA transporter
EOAD Early-onset AD GFAP Glial fibrillary acidic protein
PSEN Presenilin ACSF Artificial cerebrospinal fluid
APP Amyloid precursor protein EPM Elevated plus maze
LOAD Late-onset AD OF Open field
APOE Apolipoprotein E CA1 Cornu Ammonis 1
mIPSCs
Miniature inhibitory
postsynaptic currents
DG Dentate Gyrus
CNS Central nervous system RI Recognition index
AChE Acetylcholinesterase NCR Novel context recognition
BDNF
Brain-derived neurotrophic
factor
GABAARs GABAA receptors
NGF Nerve growth factor ORI Object recognition index
GDNF
Glia-derived neurotrophic
factor
NOR Novel object recognition

vi
Abstract
The early onset of synaptic dysfunction accompanied by anxiety is usually found in
Alzheimer’s disease (AD) [1, 2]. An increasing incidence of individuals with anxiety
disorders will likely occur due to exacerbation from the long-term COVID-19 quarantine
[3, 4]. Using a social isolation mouse model, we observed that surrounding synapses, the
length of astrocyte branches, numbers of astrocyte all increased after 4 weeks of social
isolation. In parallel, the cognitive and memory abilities of the isolated mice worsened,
suggesting the synaptic loss is a mechanism of short-term social isolation induced anxiety.
We also found that dihydromyricetin (DHM) can restore the morphology of overall
astrocyte complexity by shortening their branches and decreasing branch numbers per
cell, suggesting DHM’s therapeutic effects on reducing astrogliosis. Astrocyte branches
stuck on neuron axons are also reduced following DHM treatment, indicating its effect of
preventing and repairing synaptic structure. Combined with our previous discovery of
silent inhibitory synapses in isolated mice [5], we have found a promising therapeutic
candidate. Our studies suggest DHM can be a novel early-stage intervention and
prevention strategy for AD.

1
Introduction
Alzheimer’s Disease progression and clinical diagnostic criteria
AD is a progressive neurodegenerative disorder and the most common type of
dementia among senior citizens [6]. AD mainly experience 3 stages: the first stage has
mild symptoms with anxiety, impaired concentration but no evident memory loss; the
second one may run into troubles with remembering important things and establishing
short-term memory; the last stage is considerably severe with significant behavior change
and is largely in need of assistance [7]. Three criteria are commonly used for clinical
diagnosis and management, including the 2011 NIA-AA criteria [7], the revised NINCDS-
ADRDA criteria [8], and the 5
th
edition Diagnostic and Statistical Manual of Mental
Disorders [9]. By evaluating levels of AD various biomarkers, AD-dementia can be
diagnosed accurately. Valuable AD biomarkers (amyloid/phosphorylated tau levels in
cerebrospinal fluid, amyloid/tau PET, and structural MRI) can give providers good
references [6]. However, AD progression may occur long before any characterizable
symptoms, for example, without any cognitive dysfunction. Neuropsychiatric symptoms
like anxiety and depression are most common in AD undetectable early stages [6]. In
clinical practice, symptom heterogeneity, especially in young adults, makes it hard to
diagnose and categorize. Those atypical cases usually progress rapidly and cannot be
diagnosed easily by a single test.

Alzheimer’s Disease risk factors
Since the 1990s, several risk factors have been identified to increase AD incidence
or accelerate AD progression. In earlier understanding, aging, as a major factor of AD
2
progression, leads to enlarged ventricular, shrunk overall brain volume and weight, and
massive synaptic loss in the brain. With the continuous deepening of research, compared
to the normal aging process, AD has been observed to share similar pathological changes
with earlier onset [10]. Concerning the difference between aging and AD, some
researchers believe that it is not qualitatively, while others think AD comes with no
retaining of personalities and interests [6]. Despite this, sleep has been highly associated
with aging-related diseases, while other lifestyle changes remain debatable. Sleep
disruption can speed up the accumulation of AD hallmarks—amyloid β and
phosphorylated tau protein [11]. Despite aging, genetic abnormality remains the strongest
risk factor for AD generation. Early-onset AD (EOAD) is characterized as a rare familial
genetic disease highly correlated to presenilin 1 (PSEN1), presenilin 2 (PSEN2), and
amyloid precursor protein (APP) mutations [12-14]. EOAD patients usually have AD
family history and present cognitive decline or even dementia before 65 years old. By
contrast, sporadic late-onset AD (LOAD), the most common one of all AD types, has a
later onset of average age at 74-year-old with the apolipoprotein E (APOE) ε4 allele being
a primary genetic risk factor. Various environmental risk factors have been related to AD
generation and processing, for example, chronic metal exposure particularly aluminum
[15], and brain trauma; cardiovascular diseases caused by blood vessel degeneration
also links to AD [10]. Neuropsychiatric symptoms were also shown to be significant risk
factors for AD [16]. Recent two decades, studies have found that post-traumatic stress
disorder (PTSD) after the diagnosis of PTSD shows a higher five-year incidence of AD
[17].

3
Dementia, anxiety, and social isolation
Dementia, one of the severe symptoms of neurodegenerative diseases,
particularly AD, usually has an earlier onset long before presenting distinct cognitive
decline. Several studies showed that synaptic loss occurred before fibrillary tau tangles
[18-20]. At early stages, patients exhibit synaptic dysfunction, or even complete loss to
various degrees [1, 2]. Anxiety disorders that appeared before cognitive symptoms are
commonly regarded as a precursor for mild cognitive decline. Long-term and/or high-level
exposure to stressors can contribute to anxiety occurrence [21]. Quarantine and isolation
greatly increase the incidence of mental health problems. The explosive growth of anxiety
orders prevalence is predicted in the post-pandemic period due to unavoidably COVID
policies [3, 4]. Social isolation, part of people’s life during COVID-19 shutdowns, is one
risk factor of AD [22]. Under socially isolated conditions, brains undergo a series of
physical and mental alterations triggered by chronic stressors, especially in the
hippocampus [23]. The hippocampus plays a major role in long-term memory, memory
consolidation, and emotions like fear, anxiety, and depression [24]. The hippocampus
circuits are highly plastic and vulnerable under stressful conditions [25]. The synaptic
connections, chemical and electrical signaling transduction underlie neuroplasticity and
functions [26]. Their dysfunction impairs learning, memory, and cognition.

4
Figure 1. DHM ameliorates reduced the frequency and amplitude of mIPSCs after (A) social isolation or in (B) AD
animal model.

In our previous studies [5, 27], we have demonstrated that after social isolation
(Figure. 1A) or in animal model of AD (Figure. 1B), miniature Inhibitory Postsynaptic
Currents (mIPSCs, A from Silva et al., 2020: red circle) both frequency and amplitude
were greatly reduced. The synapse became ‘silent’. The mIPSCs’ changes after the social
isolation are very similar to the pathological changes in transgenetic AD animal (B from
Liang et al., 2014, red circles). Also, both social isolated animals and AD animals show
behavioral memory impairment. Thus, we hypothesis that the ‘silent inhibitory synapses’
is critical mechanism underlying cognition/memory loss and anxiety behaviors. Therefore,
if the early synaptic deficits are intervened, the disease progression can be slowed down
or stopped, patients may be rendered 15 to 20 years without severe symptoms. Therefore,
these results indicate that silent inhibitory synapses can be a primary the target for the
treatment.

Glial cells in central nervous system (CNS)
Glia composes a large, ubiquitous, and interactive network in CNS to buffer ionic
concentration and maintain microenvironment balance and neuronal function [28]. Three
primary glial cells have been investigated to support the nervous system: microglia are
resident immune cells that involve in neuron formation, maturation, and fate [29];
astrocyte also play a critical role in neuroinflammation, and charge of vessel interaction,
and synaptic formation and function [30]; oligodendrocyte, whose myelination controlled
by proliferative oligodendrocyte progenitor cells, are dynamically wrap around axons and
regulate neuronal signal transduction [28]. In this study, we investigated the effect of
5
short-term social isolation to astrocyte and astrocyte-synapse interaction. Astrocyte is
extremely essential to synapse health by prolonging their branches to spines, and
promoting synaptic formation and neurotransmitter signaling [28]. Astrocyte-neuron
tripartite is a sophisticated structure that remain further exploration. Nevertheless, based
on current research findings, neurons rely on the metabolic and nutrient support from
astrocyte, whereas astrocyte release signals to stimulate or reshape synapse [31].

Current AD and neuroprotective therapeutic strategies
Speaking of traditional therapies, early therapeutic strategies for synaptic
maintenance include improving synapse efficacy by inhibiting acetylcholinesterase
(AChE) [32], inhibiting selective serotonin reuptake [33], and antagonizing NMDA [34].
Though AChE inhibitors and NMDAR antagonist memantine are approved by FDA for AD
treatment, they may still have limitations in intervening ongoing synaptic loss. Current
novel treatments are mainly focusing on enhancing brain compensatory mechanisms
which also known as the concept of cognitive conserve [35]. This strategy is able to fulfill
the demand on repairing early-phase synaptic dysfunction with accelerated synthesis of
various neurotrophic factors including brain-derived neurotrophic factor (BDNF), nerve
growth factor (NGF), and glia-derived neurotrophic factor (GDNF) [36]. In recent years,
anti-amyloid agents have attracted attention among potential AD drug development [37].
Notably, among several amyloid-target agents with promising safety and efficacy profile
in late clinical trials, the antibody aducanumab (Aduhelm) was firstly approved by FDA in
June 2021 [38, 39], and its mechanism is mainly to reduce insoluble amyloid plaques.
Other promising agents in phase 3 trials, including injectable antibodies like
6
gantenerumab and BAN2401 and small-molecule compound ALZ-801, also presented
remarkable efficacy in cleaning Aβ [37]. Undoubtedly, we are still in the long haul of
developing novel and superior drugs for AD, and gladly we keep seeing new
breakthroughs.

Dihydromyricetin (DHM)
Dihydromyricetin [(2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-
dihydrochromen-4-one (DHM) is an active flavonoid extracted from medicinal plant
Ampelopsis grossedentata. It has been widely applied in Traditional Chinese Medicine,
and has been reported to have anti-tumor, anti-inflammatory and anti-oxidative properties
[40]. The solubility of this compound is poor in water but nearly 1000-fold in ethanol, and
it tends to be oxidated and degraded within PH 6.0-8.0 value range [40].
In this study, we introduce DHM as a treatment targeting silent inhibitory
synapses. Our previous studies have demonstrated that DHM has anxiolytic properties
by restoring GABAA receptors (GABAARs) function and gephyrin expression levels [41-
43]. Reported as a regulator in the immune system [44], the downregulation of GABA can
result in the initiation of neuroinflammatory changes. Overactivation of the immune
system disturbs the brain’s homeostasis and contributes to neurotoxicity, particularly
synaptic toxicity [45]. A possible mechanism of cognitive/memory decline in socially
isolated mice will be illustrated in this study. To maintain health and integrity of brain
synapses, glial cells, especially astrocytes, are essential in balancing the synaptic
microenvironment. Overactivated astrocytes with long branches can be toxic and cause
a series of pathological processes. Our current results showed that DHM could ameliorate
7
toxic astrocyte overactivation and synaptic dysfunction in 4-week socially isolated mice.
We found that DHM has excellent potential in intervening early synaptic dysfunction.
Therefore, it is promising to further develop DHM as a novel pharmacological therapy in
anxiety and AD.

8
Chapter1: Materials and method
Animal housing and social isolation model
All six-week-old C57BL/6J mice were purchased from The Jackson Laboratories
(Bar Harbor, Maine, United States). They were all housed in vivarium with preset 12-hour
light/dark cycle, proper humidity, and room temperature. 2 or 3 mice were allocated in
each cage for group housing, and all those cages are placed in one space of the shelf
without obstruction between transparent cages. For social isolation groups, each mouse
was separated into single cages wrapped with black plastic bag for depriving any social
interaction with other mice upon arrival.
Groups are listed below:
a. 2-week group housing plus 2-week group housing with vehicle treatment.
b. 2-week social isolation plus 2-week social isolation with vehicle treatment.
c. 2-week social isolation plus 2-week social isolation with DHM treatment.
All experimental protocols were approved by the USC IACUC, and all methods
were carried out in accordance with relevant guidelines and regulations.

Drug preparation and administration
DHM (HPLC purified ≥ 98%, Master Herbs Inc., Pomona, CA) was prepared in
0.5 x 0.5 x 0.5 cm
3
cubic agars with sucrose. Agarose powder (Select Agar, Invitrogen)
was dissolved in 90-degree DI water to make 3% agar solution, then 0.8% sucrose or
mixture of sucrose and DHM (2 mg/kg) was weighted and added into agar solution. After
well mixing, the solution was poured in modes and given time to cool. Both vehicle and
9
DHM agar (2 mg/kg) cubes were administrated orally once a day for two weeks during
the dark period of the 12-hour light/dark cycle.

Behavioral test
Elevated plus maze
As described in our previous study, we followed a generally used protocol to
evaluate mice anxiety level after chronic social isolation with or without treatment [46].
The plastic apparatus of elevated plus maze is 50 cm above floor with axisymmetric two
open arms and two closed arms. The connection of all arms is an 8 x 8 cm
2
central
platform. Plastic boards Arms are all in 25 x 8 cm
2
(Length x Wide) size: open arms have
no walls so that mice can directly observe surrounding environment without obstacles;
while closed arms have one U-shape 20 cm-high black opaque wall with only on entrance
to central area. Mice behavior throughout the test was recorded by a ceiling-mounted
camera with a clear view of all arm entrances. When turning on recording, mice were
placed on the central area facing one open arm. The whole test session lasts for 5 mins
in quiet dark vivarium without any disturbance or noise from lab experimenters. Time
measuring or entry counting was started when at least three paws of mice entered any
arm or central space. Various parameters were collected as listed: number of entries into
open/closed arms/central platform and separate time spent in each of these areas. All
measuring steps were completed after clip recording in a double-blind manner.

Open field test
10
We applied a previously used open field test protocol [46, 47] to track mice
locomotor activity and exploratory behaviors. The open field apparatus we used is a 50
cm x 50 cm x 38 cm white acrylic plastic chamber. The 50 x 50 cm
2
flat floor was divided
into 5 x 5 grids by drawn lines, and each square is in 10 x 10 cm
2
size. We manually
defined the inner area as the central nine squares while the outer area was defined as
the rest of 16 side squares. Mice behavior throughout the test was recorded by a ceiling-
mounted camera with a clear view of all squares and chamber walls. When turning on
recording, mice were placed at the center square. The whole test session lasts for 10
mins in a dark vivarium without interruption or noise from lab experimenters. The
parameters collected are listed as follows: initial time (the duration time from when the
mouse was placed on the maze till the mouse start moving), tail-up time (the total time
of the mice lift their tails), the time spent in the central zone, distance (cm) traveled in the
apparatus (determined by measuring the full distance of the mouse nose moved relative
to the drawn 10 x 10 cm square on the floor of the open field chamber), and the numbers
of times of rears were counted for each animal during the 10-min session. Each recording
was observed three times by different experimenters to minimize errors in a double-blind
manner.

Novel Object Recognition (NOR)-- cortex
The protocol used here was according to previous reports [42, 48]. In short, rodents
spontaneously tend to spend more time on exploring a novel object longer than a familiar
one. On the first day of test, the animals could familiarize themselves with the vacant
open field for 5 mins. On the following day, they were rendered a 5-min session to explore
11
two identical, symmetrically placed objects. After twenty-four hours, the animals were
exposed to subjected to one familiar object (used before) and one novel object for a 3-
min retention test. Mice behavior throughout the test was recorded by a ceiling-mounted
camera with a clear view of all sides of items. We recorded the times of mice exploration
and calculated object recognition index [ORI% = (tn - tf)/ (tn + tf)], where tf and tn represent
times of exploring the familiar and novel objects, respectively.

Novel Context Recognition (NCR)--hippocampus
The protocol used here was previously described in reports [46]. In short, animals
were firstly exposed to two identical objects (i.e., two toy balls) in a round cage for 5 mins
and then in a rectangular cage for another 5 mins. After twenty-four hours, animals were
placed into either the round or the rectangular cage in which one of the objects was novel
for that context (i.e., a toy ball and a small cube are placed into the round cage). The
proportion of time spent investigating the novel “out of context” object versus the in
context object was calculated as recognition index [RI% = tnovel/(tnovel + tsample)] *100 by a
blinded scorer.

Immunohistochemistry fluorescent staining
Frozen brain sectioning and staining
Mice were dissected the next day after all 4-week experiments. Half brains were
collected and then fixed by 10% paraformaldehyde (PFA, Sigma-Aldrich) at room
temperature for overnight. Brain tissues were then rinsed by PBS solution (ROCHE) three
times and were transferred into 4°C 30% sucrose solution (Sigma, USA) at 4°C for 2-3
12
days till the floating tissue sink to tube bottom. Brains were washed three times by cold
PBS, and then were flash frozen in isopentane chilled with liquid nitrogen-precooled
isopentane. Samples were immediately stored at -80℃ for future experiments. For micro
sectioning, frozen brains were embedded in a mold with O.C.T. compound (Sakura) on
dry ice. 30 μm sagittal brain slices were sectioned by freezing microtome (Microm HM525
Cryostat) and placed on SuperFrost slides (VWR). We incubated slides in mouse anti-
GFAP primary antibody (1:500, Cell signaling #3670) and goat-host anti-mouse 550 nm
fluorescent antibody (1:250, Vector DyLight 550) in humidifier for two days and one day
separately at 4°C. Stained slides were finally mounted by DAPI mounting medium
(Abcam).

Fresh brain sectioning and staining
For inhibitory synaptic imaging, we gained fresh mice brain slices from vibratome
(Campden Instrument Inc., 7000smz) immediately after mice necropsy. 37°C Chamber
perfused with temperature-controlled artificial cerebrospinal fluid (ACSF) bubbled with 95%
oxygen/5% carbon dioxide. ACSF recipe was described before in previous study [41].
Fresh sections are immediately fixed by 4% PFA/4% sucrose solution for 30 minutes at
room temperature [49]. Then brain slices are incubated in guinea pig anti-VGAT (1:500,
Synaptic System), mouse anti-gephyrin (1:500, Synaptic Systems) and rabbit anti-GFAP
(1:500, Cell Signaling) primary antibodies for 3 days at 4°C. They are then incubated in
goat host anti-guinea pig Alexa fluor
®
594 (Thermo Fisher Scientific), anti-rabbit DyLight
550 (Vector Labs) and anti-mouse DyLight 488 (Vector Labs) secondary antibodies.

13
Image acquisition and analysis
All images were obtained using z-stack function (1 μm each step) of Cytation 5
Cell Imaging multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) except
figure 4D, then merged by Photoshop (Adobe). Quantification analysis of cell density and
morphological complexity was performed by Photoshop and Sholl analysis (Image J
plugin). Sholl analysis was done after changing RGB images to binary. Data showed in
mean ± SEM. All statistical analysis were operated using Prism (GraphPad Software, Inc.,
La Jolla, California, United States) two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001,
#
P<0.05,
+
P<0.05.

14
Chapter2: Results
Mice declined cognition/memory and elevated anxiety level caused by social
isolation can be reversed by DHM treatment
Using a previously described social isolation mouse model [50], We have examined
social isolation stress induced anxiety levels and the anxiolytic effects of DHM with the
elevated plus-maze (EPM) and open field (OF) tests. Group-housed mice treated with
vehicle agars (G2+Veh2) spent 2.49± 0.41 mins of a total 5 mins in open arms of the
elevated plus-maze (Figure 2A) and 2.23±0.34 mins in the closed arms. Vehicle-treated
socially isolated mice (Iso2+Veh2) spent significantly less time (1.61±0.28 min) in the
open arms compared to the closed arms (3.33±0.29 min). DHM-treated mice after 4-week
social isolation (Iso2+D2) resulted in greater time spent in the open arms (2.29±0.26 min)
when compared with untreated socially isolated mice. Based on these results, we suggest
that 4-week social isolation increases mice anxiety levels, and DHM administration
ameliorates this type of anxiety-like behavior, as observed with increased entry into and
longer time stay in the open arms.
To further investigate mice anxiety behavior, we analyzed the time and distance
mice traveled in the open field test (Figure 2B). Group-housed mice (G2+Veh2) traveled
further distances (3404 ± 178 cm), whereas isolated mice (Iso2+Veh2) demonstrated a
significant reduction (2333±117 cm) in average pathlength, suggesting that social
isolation compromised mice locomotive activity. In contrast, DHM-treated isolated mice
(Iso2+D2) resulted in recovered running distance (2904 ± 286 cm). The number of rearing
and time spent in the center of the open field was significantly decreased in mice housed
in isolation compared to that of group-housed mice (29.7 ± 1.9 times vs 49.3 ± 2.3 times).
15
Time mice spent in the outer space were increased compared to group-housed control
mice (71.1 ± 6.7 sec vs 27.3 ± 2.2 sec). Administration of DHM in mouse groups Iso2+D2
increased the number of rearing (42.6 ± 6.4 times) and the time in the center (17.4 ± 2.6
sec) while decreasing stay in the corner duration (38.7 ± 5.6 sec). Collectively, these
results suggest that isolation decreases exploratory/locomotor activity in adult male
C57BL/6J mice and that DHM treatment ameliorates these behavioral responses in
socially isolated mice.
The knowledge that something has been previously experienced is called
recognition memory [51]. This process is composed of at least two elements: the
familiarity of items; and the contextual information (spatial and/or temporal) in which items
were encountered. Experimental evidence indicates that peripheral and insular cortices
are required to consolidate familiar objects, and the hippocampus is necessary for the
consolidation of contextual information of recognition memory. In this study, we use
behavioral tests for evaluating the effects of dihydromyricetin (DHM) in both components
of recognition memory. We used the novel object recognition (NOR) test for familiarity of
items, and we used novel context recognition (NCR) for contextual memory.
After two weeks of social isolation and two weeks of treatment, the recognition
memory level of mice was evaluated with NOR tests (Figure 2C). G2+Veh2 mice spent
more time exploring the novel objects (ORI = 66.3 ± 4.7 %) than Iso2+Veh2 mice (ORI =
55.3 ± 4.1 %). DHM significantly improved isolated mice recognition memory by around
9% (ORI = 64.2 ± 3.8 %). Similarly, RI of NCR was calculated in all groups of mice (Figure
2D). Compared with G2+Veh2, Iso2 +Veh2 mice exhibited reduced RI (51.5 ± 6.5 %).
DHM treatment reversed the RI in Iso2+D2 mice and presented significant improvement
16
on contextual memory. These results indicate that daily oral-application of DHM improves
cognition memory in isolation induced anxiety mice model.

Figure 2. DHM reduces 4-week social isolation-induced anxiety and improves cognition/memory. (A) Effects of social
isolation and DHM treatment on anxiety-like behavior as measured by the time (sec) spent in the open, closed arms,
and the area between cross arms (called ‘intersection’) of the elevated plus maze. one-way ANOVA followed by multiple
comparison, Holm-Sidak method. For open arm, p < 0.001. For close arms; p < 0.001. For intersection, p = 0.78. (B)
Effects of social isolation and DHM treatment on locomotor activity, exploratory behavior as measured by initial time
(the time duration from when the mouse first placed into the center of the apparatus to start moving), rearing (total
number of times of rearing), running distance (total distance of moving), center time (the total time duration the mouse
stayed in the center 20 x 20 cm square), and corner (the total duration the mouse stayed in the 4 corner 10x10 cm
squares) in the open field assay. One-way ANOVA followed by multiple comparison, Holm-Sidak method. For initial
time, p < 0.001. For stay in corners P < 0.001. For running length, P < 0.001; For numbers of rearing, P < 0.001. For
17
stay in the center, P < 0.001. *, p ≤ 0.05 vs. vehicle group housing control (G2+Veh2). n = 8 mice per group. Effects of
social isolation and DHM treatment on mice recognition memory as measured by (C) object recognition index (ORI) of
novel object recognition test and (D) recognition index (RI) of novel context recognition test. n = 8 mice per group.
*p<0.01,
+
p<0.01, a one-way ANOVA followed by post hoc multiple comparison Tukey method.

Social isolation anxiety influenced GFAP-positive hippocampal astrocyte density
can be adjusted by DHM
Astrocyte, one major type of glial cells in central nervous system (CNS), responses
to various stressors rapidly. It also serves as a signature for AD pathologies [52]. Reactive
astrocytes overexpress glial fibrillary acidic protein (GFAP) all over the cell body,
therefore this protein is commonly used as a reliable marker for identifying astrocyte
morphology [53]. We suggest that social isolation induced anxiety disrupts brain
homeostasis and further activates glial cells as one of its protective mechanism, which
can also be neurotoxic when being overactivated. We double stained 30 μm-thick mice
brain slices with GFAP primary antibody (orange) and DAPI for recognizing each single
cell (Figure 3). The measurements were performed by calculating cell numbers per
10000 pixels using Photoshop. After 4-week isolation, astrocyte density showed an
increasing trend in both hippocampal CA1 and DG regions, indicating higher level of
neuroinflammation triggered by social isolation (Figure 3). 2-week DHM treatment,
compared to vehicle, lowered the rising level of activated astrocytes (Figure 3).

18

19
Figure 3. DHM decreases 4-week social isolation-induced higher astrocyte density. Representative 20x magnification
images of GFAP-positive astrocytes double-immunostaining in C57/BJ mice (A) Cornu Ammonis 1 (CA1) and (B)
Dentate Gyrus (DG). Blue, DAPI staining; orange, GFAP staining. Scale bar, 100 μm. (C) Quantification of
immunofluorescence for GFAP-positive astrocytes. G2+Veh2: 2-week grouping plus 2-week grouping with vehicle
treatment; Iso2+Veh2: 2-week isolation plus 2-week isolation with vehicle treatment; Iso2+D2: 2-week isolation plus 2-
week isolation with DHM treatment. n = 3-4 mice per group. *P<0.05, one-way ANOVA followed by multiple comparison.
Data showed as mean ± SEM.

Social isolation induced astrocyte morphological changes can be modified by DHM
Affected by deleterious stressors, the major damaged regions in brain are synapses.
The activated immune system can lead to a decline in synaptic function [54]. To maintain
synaptic structures, astrocytes increase the number and length of branch arms and bring
more neurotrophic factors and nutrients to the synapse. However, this process
(astrogliosis) may also be a trigger of scar formation and AD-like pathologies [55]. To
determine if there is any morphological changes of astrocytic arm numbers and branch
length, we applied Sholl analysis function, one common comprehensive branch
complexity parameter, from Image J (Fiji). We counted the number of interactions at a
series of distance from astrocyte nucleus and measured how those glial branches stretch
out as a pathological alteration. GFAP-stained images are adjusted to binary form for
further analysis (Figure 4A), and numbers of interactions are gained by defining the step
as 0.1 pixel (Figure 4B). Graphs show that more astrocytes are significantly activated
with elongated branches and increasing number of arms in the hippocampus of socially
isolated mice than in the group-housed mice. Notably, DHM lowered the elevated level of
inflammatory astrocyte activation. In 2-week DHM treatment group of mice, the
complexity of astrocytic branches in CA1 and DG regions fell to a similar level of grouped
mice with vehicle treatment (Figure 4C, 4D). These results indicate that when stimulated
by social isolation stressor, astrocytes increase their cell size with more numerous and
longer branches, and this sort of activation can be relieved by DHM 2 mg/kg treatment.
20

Figure 4. GFAP-positive astrocyte morphological analysis. (A) Representative binary images of single GFAP-positive
astrocytes in C57/BJ mice Cornu Ammonis 1 (CA1) and Dentate Gyrus (DG). (B) Representative of Sholl analysis for
astrocytes. Quantification for morphological complexity from (C) CA1 region and (D) DG region astrocyte. Group
Vehicle: 2-week grouping plus 2-week grouping with vehicle treatment; Isolation Vehicle: 2-week isolation plus 2-week
isolation with vehicle treatment; Isolation DHM: 2-week isolation plus 2-week isolation with DHM treatment (2 mg/kg).
n = 4. *, G+V vs. I+V;
#
, I+V vs. I+D;
+
, G+V vs. I+D. *P<0.05, **P<0.01, ***P<0.001,
#
P<0.05,
+
P<0.05, two-way ANOVA.
Graphs are shown as mean ± SEM.

Social isolation anxiety induced synaptic failure restored by DHM
Previous studies reported that a major component of the central nervous system –
synapses can also be seriously damaged resulting in declined cognition level and
memory deficits. Mice hippocampal astrocytes in both CA1 and DG regions are proved
to be overactivated affected by chronic social isolation stressors in this paper before. We
considered that astrocytic activation at the normal physiological standard is one main
compensatory mechanism to maintain sustainable synaptic function. However, when the
level of astrocytic activation exceeds tolerable range, this sort of neuroinflammatory
change leads to negative consequences, presenting a significant shortage in neurotrophic
G2+Veh2 Iso2+Veh2 Iso2+D2
CA1
DG
0.0 0.5 1.0 1.5 2.0
0
2
4
6
Distance from nucleus (pixels)
Number of interactions
G+V
I+V
I+D

,#

0.0 0.5 1.0 1.5
0
2
4
6
Distance from nucleus (pixels)
Number of interactions
G+V
I+V
I+D
, #
, #
,
A
B
C D
21
factors like brain-derived neurotrophic factor (BDNF). Loss of sufficient nutrients can
cause disastrous effects in the overall hippocampus including synapses which demand a
higher level of energy and support. At the same time, overactivated astrocytes turn this
protective mechanism to harmful, leading to the loosen synaptic cleft with fewer GABA
receptors potentiated (Figure 5A). According to what is been described previously
(Figure 3,4), we assumed elongated astrocyte branches approach synapse positions for
separating pre-and post-synaptic membrane (Figure 5A).
Therefore, to investigate if DHM can stimulate neuroplasticity and restoring
synaptic function, we detected the colocalization of tripart astrocyte-synapse structure.
Neuron-astrocyte double staining reveled that a higher intensity of hippocampal GFAP
expression in socially isolated mice, suggesting more activated astrocytes are sticked on
axons after 4-week isolation (Figure 5B, 5C). Interestingly, DHM significantly
downregulated overall GFAP expression with decreasing number and length of astrocytic
branches in hippocampus different subregions, proving its potential mechanism in
repairing neuronal damage (Figure 5B, 5C). To further illustrate the impact of DHM to
synapses, sagittal fresh mice brain slices were sectioned immediately after sacrificing in
37℃ ACSF perfusion for better keeping the synapse integrity. We stained vesicular GABA
transporter (VGAT, one inhibitory pre-synaptic marker protein), gephyrin (post-synaptic
marker protein) and GFAP (astrocytic marker) to visualize the relationship between
synapses and astrocytes. We found that there is less gephyrin expression in isolated mice
with more astrocyte branches around pre- or post- synapses (Figure 5D). Interestingly,
mice treated with DHM are found to have recovered synaptic cleft tightness, suggesting
22
DHM to be effective in ameliorating astrogliosis and reserve synaptic normal function after
four weeks of social isolation.

A
23
Figure 5. Images of Synapse-astrocyte tripartite after 4-week social isolation. (A) Schematic diagram of hippocampal
synaptopathy after 4-week social isolation. Astrocytes are overactivated after 4-week social isolation stress to damage
synaptic cleft further destroy synaptic neurotransmission. 2-week DHM treatment reversed this pathological process by
restoring GABA receptor function and reducing astrocyte activation level. Representative 20x magnification images of
GFAP-positive astrocytes and neuron double-immunostaining in C57/BJ mice (B) Cornu Ammonis 1 (CA1) and (C)
Dentate Gyrus (DG). Green, neuron staining; orange, astrocyte staining. Scale bar, 100 μm. (D) Representative 40x
magnification images of astrocyte and synapses in DG region. Red, vGAT, presynaptic marker; green, gephyrin, post-
synaptic marker; white, GFAP, astrocytic marker. Scale bar, 10 μm. Group Vehicle: 2-week grouping plus2-week
grouping with vehicle treatment; Isolation Vehicle: 2-week isolation plus 2-week isolation with vehicle treatment;
Isolation DHM: 2-week isolation plus 2-week isolation with DHM treatment (2 mg/kg).

24
Chapter3: Discussion
This study aims to evaluate the therapeutic effects and possible mechanism of DHM
in ameliorating cognitive symptoms. We found that DHM can improve recognition and
memory levels by reducing the activation level of hippocampal astrocytes and sticking
astrocytes on axons in Iso2+Veh2 mice. Astrocyte activation, or astrogliosis, has been
reported to protect acute neurodegeneration and brain injury depending on STAT3
signaling pathway [56-58], whereas exhibiting neurotoxic properties after chronic stress
[59]. In this case, abnormal long-term astrocyte activation does harm to neurogenesis
and probably synapse function. In our previous study [5, 27], we found that after social
isolation, miniature mIPSCs were significantly reduced. The synapse became ‘silent’. The
‘silent mIPSCs’ is related to cognition/memory loss and anxiety behaviors. In this
continuing study, we found that fewer astrocytic branches aggregated on neuron axons
after DHM treatment, indicating DHM effect on reducing astrogliosis (Figure 5B, 5C).
Further evidence is needed to illustrate the molecular mechanism of astrocyte activation,
including inflammatory cytokines changes. Astrocyte activation has long been
characterized as part of neuroinflammatory changes; astrocytes can still function by
establishing crosstalk with other cells like microglia. The further experimental plan should
focus more on uncovering the underlying mechanism of DHM therapeutic effects.
Neuropsychiatric symptoms are highly correlated to various dementia-related
illnesses, among which anxiety is the most common in AD patients, especially those with
more severe cognitive/memory decline and an earlier age when onset [60]. Loneliness
serves as an early predictor of psychological problems like anxiety disorder [61]. Further
progression of those problems increases suicidal thoughts and morbidity of severe
25
diseases. Loneliness and social isolation are also suggested to be closely associated with
later-life cognitive decline [62]. Previously, our results showed that 4-week social isolation
could significantly elevate C57/BJ mice anxiety level, while two weeks of DHM treatment
could reduce that
16
. In this study, we further illustrate the therapeutic effect of DHM in
improving 4-week social isolation-induced recognition and memory loss. Applying NOR
and NCR to test recognition and memory separately, we found a significant fall in mice
cognitive function resulted from a relatively short duration of social isolation. At the same
time, DHM exhibited its neuroprotective feature by preventing cognitive dysfunction. To
understand their underlying mechanisms, we analyzed both cell density and
morphological changes of astrocytes, one of the major components of the synapse
microenvironment. Our results presented that astrocyte increased their density per area
and average cell size in the hippocampus (CA and DG region) (Figure 3, 4). We
supposed that these neuroinflammatory alterations might contribute to neuronal damage
due to the increased GFAP fluorescent staining intensity around axons in socially isolated
mice hippocampus (Figure 5B, 5C). For stepping closer to synaptic clefts changes, we
chose one pre-synaptic marker (vGAT) and one post-synaptic marker (gephyrin) for
inhibitory synapses to visualize the association between astrocytes and synaptic failure.
Images showed that after social isolation, pre- and post-synaptic areas lose their
tightness, resulting in reduced GABAergic signal transduction (Figure 5D). This finding is
consistent with our previous result that the expression of gephyrin, the scaffold protein for
GABAergic neurotransmission, decreased in mice hippocampus. Based on these findings,
most importantly, we found that DHM has the ability to reverse pathological astrocyte
activation and reserve synaptic normal function.
26
Collectively, this study revealed a possible underlying mechanism of chronic social
isolation damage to mice cognition and memory. Astrogliosis could be toxic to the
synaptic junction, and the tightness of synapse are therefore lost with the abnormality of
signal transduction, especially in GABAergic neurons. DHM showed an excellent
therapeutic effect in reversing this kind of damage at the dose of 2 mg/kg. Therefore, we
suggest DHM be a potential candidate compound in early intervention strategy of delaying
dementia progression.

27
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Abstract (if available)

Abstract The early onset of synaptic dysfunction accompanied by anxiety is usually found in Alzheimer’s disease (AD) [1, 2]. An increasing incidence of individuals with anxiety disorders will likely occur due to exacerbation from the long-term COVID-19 quarantine [3, 4]. Using a social isolation mouse model, we observed that surrounding synapses, the length of astrocyte branches, numbers of astrocyte all increased after 4 weeks of social isolation. In parallel, the cognitive and memory abilities of the isolated mice worsened, suggesting the synaptic loss is a mechanism of short-term social isolation induced anxiety. We also found that dihydromyricetin (DHM) can restore the morphology of overall astrocyte complexity by shortening their branches and decreasing branch numbers per cell, suggesting DHM’s therapeutic effects on reducing astrogliosis. Astrocyte branches stuck on neuron axons are also reduced following DHM treatment, indicating its effect of preventing and repairing synaptic structure. Combined with our previous discovery of silent inhibitory synapses in isolated mice [5], we have found a promising therapeutic candidate. Our studies suggest DHM can be a novel early-stage intervention and prevention strategy for AD. ? 1. Terry, R.D., et al., Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol, 1991. 30(4): p. 572-80. ? 2. Scheff, S.W., et al., Hippocampal synaptic loss in early Alzheimer's disease and mild cognitive impairment. Neurobiol Aging, 2006. 27(10): p. 1372-84. ? 3. Hossain, M.M., A. Sultana, and N. Purohit, Mental health outcomes of quarantine and isolation for infection prevention: a systematic umbrella review of the global evidence. Epidemiol Health, 2020. 42: p. e2020038. ? 4. Hossain, M.M., et al., Prevalence of anxiety and depression in South Asia during COVID-19: A systematic review and meta-analysis. Heliyon, 2021. 7(4): p. e06677. ? 5. Silva, J., et al., Modulation of Hippocampal GABAergic Neurotransmission and Gephyrin Levels by Dihydromyricetin Improves Anxiety. Front. Pharmacol., https://doi.org/10.3389/fphar.2020.01008, 2020.

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Creator Xue, Chen (author)

Core Title Dihydromyricetin improves social isolation anxiety induced synaptic loss and astrogliosis in mice hippocampus

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Degree Conferral Date 2021-08

Publication Date 07/24/2021

Defense Date 07/22/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Alzheimer's disease,anxiety,astrocyte,cognition,dihydromyricetin,hippocampus,OAI-PMH Harvest,social isolation,synapses

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Advisor Liang, Jing (committee chair), Culty, Martine (committee member), Davies, Daryl (committee member)

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