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The therapeutic effects of dihydromyricetin (DHM) on anxiety disorders

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The Therapeutic Effects of Dihydromyricetin (DHM) on Anxiety Disorders

By

Alzahra J Al Omran

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSPHY
(CLINICAL AND EXPERIMENTAL THERAPEUTICS)

August 2022

ii
ACKNOWLEDGEMENTS

Undertaking the Ph.D. journey was a life-changing experience that I couldn’t have completed
without the endless help and support that I received from many people.
First of all, I would like to express my deepest gratitude to my advisor, Dr. Jing Liang, for
allowing me to work on this exciting project and for her invaluable guidance throughout my
research and professional development. Her continued encouragement, motivation, vision, and
creativity helped shape my scientific ideas and advance my research skills. I would also like to
extend my sincere thanks to my co-advisor, Dr. Daryl L Davies, his expertise, mentorship, and
advice guided me throughout the stages of my studies.
I am also grateful to my dissertation committee member, Dr. Enrique Cadenas, and my Ph.D.
proposal committee members, Dr. Culty and Dr. Beringer, for their insightful comments and
suggestions. I sincerely thank you all for your time and guidance that helped me shape my
investigations, without it, this project endeavor would not have been possible. I would like to
give a special thanks to Dr. Xuesi M. Shao (our long-term collaborator at UCLA) for his
countless guidance and for sharing his immense knowledge, especially during manuscript
preparation and proofreading. It was a great privilege and honor to work with him.
I would like to acknowledge my lab mates for their help, support, friendship, empathy, and
great sense of humor that created an excellent environment to work and thrive. I am deeply
indebted to my little angel (Jana) for putting up with the endless hours in the lab and the office
working on my research and dissertation. Words cannot express my gratitude to my husband for
his caring and endless sacrifices. Lastly, I would like to thank my parent and the rest of my
family for their continuous support, love, and prayers in the good and bad times. Without such a
team behind me, I doubt that I would be in this place today.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................ II
LIST OF FIGURES ..................................................................................................................... V
ABSTRACT ................................................................................................................................. VI
CHAPTER 1 .................................................................................................................................. 1
INTRODUCTION......................................................................................................................... 1
1.1 ANXIETY DISORDERS PREVALENCE AND MORBIDITY ............................................................ 1
1.2 TYPES OF ANXIETY DISORDERS ............................................................................................. 2
1.3 RISK FACTORS ....................................................................................................................... 3
1.4 ANXIETY DISORDERS COMORBIDITIES ................................................................................... 4
1.5 NEUROCIRCUITRY OF ANXIETY DISORDERS ........................................................................... 4
1.6 STRESS INDUCES NEURONAL FUNCTIONAL MORPHOLOGICAL CHANGES ............................... 6
1.7 PATHOGENESIS OF ANXIETY DISORDERS ............................................................................... 8
1.7.1 Neurotransmitters Imbalance ......................................................................................... 8
1.7.2 HPA Axis Dysregulation ............................................................................................. 10
1.7.3 Neuroinflammation ...................................................................................................... 11
1.7.5 Oxidative Stress ........................................................................................................... 15
1.7.6 Autophagy .................................................................................................................... 17
1.7.7 Neurotrophic Factors ................................................................................................... 20
1.8 ANXIETY DISORDERS PHARMACOTHERAPY ......................................................................... 21
1.9 DIHYDROMYRICETIN A NATURAL FLAVONOID ..................................................................... 23
1.9.1 Flavonoids .................................................................................................................... 23
1.9.2 Dihydromyricetin ......................................................................................................... 23
1.9. 3 DHM Neuroprotection (Therapeutics Benefits) ......................................................... 24
CHAPTER 2 ................................................................................................................................ 27
SOCIAL ISOLATION INDUCES NEUROINFLAMMATION AND MICROGLIA
OVERACTIVATION, WHILE DIHYDROMYRICETIN PREVENTS AND IMPROVES
THEM .......................................................................................................................................... 27
ABSTRACT ................................................................................................................................. 28
INTRODUCTION .......................................................................................................................... 29
METHODS .................................................................................................................................. 32
RESULTS .................................................................................................................................... 40
DISCUSSION ............................................................................................................................... 49
CHAPTER 3 ................................................................................................................................ 62

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DIHYDROMYRICETIN IMPROVES SOCIAL ISOLATION-INDUCED COGNITIVE
IMPAIRMENTS AND ASTROCYTIC CHANGES IN MICE .............................................. 62
ABSTRACT ................................................................................................................................. 63
INTRODUCTION .......................................................................................................................... 64
MATERIALS AND METHODS ....................................................................................................... 66
RESULTS .................................................................................................................................... 70
DISCUSSION ............................................................................................................................... 76
CHAPTER 4 ................................................................................................................................ 83
DIHYDROMYRICETIN AMELIORATES SOCIAL ISOLATION-INDUCED ANXIETY
BY MODULATING MITOCHONDRIAL FUNCTION, ANTIOXIDANT ENZYMES,
AND BDNF .................................................................................................................................. 83
ABSTRACT: ................................................................................................................................ 83
INTRODUCTION: ......................................................................................................................... 84
METHODS: ................................................................................................................................. 87
RESULTS .................................................................................................................................... 93
DISCUSSION: ............................................................................................................................ 102
CHAPTER 5 .............................................................................................................................. 109
CONCLUSIONS ....................................................................................................................... 109
BIBLIOGRAPHY ..................................................................................................................... 115

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LIST OF FIGURES
Figure 1. 1 The classification of anxiety disorders ......................................................................... 3
Figure 1. 2 Neural circuits involved in anxiety disorders. (ACC) anterior cingulate cortex, (BLA)
basolateral amygdala, (BNST) bed nucleus of the stria terminalis, (CeA) central nucleus of the
amygdala, (PFC) prefrontal cortex. Green arrows: principal inputs to the BLA; orange arrows:
principal outputs of the BLA; blue arrows: principal outputs of the CeA and BNST (Adapted
from (Nuss, 2015). .......................................................................................................................... 6
Figure 1. 3 Structure and function of neurons within the mesocorticolimbic brain systems.......... 7
Figure 1. 4 Mitochondrial antioxidative enzymes. ....................................................................... 17
Figure 1. 5 Schematic diagram of autophagy process. ................................................................. 19
Figure 1. 6 BDNF-TrkB signaling pathway in pre- and postsynaptic. ......................................... 21
Figure 1. 7 Chemical structure of dihydromyricetin (DHM). ....................................................... 24
Figure 2. 1 DHM reduces social isolation-induced anxiety 40
Figure 2. 2 Changes in gephyrin protein expression after social isolation and the effect of DHM
treatment. ...................................................................................................................................... 43
Figure 2. 3 The effects of social isolation-induced anxiety and DHM treatment on microglia
activation and proliferation in the hippocampal CA area. ............................................................ 44
Figure 2. 4 DHM treatment modulates microglia morphology in the CA1 and CA2 area of
hippocampus. ................................................................................................................................ 45
Figure 2. 5 The effect of DHM on the levels of serum corticosterone, p-NF-kB p65 protein
expression, and proinflammatory cytokines expression after social isolation. ............................. 47
Figure 2. 6 Schematic summary of the various pathways involved in social isolation induced
neuroinflammation and anxiety-like behaviors. ........................................................................... 50
Figure 3. 1 Cognition-related behaviors. ...................................................................................... 71
Figure 3. 2 Astrocyte density and size in the dentate gyrus.......................................................... 73
Figure 3. 3 Astrocyte morphology in the dentate gyrus................................................................ 75
Figure 4. 1 Study design and timeline of the experiments. 89
Figure 4. 2 SI mice exhibit an increase in H2O2 Levels and a decrease in mitochondrial
complexes I, II, and IV, while DHM normalizes them. ................................................................ 93
Figure 4. 3 Modulation of mitochondrial antioxidant enzymes activity and protein levels induced
by SI, repeated SI, and DHM treatment. ....................................................................................... 95
Figure 4. 4 Modulation of Bcl-1 and SQSTM1/p62 after SI, repeated SI, and DHM treatment. . 98
Figure 4. 5 Reduction of BNDF, p-TrkB, p-Erk p42, and p-Erk p44 protein levels after SI and
repeated SI; DHM improves them. ............................................................................................. 101

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ABSTRACT

The Therapeutic Effects of Dihydromyricetin (DHM) on Ameliorating Anxiety Behaviors

By

Alzahra J Al Omran

Anxiety disorders affect over 20% of the US population and 3.8% worldwide. Anxiety disorders
are considered the most prevalent mental disease and the sixth leading cause of disability. They
dramatically impact an individual’s quality of life, work performance, and well-being. Despite
the availability of several anxiolytic medications, many patients are struggling with treatment
resistance, disease relapse, or debilitating adverse effects. Regardless of the distinctive difference
in clinical presentation among the various types of anxiety disorders, they share various
pathogenesis pathways, including disruption in GABAergic neurotransmission,
neuroinflammation, oxidative stress, autophagy impairment, and BDNF reduction.
Dihydromyricetin (DHM), the major bioactive flavonoid extracted from Hovenia dulcis, exhibits
anti-anxiety effects and enhances cognitive and memory properties. Even though DHM's effect
in modulating anxiety behaviors and enhancing cognition was previously reported in multiple
disease models, a lack of thorough understanding of DHM's anxiolytics and enhancing
cognition/memory, the mechanisms remain to be elucidated. Using social isolation-induced
stress in C57BL/6J mouse model, I found that oral administration of DHM (2 mg/kg) restores
GABAergic function by up-regulating gephyrin levels. DHM ameliorates neuroinflammation by
inhibiting the nuclear factor kappa B (NFκB) pathway activation, thereby reducing the
production of proinflammatory cytokines and the overactivation of microglia and astrocytes.

vii
Moreover, DHM modulates alterations in mitochondrial function by enhancing the performance
of multiple mitochondrial antioxidative enzymes, including SOD2, HO-1, PRDX3, and GPX4,
thereby reducing oxidative stress. DHM improves the homeostasis of synaptic plasticity not only
by providing a neuroprotective effect and maintaining the viability of microglia and astrocytes
but also by reversing the stress impact on neuronal autophagy and the BNDF-TRkB signaling
pathway. My dissertation work collectively supports DHM's use as a novel therapy to treat
anxiety disorders and improve cognition and memory.

1

Chapter 1
Introduction

1.1 Anxiety Disorders Prevalence and Morbidity

Anxiety disorders have the highest prevalence rate of mental illnesses; approximately 42 million
(20%) individuals in the US and 300 million (3.8%) worldwide are affected annually (Saloni
Dattani, 2021). The lifetime prevalence of anxiety disorders in the Western world is 20-30%, the
rate of incidence is twice as high in women, then it is in men (Agarwal & Landon, 2019;
Bandelow & Michaelis, 2015; Collaborators, 2018; Kessler et al., 2005). Anxiety disorders elicit
a significant burden on societies due to their early onset (usually starting during childhood) and
as symptoms persist, it impacts patients’ quality of life, work performance, and overall well-
being (Costello et al., 2005). Anxiety disorders are considered the sixth leading cause of
disability, with adolescents and young adults having the highest incidence rate (Baxter et al.,
2014). The disorders are associated with a hefty economic cost, the amount spent on mental
health treatments in the US exceed $225 billion annually; not considering the indirect costs such
as reduction in work productivity and treatments associated with other comorbidities (Revicki et
al., 2012). These costs were raised even more during COVID-19, with clustering of various
emotional stressors such as fear, anxiety, bereavement, and loneliness due to social isolation,
which generated and exacerbated mental illnesses. The rate of anxiety disorders increased by
40% following the pandemic and social isolation orders, bringing the estimated cost of mental
health treatment to $1.6 trillion (CDC, 2020; Cutler & Summers, 2020).

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Anxiety is a state of increased apprehension and enhanced vigilance in the absence of an
immediate threat (Santos et al., 2019). It involves a range of behavioral reactions (avoidance and
scanning), emotional reactions (excessive or sustained fear and worry), and physiological
reactions (increased heart rate, blood pressure, and sweating). Anxiety and fear are commonly
regarded as related emotions, sharing many subjective and physiological symptoms, yet they are
different responses. Fear is a response to clear and present danger, while anxiety is a response to
an unknown or anticipated threat. Even though some sporadic anxiety responses are normal as an
evolutionary adaptation to alerts or threats, when anxiety responses are excessive, persistent,
uncontrollable, maladaptive, or negatively affecting daily life, they become pathological.
Likewise, fear is a critical response for survival during situations involving immediate threats,
however can lead to anxiety when it becomes chronic and abnormal (Sartori & Singewald,
2019).

1.2 Types of Anxiety Disorders

According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5)
and The International Classification of Diseases (ICD-11), anxiety disorders are classified into
the following categories: panic disorder (PD), agoraphobia, generalized anxiety disorder (GAD),
social anxiety disorder (SAD), specific phobia, selective mutism, separation anxiety disorder,
substance/medication-induced anxiety disorder, and anxiety disorder due to another medical
condition (Figure 1. 1) (DSM-5, 2013; ICD-11, 2022). Post-traumatic stress disorder (PTSD) and
obsessive-compulsive disorder (OCD) have been placed in separate diagnostic chapters in DSM-
5 and ICD-11 based on phenomenological and neurobiological differences. Despite the
differences in clinical presentation and diagnostic definitions among anxiety disorders, anxiety

3
and fear are the core features of all these diseases. They are often highly comorbid, indicating an
overlap in the neurobiological and pathological mechanisms (Reed et al., 2019). Therefore,
antianxiety drug development is frequently targeting these disorders concurrently.

Figure 1. 1 The classification of anxiety disorders

1.3 Risk Factors

Genetics and environmental factors, independently or combined, contribute to the increasing
vulnerability and susceptibility to anxiety disorders. Anxiety disorders aggregate in families;
however, the specific genetic variants and heritable components correlated with each anxiety
disorder- up to this point, are not well studied (Smoller, 2016). Thus, several meta-analytical
studies demonstrated that the offspring of individuals with anxiety disorders have a two-to four-
fold increased risk of developing one or more of anxiety disorders (Chantarujikapong et al.,
2001; Hettema et al., 2001; Lieb et al., 2002). The other risk factor is psychological stress,
childhood trauma and adulthood stressful life events are associated with various anxiety
disorders (Gilman et al., 2015; Matheson et al., 2013). Studies show that many forms of adverse
childhood experiences, such as disturbances in familial environments, sexual and physical abuse,

4
and low socioeconomic status are correlated with adult GAD, panic disorder, as well as
substance use disorders (Fergusson et al., 1996; Lähdepuro et al., 2019).

1.4 Anxiety Disorders Comorbidities

Mood disorders, particularly major depressive disorder (MDD), are frequently comorbid with
anxiety disorders; correspondingly, 50-90% of anxiety patients are also diagnosed with
depression (Assmann et al., 2018; Belzer & Schneier, 2004; Lenze, 2003; Y. Liu et al., 2018;
Olfson et al., 2017). MDD is characterized by distinctive symptoms like depressed mood, lack of
interest, and sadness; however, several other symptoms are overlapped with anxiety features,
such as sleep disturbances, attention deficits, and fatigue (Y. Liu et al., 2018). Studies also
showed that anxiety patients with comorbid MDD have a higher rate of anxiety recurrence,
persistent symptoms, and a lower recovery rate (Bruce et al., 2005). Additionally, alcohol
misuse, drug addiction, and medical conditions like chronic pain, diabetes, and heart disease are
frequently associated with anxiety disorders and contribute to the overall increase in disease
burden and severity (Andrew H. Rogers, 2019).

1.5 Neurocircuitry of Anxiety Disorders

Mood and anxiety disorders are believed to occur due to functional and anatomical disturbances
in the brain's emotion centers (Martin et al., 2009). This region mainly includes the
hypothalamus, hippocampus, nucleus accumbens (NAc), bed nucleus of the stria terminalis
(BNST), and amygdala, which communicate with each other along with the prefrontal cortex
(PFC) and insular cortex, to regulate behavioral and emotional responses. In general, the
neuronal activation during anxiety disorders is considered either fear-driven, associated with

5
social anxiety disorder and specific phobias; or worry-driven, associated with generalized
anxiety disorder and panic disorder (Duval et al., 2015). The amygdala, insular cortex, and
BNST are the primary emotion-processing regions and are believed to be overactivated during
fear-driven anxiety disorders, while the hippocampus and the PFC are more involved in emotion
regulation and become defective during worry-driven anxiety disorders (Daffre, 2020). Overall,
these brain regions are interconnected and contribute to anxiety pathogenesis (Figure 1. 2). To
elucidate the connectivity of the anxiety-involved brain regions, the amygdala receives negative
emotional signals from the thalamus, then activates the BNST and subsequently the
hypothalamus leading to the somatic manifestations of anxiety. The amygdala is a critical player
in processing anticipatory, avoidant, and fear-related behaviors; therefore, amygdala
overactivation causes anxiety pathogenesis (Daffre, 2020). The hippocampus mainly regulates
the conditioning of fear to contextual memories through projecting to the PFC (Kjelstrup et al.,
2002). The PFC provides top-down regulation of emotional response by receiving inputs from
the hippocampus and thalamus and projecting to the amygdala. Under physiological conditions,
the PFC exhibits both inhibitory and excitatory control over the amygdala activity. However, in
pathological conditions such as anxiety, this modulation is defective, leading to the chronic
overactivation of the amygdala and pathological behaviors (Liu et al., 2020).

6

Figure 1. 2 Neural circuits involved in anxiety disorders. (ACC) anterior cingulate cortex, (BLA)
basolateral amygdala, (BNST) bed nucleus of the stria terminalis, (CeA) central nucleus of the
amygdala, (PFC) prefrontal cortex. Green arrows: principal inputs to the BLA; orange arrows:
principal outputs of the BLA; blue arrows: principal outputs of the CeA and BNST (Adapted
from (Nuss, 2015).

1.6 Stress Induced Changes in Neuron Function and Morphology

Stress can have a long-lasting impact on brain circuitry, function and structure, that lead to
behavioral abnormalities as observed in neuropsychological disorders (Hoehn et al., 1997).
Synaptic plasticity is described as having activity-dependent changes in the strength and efficacy
of synaptic transmission between neurons. It governs the storage and perpetuation of information
in synapses, neurons, and neuronal circuits to regulate behaviors, memories, and learning
(Christoffel et al., 2011). Dysregulated synapse plasticity, under prolonged psychological stress
induces alterations to neuronal structure in the emotion processing/regulation regions including,
dendritic atrophy in the hippocampus and PFC, and dendritic spine density increases in the
amygdala and NAc (Figure 1. 3) (Becker et al., 2018; Christoffel et al., 2011; Magariños et al.,
1997; McKlveen et al., 2013; Mitra et al., 2005; Wellman, 2001). Consequently, the loss of these

7
neurons’ surface and dendritic materials, leads to synapse loss and deterioration in overall
neuroplasticity (Csabai et al., 2018). These morphological transformations are the core cause of
the brain’s structural changes that cause plasticity impairment leading to behavioral
abnormalities and cognitive decline (Csabai et al., 2018).

Figure 1. 3 Structure and function of neurons within the mesocorticolimbic brain systems.
Sagittal brain slice showing the neural circuits of the brain, highlighting the function and
pathophysiology of the major neuron types of each region. The solid red lines represent
dopamine neurons projections on NAc, PFC, amygdala, and hippocampal neurons. The solid
purple line represents GABAergic afferents from NAc to VTA. The dotted purple lines represent
glutamatergic afferents to the NAC from PFC, amygdala, and hippocampus. Each region has a
specialized neuron structure that contribute to the complex role of the region to modulate the
behavioral phenotypes (adapted from Christoffel et al., 2011).

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1.7 Pathogenesis of Anxiety Disorders

1.7.1 Neurotransmitters Imbalance

Neurotransmitters modulate the communication between the brain regions to regulate emotional
processing. γ-amino-butyric-acid (GABA) is the predominant inhibitory neurotransmitter in the
CNS, while glutamate is the primary excitatory neurotransmitter as well as the metabolic
precursor of GABA synthesis (Del Arco & Mora, 2009). The maintenance of both
neurotransmitter levels is strictly dependent on the synthesis of glutamine in astrocytes
(Mahmoud et al., 2019; Schousboe et al., 2014). Decarboxylation of glutamate in GABAergic
neurons by glutamic acid decarboxylase (GAD) enzyme is the rate-limiting step in GABA
synthesis (Schousboe et al., 2014). GABA is critical in maintaining the balance between
neuronal excitation and inhibition (Klausberger & Somogyi, 2008).

GABA receptors consist of two main superfamilies, GABAA and GABAB. GABAA is a fast-
acting ionotropic, ligand-gated chloride channel/receptor and is responsible for rapid signal
transmission inhibition. GABAB, on the other hand, is a metabotropic G-protein, coupled
receptor that results in slow and prolonged inhibitory action (Nuss, 2015). GABAARs are
composed of five different subunits from eight families (α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3);
the major and most common isomers are α1, β2, and γ2 that are arranged as α1γ2β2α1β2 in 40%
of the brain and as α2β3γ2 in the limbic system and cerebral cortex (Baumann et al., 2002; Baur
et al., 2006). GABAA has been considered a primary target for antianxiety, anesthesia, and
anticonvulsant medications due to the allosteric site that allows for superior and effective

9
anxiolytics and sedative effects. Benzodiazepines, barbiturates, and picrotoxin are among the
medications that function by acting on the allosteric site of GABAARs (Olsen et al., 1986).

GABAergic neurotransmission has been recognized as a prominent part of modulating anxiety
behaviors during the physiological and pathological states. An imbalance in the glutamate
excitatory signaling and the GABA inhibitory signaling could increase the activity in the
emotion-processing brain regions. Hence, effective tuning down of the excitatory
neurotransmission by GABA is essential for controlling and maintaining GABA homeostasis in
the brain. For this reason, the dysfunction of GABAARs play a critical role in the pathogenesis of
anxiety disorders (Sarup et al., 2003). Several clinical studies suggested a correlation between
GAD and genetic polymorphs of the proteins involved in the transcriptions of GABAARs
(Haxhibeqiri et al., 2019; Hettema et al., 2006; Kalueff & Nutt, 2007; Lacerda-Pinheiro et al.,
2014; Pham et al., 2009). Furthermore, panic disorder patients demonstrate a reduction in GABA
concentration levels along with impairment of GABA neuronal responses in the amygdala and
cerebral cortex (Goddard et al., 2004; Ham et al., 2007). Altogether, this clearly demonstrates the
role of GABA dysfunction in the pathology of anxiety disorders.

Disturbance in monoaminergic neurotransmitters, including serotonin, dopamine, and
noradrenaline is implicated in the etiology of anxiety disorders (Hamon & Blier, 2013). The
serotonin system is essential in modulating fear, mood, cognitive function, and is involved in
processing anxiety behaviors (Akimova et al., 2009). Several clinical studies highlighted the
reduction in serotonergic activity due to the downregulation of serotonin receptors' 5-
hydroxytryptamine (5-HT) binding site, in particular 5-HT1A, the primary inhibitory serotonergic

10
receptor (Akimova et al., 2009; Lanzenberger et al., 2007; Nash et al., 2008). Furthermore,
noradrenaline neurotransmission is a vital regulator of stress-induced responses in order to adapt
to both internal and environmental stimuli. Chronic increase of noradrenaline during chronic
stress leads to a persistent increases in sympathetic neuronal activity and turns the homeostasis
function of noradrenaline into pathological (Goddard et al., 2010). An additional
neurotransmitter that plays a key role in anxiety pathology is dopamine. Dopamine is known for
its primary role in modulating emotions by enhancing reward, motivation, concentration, and the
ability to experience pleasure (Coppen, 1967). Impairment of dopaminergic neuronal activity,
particularly in the mesolimbic system, aggravates anxiety symptoms (Tye et al., 2013). The
downregulation of the dopamine 2 (D2) receptor binding potential has been associated with
GAD and social phobia (Schneier et al., 2000). In general, monoaminergic neurotransmitters are
directly involved in the pathogenesis of anxiety disorders (Y. Liu et al., 2018).

1.7.2 HPA Axis Dysregulation

Fear and stress trigger the activation of the HPA axis and the secretion of corticotrophin-
releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and glucocorticoids, followed
by activating the autonomic sympathetic system to facilitate individual adjustment to unpredicted
stress (Phelps & LeDoux, 2005) (Bulfin et al., 2011). This physiological process is highly
efficient in maintaining the allostasis state in the body. However, chronic activation of this
defense system can lead to hypersecretion of glucocorticoids and subsequent glucocorticoid
receptors resistance. These changes result in allostasis load and overload, followed by
pathological behaviors of anxiety disorders (Chen et al., 2015; McEwen & Wingfield, 2003).
Various studies showed hyperactivity of the HPA axis in patients with different types of anxiety

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disorders indicated as an increase in cortisol serum levels and hippocampal glucocorticoid
receptors downregulation and resistance (Licznerski & Duman, 2013).

1.7.3 Neuroinflammation

Stress induces overactivation of the HPA axis and the autonomic nervous system, which in turn
provokes immune responses (Hoge et al., 2009; Won & Kim, 2020). Accumulating evidence
suggests that cytokines and chemokines mediate this network of communication, linking the bi-
directional relation between the immune system and the brain (Won & Kim, 2020). The release
of proinflammatory cytokines during an immune response is usually brief. However, systemic
inflammation during prolonged psychological stress can access the brain, which triggers several
pathological changes in neurons, microglia, and astrocytes. Persistent neuroinflammation impairs
neuroplasticity and induces structural and functional brain changes, specifically in the emotion
processing/regulation of the brain regions, leading to cognition and emotional behavior
abnormalities that eventually contribute to the pathogenesis of affective disorders and
Alzheimer's disease (Calcia et al., 2016; Salim et al., 2012; Won & Kim, 2020). Various clinical
studies have confirmed the signs of inflammation in anxiety patients demonstrated as an increase
in the serum proinflammatory cytokines levels such as tumor necrosis factor-alpha (TNF-α),
(Interleukin 6) IL-1β, Interleukin 6 (IL-6), and interferon-gamma (IFN-γ), and a reduction in
anti-inflammatory cytokines IL-10 and IL-4 in anxiety disorders patients (Hou et al., 2017;
Vieira et al., 2010; Vogelzangs et al., 2013).

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Systemic Inflammation and Neuroinflammation
Systemic inflammation can access the brain via multiple pathways, including afferent vagus
nerve signaling, cytokine transporter across the blood-brain barrier (BBB), and circumventricular
organs (Walker et al., 2019). Serval studies suggested that the vagus nerve communicates
peripheral immune information to the brain without interfering with the BBB to provoke
inflammation signaling (Bluthé et al., 1994; Ericsson et al., 1994). The other neuroimmune
communication pathway is circumventricular organs, brain areas that do not form BBB, where
circulating cytokines can access the brain (De Luca et al., 2014). Cytokines also can cross to the
brain through saturable transporters in a disease states-dependence manner (Xiang et al., 2005).
Moreover, numerous cytokines can be secreted by the BBB cells (Quan & Banks, 2007). This
systemic-central inflammation communication elicits neuroinflammatory responses that are
illustrated as persistent activation of microglia and astrocytes (Gyoneva et al., 2014).

Microglia Functions
Microglia are the first line of innate immune defense in the brain and a crucial component in
neuroinflammation. They are the most abundant myeloid cells, accounting for 10% of brain
cells; they are vital for brain homeostasis and involved in diverse physiological and pathological
processes (Prinz & Priller, 2014). Microglia are members of the monocyte macrophage family,
and their well-known function is the phagocytosis of endogenous and exogenous debris and
chemokine and cytokine release (von Bernhardi et al., 2016). Beyond the immune response,
microglia contribute to brain development and survival by stimulating neurogenesis and
removing excess neurons; they also support the BBB by actively regulating its permeability and
neurovascular unit integrity (Szepesi et al., 2018). Microglia have a vital role in synaptic

13
remodeling achieved by engulfing synaptic materials and forming dendritic spines. They also
promote synaptic plasticity by producing neurotrophic factors and neuromodulators (Paolicelli et
al., 2011; Parkhurst et al., 2013). In addition, microglia closely communicate with astrocytes by
releasing diverse signaling molecules necessary for astrocytes development and regulating the
innate immune functions and reactivity states (astrogliosis) (Jha et al., 2019).

In a physiological state, “resting microglia” are characterized by a ramified cell, small soma, and
long and defined processes phenotypes (Davalos et al., 2005; Jurga et al., 2020). They are
dynamic and highly active, consistently surveying the brain environment for pathogens and
cellular damage. Microglia are considered the most sensitive sensor of brain injuries. Once these
cells detect any signal of neuronal distress or damage, microglia undergo multistage
morphological changes and convert to “active microglia”. These complex structural alterations
include larger soma, shorter and thicker processes, and overall amoeboid and de-ramified shapes
that prepare microglia to execute their functions (Jurga et al., 2020). Given the fundamental
physiological and pathophysiological functions of microglia, it is not surprising that stress-
mediates microglial overactivation is associated with impaired neuroplasticity resulting in
neuropsychiatric and neurodegenerative disorders (Piirainen et al., 2017; Y. L. Wang et al.,
2018).

Astrocytes Functions
Astrocytes are considered the largest glial cell group in the brain; a single astrocyte is connected
to an average of 2 million synapses (Matejuk & Ransohoff, 2020). Astrocytes are pivotal in
maintaining brain homeostasis by providing the essential neuronal nutritious and neurotrophic

14
factors, eliminating waste metabolites, regulating blood flow perfusion, and maintaining the
integrity of the BBB (Sofroniew & Vinters, 2010). Moreover, they regulate synaptogenesis via
maintaining, pruning, and remolding of the synapses during health and diseases (Sofroniew &
Vinters, 2010; Spampinato et al., 2019). Astrocytes have a direct role in controlling
neurotransmitter levels by regulating the uptake of synaptic released glutamate and GABA,
metabolizing them, and then releasing their precursors back to neurons (Christopherson et al.,
2005; Garcia-Segura & Melcangi, 2006; Mahmoud et al., 2019; Perea et al., 2009).

Astrocytes become activated "astrogliosis" upon acute or chronic stress and undergo
morphological and physiological alterations, which could contribute to behavioral deficits
(Blackburn et al., 2009). During mild to moderate astrogliosis, astrocyte is characterized by
hypertrophy of the overall cell body size and shorter processes (Wilhelmsson et al., 2006).
However, in severe astrogliosis, a pronounced increase in proliferation rate, hypertrophy of the
cell body, thicker processes, and an increase in astrocyte functional gene expression are observed
(Sofroniew & Vinters, 2010). Several mechanisms and molecular pathways can trigger the
reactivity of astrocytes, including growth factors, cytokines, neurotransmitters such as glutamate
and noradrenaline, ATP, ROS, and β-amyloid (Sofroniew, 2009). Provided the essential
functions of astrocytes in brain viability, and most importantly, the astrocyte's role in synaptic
activity and neuronal circuits, astrocyte dysfunction significantly influences behaviors and
contributes to the pathogeneses of anxiety disorders and cognitive impairment (Volterra &
Meldolesi, 2005).

15

1.7.5 Oxidative Stress

Oxidative stress occurs when redox activity is imbalanced, resulting in excessive production of
free radicals, causing damage to several cellular components like proteins, lipids, and nucleic
acids (Lichtenberg & Pinchuk, 2015). The brain is vulnerable to oxidative stress damage owing
to the high oxygen consumption, which accounts for 20% of the total body's oxygen (Fedoce et
al., 2018). In addition, the regenerative capacity of neurons is limited, and the antioxidative
defense mechanism is relatively modest compared to various other cells throughout the body
(Fedoce et al., 2018; Yang & Herrup, 2007). Furthermore, neurotransmitters including
dopamine, norepinephrine, and serotonin are relatively easily auto-oxidized, while glutamate has
a high excitotoxic nature that increases generation of free radicals (C. T. Chen et al., 2008;
Hermida-Ameijeiras et al., 2004; Mailly et al., 1999). Finally, overactivated microglia and
astrocytes, as a response to brain homeostatic alteration, produce a massive amount of reactive
oxygen and nitrogen species (Nimmerjahn et al., 2005; Wilkinson & Landreth, 2006).

After four consecutive single-electron reduction processes, oxygen is converted to water by
mitochondrial complex IV to generate an electrochemical energy gradient to form ATP.
Approximately 1-2% of the oxygen used during mitochondrial respiration is reduced to
superoxide anions (O2
•−
), mainly in complexes I and III. Next, O2
•−
is immediately dismutated
into hydrogen peroxide (H2O2) by the superoxide dismutase (SOD) enzyme and subsequently
converted into water by the enzyme glutathione peroxidase (GPX) (Fedoce et al., 2018). Under
certain conditions such as stress, the mitochondria produce a larger amount of O2
•−
, which leads

16
to the depletion of antioxidative enzymes and the accumulation of H2O2, causing a state of
oxidative stress. An extremely oxidizing hydroxyl radical (OH
•
) is generated as a result of the
interaction of H2O2 with Fe
2+
, leading to various cellular structural and functional impairments
(Lin & Beal, 2006) (Figure 1.4). ROS-mediated damages impact the function of neuronal
circuity, neurotransmitter, and synapse plasticity, which eventually induce behavioral
abnormalities and cognitive decline commonly observed in neuropsychological disorders (Sarter
et al., 2007).

Increasing evidence points toward the link between impairment of cellular redox activity and the
pathology of neuropsychiatric and neurodegenerative disorders (Kasote et al., 2013; Lin & Beal,
2006). Maintaining the viability of mitochondrial antioxidative enzymatic activity is fundamental
to sustain oxidative phosphorylation (OXPHOS) activity. In the mitochondria, the first step in
the antioxidative defense system begins with Manganese superoxide dismutase (SOD2) , it
reduces O2
•−
to H2O2; followed by peroxiredoxins 3 (PRDX3), a mitochondrial-specific enzyme
mediating the conversion of H2O2 to water by the oxidation of thioredoxin (Trx) (Andreyev et al.,
2005)(Chang et al., 2004). Another antioxidative enzyme that plays a significant role in the
mitochondrial antioxidative system is glutathione peroxidase-4 (GPX-4), it primarily reduces the
phospholipid hydroperoxides by reducing H2O2 to water (Brigelius-Flohé & Maiorino, 2013)
(Figure 1.4). Redox status can also be evaluated by measuring the activity of heme oxygenase
(HO), this enzyme executes antioxidative action by catalyzing the pro-oxidant heme to free iron,
carbon monoxide, and biliverdin. Biliverdin is subsequently converted to bilirubin and acts as an
antioxidant by scavenging and neutralizing ROS (Schipper et al., 2019). HO enzymes have two
isoforms, HO-1 and HO-2. HO-1 is an inducible enzyme that is activated under stress,

17
inflammation, and oxidative challenges, while HO-2 is abundant and ubiquitous under
homeostatic physiological conditions (Neis et al., 2018). Altogether, variable changes in the
expression or activities of one or more of the mitochondrial antioxidant systems contribute to
psychiatric and neurodegenerative diseases, as demonstrated in human and animal models
(Kasote et al., 2013; Ruszkiewicz & Albrecht, 2015).

Figure 1. 4 Mitochondrial antioxidative enzymes.
Abbreviations: ETC: electron transport chain, GSH: reduced glutathione, GPx: glutathione
peroxidase 1, GPx4: glutathione peroxidase 4, GR: glutathione reductase, Grx2: glutaredoxin 2,
Grx5: glutaredoxin 5, GSSG: oxidized glutathione, NADP+: oxidized nicotinamide adenine
dinucleotide phosphate, NADPH: reduced nicotinamide adenine dinucleotide phosphate,
Prx3:peroxiredoxin 3, Prx5: peroxiredoxin 5, SOD1: superoxide dismutase 1, SOD2: superoxide
dismutase 1, Trx2OX: oxidized thioredoxin 2, Trx2RED: reduced thioredoxin 2, TrxR2:
thioredoxin reductase 2.

1.7.6 Autophagy

18
Autophagy is a crucial catabolic process responsible for maintaining cellular quality control by
degrading damaged organelles such as mitochondria, eliminating misfolded and aggregated
proteins, as well as recycling nucleic acids, lipids, and polysaccharides (Mizushima, 2018).
There are three types of autophagy: macroautophagy (the main type), microautophagy, and
chaperone-mediated autophagy (Hu et al., 2015). Macroautophagy is either nonselective -
targeting random and bulk cytoplasmic proteins or selective- targeting tagged cargos of damaged
mitochondria and ubiquitinated protein aggregates. Both pathways share a core autophagic
machinery thus, selective autophagy has unique receptors that tag the specific cargo to initiate
the processes (Lamark et al., 2017). The process of autophagy is initiated by sac formation, from
membrane material most likely originating from the endoplasmic reticulum. This sac is further
expanded to form an autophagosome, a double-membraned vesicle that fuses to lysosomes,
forming autolysosomes, where the autolysosomal contents are degraded by acidic lysosomal
hydrolases. Amino acids and lipid from such degradation events can be recycled and reused for
energy biosynthesis and the generation of essential macromolecules that contribute to cellular
rejuvenation (Ariosa & Klionsky, 2016). From sac to autolysosome formation, the autophagy
pathway involves several critical autophagic regulatory proteins controlling the dynamics of the
autophagy process (Figure 1.5).

Autophagy is induced by psychological stress and the associated conditions like nutrient
deprivation, low oxygen levels, reduction in growth factors, and oxidative stress (Mazure &
Pouysségur, 2010; Schneider & Cuervo, 2014; Tomoda et al., 2020). In the CNS, autophagy
plays a vital role in supporting neuron viability, survival, and development by continuously
providing amino acids and lipids required for cellular biosynthesis- (Maday et al., 2012).

19
Neuronal autophagy is crucial in regulating structural and functional plasticity by selectively
degrading local synaptic proteins, excess neurotransmitters, neurotransmitters receptors, and
synaptic vesicles (Tomoda et al., 2020). This is particularly important to counterbalance synaptic
protein synthesis to maintain tight control of local synaptic proteome, therefore protecting the
integrity of synaptic proteins and regulating synapse remodeling and function (Alvarez-Castelao
& Schuman, 2015). That being said, autophagy dysregulation might lead to neurotoxic effects
and synaptic plasticity dysfunction that contributes to the progression of behavioral deficits in
psychological and neurodegenerative disorders (Hu et al., 2015; Tomoda et al., 2020).

Figure 1. 5 Schematic diagram of autophagy process.
Cellular stress (deprivation, oxidative stress, misfolded or aggregated proteins) suppresses
mammalian target of rapamycin (mTOR) signaling leads to cascade of events (1) induction:
assembly of autophagy regulatory protein complex on the membrane ULK1 complex
(ULK1/ULK2, FIP200, ATG13) and PI3K-III complex I (AMBRA1, BECLIN), formation of as
adaptor protein LC3/ATG8 (2) LC3 recognizes p62/sequestosome 1 to induce the elongation of
autophagosomes to completely engulf the autophagic components (3) autophagosome trafficking
to fuse with lysosome to make autolysosomes (4) maturation and degradation.

20
Abbreviations: (AMBRA1) activating molecule in Beclin-1-regulated autophagy, (ATG)
autophagy-related protein, (BECLIN1) Bcl-2-interacting myosin-like coiled-coil protein,
(FIP200) FAK family-interacting protein of 200 kDa (LC3) microtubule-associated protein
1A/1B-light chain 3, (SQSTM1) sequestosome 1, (ULK) Unc-51 like autophagy activating
kinase. (Adapted from Tomoda et al., 2020)

1.7.7 Neurotrophic Factors

Brain-Derived Neurotrophic Factor
Brain-derived neurotrophic factor (BDNF) is the predominant neurotrophin (Rosenfeld et al.,
1995). BDNF is essential in neuronal proliferation, development, differentiation, survival, and
plasticity (Davies, 1994). BDNF is stored, synthesized, and released from glutamatergic neurons,
therefore, BDNF and its receptor tyrosine kinase B receptor (TrkB) are widely distributed at
glutamate synapses (Lessmann et al., 2003). The co-localization of BDNF-TrkB in the
glutamatergic synapse makes BNDF a dynamic regulator of synapses plasticity and
neurotransmission (Bramham & Messaoudi, 2005). Mature BDNF is formed following the
cleavage of the BDNF precursor (pro-BDNF), after binding to the TrkB receptor, activating
downstream transcriptional signaling via the mitogen-activated protein kinase (MAPK) pathway
(Licznerski & Jonas, 2018) (Figure 1.6). Activation of this transcriptional signaling cascade
produces several proteins that are critical for ensuring neuronal survival.

Chronic stress contributes to the reduction in BDNF concentrations, and growing evidence
shows that disturbances in the BNDF pathway underly mental disorders and neurodegenerative
disorders (Licznerski & Jonas, 2018; Miao et al., 2020; Z. H. Wang et al., 2019). In clinical
studies, serum BDNF levels have been used as a biomarker for several psychiatric disorders
(Chen et al., 2017; Sen et al., 2008; Suliman et al., 2013). Declined expression of BDNF is
attributed to an overall reduction in neurogenesis and synaptic plasticity that eventually

21
contributes to the volumetric atrophy of particular regions in the limbic system, such as the
hippocampus (Kalisch et al., 2006; Licznerski & Duman, 2013).

Figure 1. 6 BDNF-TrkB signaling pathway in pre- and postsynaptic.
Mature BDNF binds to TrkB receptor activating signaling cascades, this supports relevant gene
transcription and protein translation necessary for neuronal differentiation, survival and synaptic
plasticity. BNDF is essential for learning, memory formation, and for stress coping strategies.
(Adapted from Licznerski & Jonas, 2018)

1.8 Anxiety Disorders Pharmacotherapy

According to anxiety therapy guidelines, the recommended treatments are psychotherapy
(cognitive behavioral therapy), pharmacotherapy, or a combination of both (CPNP, 2022; Ströhle
et al., 2018; Walter et al., 2020). The current approved antianxiety medications can be classified
based on their mode of action to selective serotonin reuptake inhibitors (SSRIs), serotonin-

22
norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), monoamine
oxidase inhibitors (MAOIs), and Benzodiazepines (BDZs) (Sartori & Singewald, 2019). SNRIs,
MAOIs, and TCAs are prescribed in a disorder-specific manner. The overall use of MAOIs and
TCAs is less common due to safety and tolerability issues such as weight gain, dry mouth,
sedation, urinary retention, arrhythmias, and risk of death with overdose that are associated with
TCAs and dietary restrictions during MAOIs use (Bandelow et al., 2017; Garakani et al., 2020).
SSRIs including (es)citalopram, sertraline, and fluoxetine, are currently considered the first-line
therapy, demonstrating a better benefit/risk ratio compared to the rest of the anxiolytic
medications (Bandelow et al., 2017). Although this class of medication tends to be relatively
well-tolerated, SSRIs are associated with several adverse effects, such as gastrointestinal
problems, insomnia, and sexual dysfunction. SSRIs are also associated with increased suicidal
ideation and behavior (Garakani et al., 2020; Stübner et al., 2018). Another major issue with
SSRIs is delayed therapeutic onset for 2-3 weeks or even more, which limits the use of this class
as an acute anxiolytic (Thompson, 2002). Taken together, the adverse effects associated with
SSRIs compromise patient compliance and contribute to the reduction of therapeutic efficacy.
BDZs have been a longstanding drug of choice for anxiety in primary care settings and the most
widely used anxiolytics class due to their rapid onset of action (Agarwal & Landon, 2019;
Torres-Bondia et al., 2020). However, BDZs are associated with a high risk of tolerance,
dependence, withdrawal symptoms, misuse, cognitive impairment, and deaths by overdose, if
combined with alcohol (Baandrup et al., 2018). Therefore, recent recommendations suggest that
only patients without an active or a history of substance use are eligible to use BDZs and if so, it
is on a short-term basis (Baandrup et al., 2018). Additionally, it is no longer considered first-line
monotherapy for anxiety disorders and can only be used as adjunctive with SSRIs or SNRIs and

23
for short-term use (Gomez et al., 2018). Despite the availability of anxiolytic medications, only
50-60% of patients achieve clinically significant improvement due to poor adherence to
treatment, treatment-resistance, and relapse (Roy-Byrne, 2015; Taylor et al., 2012). Therefore,
there is an urgent need to develop novel approaches and new medications that provide a safe,
effective, and high remission rate.

1.9 Dihydromyricetin a Natural Flavonoid

1.9.1 Flavonoids

Flavonoids are natural compounds from the polyphenolic phytochemical family, found mainly in
plants, fruits, vegetables, and some medicinal herbs (Patil et al., 2020). These flavonoids are
divided into various classes based on their chemical structure, including flavanols, flavanones,
flavonols, isoflavones, flavones, and anthocyanins (Huang et al., 2017). Flavonoids chemical
structure is essential in their pharmacological activity characterization (Ko et al., 2020). Natural
flavonoids exhibit numerous health-promoting effects such as antioxidant, anti-inflammatory,
anti-allergic, antiviral, and anticarcinogenic activity (Ko et al., 2020).

1.9.2 Dihydromyricetin

Hovenia dulcis is an herbal plant, indigenous to East Asia and commonly known as the Japanese
or Chinese Raisin Tree, it was well known as an herbal remedy in ancient Eastern medicine
(Hyun et al., 2010). H. dulcis is a flavonoid that has been recognized to possess numerous
pharmacological properties such as antioxidant, antidiabetic, anticancer, anti-inflammatory,
hepatoprotective, and mitigation alcohol intoxication (Sferrazza et al., 2021). These therapeutic
benefits are linked to several secondary metabolites produced by different parts of the plant (Ko

24
et al., 2020). Among all the flavonoids isolated from H. dulcis, dihydromyricetin (DHM) is one
of the most abundant secondary metabolites (H. Li et al., 2017). DHM, [(2R,3R)-3,5,7-
trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one], is extracted from the seeds
and fruits of H. dulcis (Figure 1.7). It is well described as a bioactive component responsible for
lowering blood glucose, regulating lipid metabolism, counteracting alcohol intoxication, having
antiangiogenic (to prevent cancer), hepatoprotective, and neuroprotective properties(Han et al.,
2017; Liang, López-Valdés, et al., 2014; L. Liu et al., 2017; J. Silva, X. Yu, et al., 2020; Xie et
al., 2016; Zhou et al., 2017). It also showed potential in the treatment of asthma, osteoporosis,
nephrotoxicity; it demonstrates cardio-protective effects in myocardial ischemia-reperfusion
injury, atherosclerosis, and diabetic cardiomyopathy (T. T. Liu et al., 2017; Ravindran et al.,
2017; Wang et al., 2017; Wu et al., 2017; Wu et al., 2016; Xu et al., 2017).

Figure 1. 7 Chemical structure of dihydromyricetin (DHM).

1.9. 3 DHM Neuroprotection (Therapeutics Benefits)

Pivotal evidence has shown that DHM can cross the BBB: therefore, it has the potential for
neuroprotective effects (Youdim et al., 2003). The versatile neuroprotective action of DHM

25
might be due to its capacity to directly scavenge ROS and reactive nitrogen species (RNS) along
with inhibiting lipid-peroxidation (Li et al., 2016; Zhang et al., 2003). DHM also upregulates the
nuclear transcription factor-erythroid 2-related factor 2 (Nrf2) signaling pathway and enhances
the activity of antioxidative enzymes such as SOD, glutathione peroxidase, and catalase (Guo et
al., 2021; Q. Hu et al., 2018; Song et al., 2017). DHM exhibits a neuroprotective effect in
hypoxia-induced memory impairment and neurodegeneration animal models via protection of
synapse structures and enhancement of mitochondrial biogenesis (Liu et al., 2016). Additionally,
DHM has been demonstrated neuroprotective activity in a depression model by promoting an
anti-inflammatory effect, decreasing the production of proinflammatory cytokines IL-1β, IL-6,
and TNF-α and increasing the anti-inflammatory cytokine IL-10, mainly through the suppression
of the activation of nuclear factor-kappa B (NF-kB) and MAPK pathways (X. L. Hou et al.,
2015; Qi et al., 2012; Ren et al., 2018; Tang et al., 2016). DHM has also been demonstrated
indirect anti-inflammatory action by increasing the expression of BDNF in the chronic
unpredicted mild stress (CUMS) mouse model of depression (Ren et al., 2018). Moreover, DHM
treatment in an Alzheimer's disease (AD) animal model inhibited macroglia overactivation,
enhanced autophagy, and reduced apoptosis through modulation of various signaling pathways,
including silent mating type information regulation 2 homolog 1 (SIRT1), mTOR, and NLRP3
inflammasomes singling pathway, which promotes neuroprotection and prevent the progression
of AD (Kou et al., 2016). Taken together, DHM exhibits neuroprotective effects through
modulating diverse pathways in neurological disorders.

DHM's ability to modulate anxiety behaviors might be linked to its effect on potentiating
GABAARs. Shen and colleagues showed that DHM is a positive modulator of GABAARs and

26
acts on the benzodiazepine site, thereby counteracting alcohol intoxication (Shen et al., 2012).
Moreover, DHM treatment in an AD model, improved GABAergic transmission, restored
gephyrin levels, reduced anxiety-related behaviors, and improved cognition and memory (Liang,
López-Valdés, et al., 2014). To date, GABAergic modulation is the only studied pathway that
explains DHM's anxiolytic effect. However, the other pathways implicated in anxiety pathology
are yet to be investigated. In my dissertation, I tested the hypothesis that DHM mitigates anxiety-
like behaviors utilizing the social isolation-induced stress mouse model. This was accomplished
by studying the effect of DHM treatment on multiple underlying anxiety pathologies, including
neuroinflammation, oxidative stress, autophagy, and neurotrophic factors.

27

Chapter 2
Social isolation induces neuroinflammation and microglia overactivation, while
dihydromyricetin prevents and improves them

Alzahra J Al Omran
1†
aalomran@usc.edu, Amy S Shao
2
amy.shao@med.wmich.edu, Saki
Watanabe
1
sakiwata@usc.edu, Zeyu Zhang
3
zeyuz@usc.edu, Jifeng Zhang
1
jifengzh@usc.edu, Chen Xue
1
xue4182@usc.edu, Junji Watanabe
3
junjiwat@usc.edu,
Daryl L. Davies
1
ddavies@usc.edu, Xuesi M. Shao
4
mshao@g.ucla.edu, Jing Liang
1*
jliang1@usc.edu

(†) First author
(*) Corresponding author

Authors' information
1. Titus Family Department of Clinical Pharmacy, University of Southern California School
of Pharmacy, Los Angeles, CA, 90033, USA.
2. Homer Stryker M.D. School of Medicine, Western Michigan University, Kalamazoo, MI
49007, USA.
3. Translational Research Lab, School of Pharmacy, University of Southern California, Los
Angeles, CA, 90033, USA.
4. Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095,
USA.

28

Abstract

Background:

Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to
consume one-third of the country's mental health treatment cost. Although anxiolytic therapies
are available, many patients still exhibit treatment-resistance, relapse, or substantial side effects.
Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the
pandemic, and unprecedented times, the incidence of anxiety has dramatically increased.
Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid
extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of
social isolation-induced anxiety. Because GABAergic transmission modulates the immune
system in addition to the inhibitory signal transmission, we investigated the effects of short-term
social isolation on the neuroimmune system.
Methods:
Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks.
The anxiety like behaviors after DHM treatment were examined using elevated plus maze and
open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB
pathway activation, cytokine level, and serum corticosterone were measured.
Results:
Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and
reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected
illustrated as a decline in microglia number and overactivation as determined by significant
morphological changes including decreases in lacunarity, perimeter, and cell size and increase in

29
cell density. Moreover, social isolation induced an increase in serum corticosterone level and
activation in NF-κB pathway. Notably, DHM treatment counteracted these changes.
Conclusion:
The results suggest that social isolation contributes to neuroinflammation, while DHM has the
ability to improve neuroinflammation induced by anxiety.

Keywords:
Social isolation, neuroinflammation, microglia, anxiety, GABAAR, dihydromyricetin (DHM).

Introduction

Anxiety disorders are the largest class of mental health diseases in the U.S. and are estimated to
consume one-third of the country’s mental health spending. Considering the ineffective
therapeutics in the clinic due to treatment resistance, inconsistent patient adherence, and other
exogenous factors, the prevalence of chronic anxiety continues to be on the rise (Devane et al.,
2005; Konnopka et al., 2009; Roy-Byrne, 2015). The rise in individuals suffering anxiety has
been particularly noticeable during the ongoing COVID-19 pandemic that has resulted in
quarantine and social isolation (Twenge & Joiner, 2020). The pandemic also has led to an
increase in posttraumatic stress disorder (PTSD) (Yuan et al., 2021). The pandemic is bringing
up similar fears and mental distress, due to loss of family member(s) and loved ones (DePierro et
al., 2020; Yuan et al., 2021). The ongoing increase in cases of individuals suffering from anxiety
disorders, coupled with the lack of effective medications for many, highlight the need for new
treatment strategies to prevent or manage anxiety disorders.

30
Clinical evidence indicates that GABAergic neurotransmission alteration involves in
pathophysiology of anxiety disorders in humans (Rudolph & Knoflach, 2011). Therefore,
modifying GABAA receptor (GABAARs) activity is a target for regulating anxiety (Rudolph &
Knoflach, 2011; Shekhar et al., 1990). We have demonstrated that dihydromyricetin (DHM)
[(2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one], a major
bioactive flavonoid extracted from Ampelopsis grossedentata, is a positive allosteric modulator
(PAM) of GABAergic transmission, and thus has the potential to regulate anxiety-like behavior
via its action on GABAergic receptors (Liang, López-Valdés, et al., 2014; Shen et al., 2012). In
addition, we found that DHM antagonizes the acute and chronic effects of alcohol on GABAARs
(Liang, López-Valdés, et al., 2014; Shen et al., 2012). Thus, the activity of DHM on GABAARs
provides one possible mechanism for its role in anxiolysis (Liang, López-Valdés, et al., 2014).
Because GABAergic transmission modulates the immune system in addition to the inhibitory
signal transmission, we investigate the effects of social isolation on the neuroimmune system.
To date, the role of neuroinflammation in anxiety pathogenesis is not well established. Several
studies had highlighted the regulatory role of GABA in neuroimmune functions. GABA involves
in inflammation regulation by modulating the production of pro-inflammatory cytokines through
activating inflammatory signaling pathways such as nuclear factor-κB (NFκB) and mitogen-
activated protein kinase (MAPK) (Bhat et al., 2010; Crowley et al., 2015). Indeed, GABAergic
system components, including GABA enzyme, transports, and receptors, are expressed in the
immune cells (Jin et al., 2011; Schleimer et al., 2004).

The extended psychological stress that occurs due to social isolation disrupts the hypothalamic-
pituitary-adrenal (HPA) axis, which is considered the primary stress adaption pathway in the

31
body (Mumtaz et al., 2018). This disruption has the potential to increase proinflammatory
cytokines, persistent microglia and astrocytes activation, and reduce synaptic plasticity (Rhie et
al., 2020). Additionally, several studies indicated that GABAergic deficits during stress
contribute to HPA hyperactivity that correlated with anxiety pathogenesis (Mody & Maguire,
2011; Shen et al., 2010). The outcome of these immunoendocrine dysregulations could lead to
the occurrence of neuroinflammation and anxiety.

Microglia, the resident macrophages of the brain’s innate system, are a key player in modulating
the neuroinflammatory response in the CNS. Microglia are not only involved in the nervous
system infection and debris phagocytosis, but also play a crucial role in the physiological
development of the brain by engaging in the shaping process of neuronal circuits and synapse
plasticity (Salter & Beggs, 2014; Wake et al., 2013). During various psychological stressors such
as social isolation, microglia undergo several changes that compromise their functions and
further lead to neurological and neurodegenerative disorders (Calcia et al., 2016). To understand
the role of microglial in social isolation-induced anxiety, we utilized a social isolation mouse
model that induces anxiety via reduction of social interaction as a stressor. The primary goal of
this study was to determine whether social isolation-induced anxiety leads to neuroinflammation,
and to understand the pharmacological mechanisms of DHM as an alternative therapy for
preventing and reducing neuroinflammation.

32
Methods

Animals and treatments
Overview:
Eight-week-old male C57BL/6 mice (Charles River Laboratories, Hollister, CA) were housed in
the vivarium under a 12 h light/dark cycle with direct bedding and free access to food and water.
All animal experiments were performed according to the protocols approved by the University of
California, Los Angeles (UCLA) and the University of Southern California (USC) Institutional
Animal Care and Use Committees, and all methods were carried out in accordance with relevant
guidelines and regulations. Animals were habituated to the vivarium for 2 days before beginning
experimentation.

Social isolation:
Social isolation is reported to elicit anxious and depressive behaviors in rodents (Cryan &
Sweeney, 2011; Hershenberg et al., 2014; Pinna et al., 2006; Pinna et al., 2004). For the present
study, we modified these methods to induce stress associated with social isolation by using opaque-
walled cages, thus depriving the animals of environmental enhancers (e.g., toys, objects, etc.). We
investigated anxiety-like behaviors after 4 and 6 weeks post-social isolation to determine
behavioral responses comparable to the established 4-6 weeks isolation that results in anxiety
(Pinna et al., 2006). We used these time points to determine the potential therapeutic effects of
DHM. Group-housed mice were housed with the standard 3-4 mice per cage. Isolated mice were
singly housed with opaque walls without human handling except to change cages once per week.
The mice were divided as follows:

33
1) Group-housed mice without any drug administration for 2 weeks, and then given daily sucrose
agar as a vehicle for an additional 2 weeks (G2+Veh2).
2) Group-housed mice without any drug administration for 2 weeks, and then given daily DHM in
sucrose+agar for an additional 2 weeks (G2+D2, 2mg/kg DHM).
3) Isolated mice without any drug administration for 2 weeks, and then given daily sucrose agar
as a vehicle for an additional 2 weeks for a total isolation period of 4 weeks (Iso2+Veh2).
4) Isolated mice without any drug administration for 2 weeks, and then given daily DHM for an
additional 2 weeks for a total isolation period of 4 weeks (Iso2+D2).
5) Isolated mice without any drug administration for 4 weeks, and then given daily vehicle for an
additional 2 weeks for a total isolation period of 6 weeks (Iso4+Veh2).
6) Isolated mice without any drug administration for 4 weeks, and then given daily oral
administration of DHM for an additional 2 weeks for a total isolation period of 6 weeks (Iso4+D2).

Drug preparations:
DHM (HPLC purified ≥ 98%, Master Herbs Inc., Pomona, CA) was given orally as agar cube once
per day (2 mg/kg) for 2-weeks (Liang, López-Valdés, et al., 2014). To prepare the DHM or vehicle
agar cube, 3% agar was prepared with water, heated to ~90 °C to dissolve the agar, then DHM +
5% sucrose or 5% sucrose only were added and mixed until cooled and solidified. Agar was
prepared for the mice by cutting it into cubes of 0.5 X 0.5 X 0.5 cm each.

34
Drug administration:
Every evening (2 PM), all food from the cages of each mouse was removed, and an agar cube was
placed in the cage for each mouse. The mouse was observed to ensure it ate the agar cube, which
ranged from 30 to 90 minutes. Afterward, 4g of regular rodent food (the recommended daily
amount for an adult mouse; was placed in the cage for each mouse for the rest of the day
(Bachmanov et al., 2002). To ensure each mouse of the group housing mice received one cube,
they were isolated, fed, and then returned to group housing.

Behavioral Testing:
Anxiety-like behavior was tested 24 h after the last treatment, at the end of the 4-week or 6-week
time points in the following evening (8 PM; in the dark phase of 12/12-hr light/dark cycle). Anxiety
tests were reliant on ethologically appropriate behavior and sensitive to ‘state’ anxiety
measurements. Because mice are nocturnal animals, behavior tests were conducted during their
active time to ensure accurate behavioral responses and minimize interference of their circadian
cycle. Behavioral tests were performed under indirect red lighting and recorded with a video
camera. Indirect red lighting was used to better assess parameters of anxiety without influencing
mouse behavior (i.e., reduced activity in the open field test) and stress (Peirson et al., 2018).
Investigators were blind to experimental groups when conducting behavioral analyses.

1. Elevated plus maze:
The elevated plus maze was conducted following a previously published protocol (Liang, Lopez-
Valdes, et al., 2014). The elevated plus-maze apparatus was made of opaque, 0.6 cm-thick plastic.

35
It comprised of two open arms 25x8 cm across from each other and perpendicular to two closed
arms 25x8 cm with a center platform of 8x8 cm. The closed arms had a 20 cm-high wall that
enclosed the arms. The walls and floors of the closed arms were black, while the open arms were
white. The apparatus was elevated 50 cm above the floor. Throughout the test, each animal was
placed in the center of the maze facing an open arm and allowed to explore for 5 mins. The
behaviors were recorded by a ceiling-mounted camera. Entry into an arm of the maze was defined
by the placement of at least 3 paws into that compartment. The following measures were
physiological scoring system: number of entries into open arms, closed arms, or center platform
and time spent in each of these areas. All scoring was performed offline in a double-blind manner.

2. Open field:
The open field test was conducted following a previously published protocol (Chen et al., 2004;
Liang, López-Valdés, et al., 2014). The open field chamber measured 50 cm (length) x 50 cm
(width) x 38 cm (height) and was made from a white acrylic plastic sheet. 4 x 4 grid lines were
drawn to divide the floor into 10 x 10 cm squares, and an additional 20 x 20 cm square zone was
drawn in the center. Mouse activity was assessed as previously reported in Chen et al., 2004 for
10 min. The following parameters were summed for each animal during the 10-min test: the time
spent in the central zone, time spent in the 4-corner square grid, pathlength (cm) traveled in the
apparatus (determined by measuring the distance of the nose of the mouse relative to the 10 x 10
cm square grid lines on the floor of the open field chamber), and the numbers of times the animal
reared. All scoring was conducted manually in a double-blind manner, with each recording being
observed three times to minimize error.

36
Immunohistochemistry analysis:
Mouse brain was fixed in 4% formaldehyde for 24 hours, and then incubated in 30% sucrose
until tissues are sink. Fixed brain was flash frozen using pre-cooled isopentane (−78 °C),
sectioned at 30 μm using Microm HM525 Cryostat (Thermo) and picked up on Superfrost Plus
slides (VWR, 48311-703). Sections were blocked with 5% normal goat serum and washed in
PBS with 1% bovine serum albumin (BSA) and incubated with rabbit- anti-mouse Iba-1 primary
antibody (FUJIFILM Wako Pure Chemical Corporation 019-19741, 1:500) or rabbit- anti-mouse
Iba-1 primary antibody Alexa Fluor
®
594 conjugate (Cell Signaling, 48934, 1:50) overnight.
Sections were washed with phosphate buffer with 1% Tween 20 (PBS-T), and then incubated in
goat anti-rabbit IgG (H+L) secondary antibody (Vector laboratory CY-1300, 1:250) at room
temperature for 1 hour when unconjugated antibody was used. Afterword, sections were washed
three times with PBS-T followed by mounting on coverslip using Vectashield DAPI (4′6-
diamidino-2-phenylindole 2HCl, Vector Labs, Burlingame, U.S.) mounting media to detect
nuclei.

Imaging and Analysis:
The immune-stained sections were scanned for high-resolution images by Cytation 5 cell
imaging multi-mode reader (BioTek, Winooski, VT, USA) or super-resolution images by Zeiss
LSM880 with Airyscan confocal laser scanning microscope (Carl Zeiss Microscopy, White
Plains, NY). The images from the CA1 and CA2 of the brain hippocampus were processed by
Zen black and blue imaging analysis software (Carl Zeiss Microscopy) and analyzed by FracLac
box counting and convex hull analysis to evaluate the morphological changes of microglia cells
using ImageJ software by following the steps published in Young & Morrison, 2018 (Young &

37
Morrison, 2018). Microglia cells count, within the region of interest ROI (0.3 x 0.4 mm) in the
CA1 and CA2 hippocampus area, was done manually blinded to the treatment group using
ImageJ cell count command and presented in cells per 1 mm
2
.

Western blot analysis:
Hippocampus was homogenized in pre-cooled Tris-EDTA with 1 % PMSF (0.1 M Tris-acetate
buffer + 2 mM EDTA, pH 7.75). The homogenate was centrifuged at 10,000 x g for 15 minutes
at 4°C and the supernatant was collected. The supernatant protein was quantified using the BCA
Protein Assay kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s
instructions. 50 μg of protein was separated on a 10 % sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and then transferred to PVDF membranes (Bio-Rad Laboratories, Hercules,
CA). The transferred membrane was blocked with a blocking buffer containing 5% skim milk in
1X Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature. The membrane
was incubated in the following primary antibody for either: rabbit anti-mouse Gephyrin (Cell
Signaling 14304, 1:1000), mouse anti-mouse β-actin (Cell Signaling 4970, 1:1000), rabbit anti-
mouse NF-κB p65 (Cell Signaling 8242, 1:1000) or rabbit anti-mouse Phospho-NF-κB p65 (Cell
Signaling 3033, 1:1000) in 1X TBST overnight at 4°C with gentle agitation. The membrane was
washed three times with 1X TBST for 10 min each and incubated with a secondary antibody goat
anti-rabbit IgG or goat anti-mouse (Bio-Rad 1706515 and 1706516) in 1X TBST for 1 hour.
Finally, the membrane was visualized with enhanced chemiluminescence detection reagent (Bio-
Rad 1705061) and Chemi-Doc (Bio-Rad) imaging device.

38

Cytokines profile arrays:
Serum samples were pooled from 4-5 mice/group (n=4 vehicle grouped housed, n=4 DHM
grouped housed, n=5 vehicle social isolated, n=5 DHM in social isolated group). According to
the manufacturer’s instructions, pooled sera were tested by Profiler Mouse Cytokine Array Kit,
Panel A (ARY006, R&D Systems, Minneapolis, MN, USA) for a quantitative measure of the
peripheral cytokines and chemokines level to determine changes in response to the social
isolation-induced anxiety. The membrane immunoreactivity was detected after adding
chemiluminescence reagent mix. Array images were obtained using (Chemi-Doc imager system
(Bio-Rad). The results were analyzed using ImageJ software and expressed as a mean change in
the gray value relative to the vehicle grouped housed groups.

ELISA analysis:
Fresh blood was collected from the right atrium quickly after the animals were sacrificed. serum
samples were collected by centrifugation at 1000 × g for 10 min at 4°C. A competitive ELISA
kit (Abcam, ab108821) was used to determine the corticosterone levels according to the
manufacturer’s protocol. The intensity of the color developed was measured using Synergy H1
Hybrid Multi-Mode Reader (BioTek).

Statistical analysis
All assays were performed at least three times. All behavioral records were observed and
analyzed in a double-blind manner. The data were presented as the mean ± standard error of the
mean using GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA). One or two-way

39
analysis of variance (ANOVA) followed by Holm-Sidak multiple comparison tests were
performed, the significance level is set at p ≤ 0.05.

40
Results

DHM treatment ameliorates social isolation induced anxiety-like behaviors

Figure 2. 1 DHM reduces social isolation-induced anxiety
0
1
2
3
4
5
Time (min)
*
0
1
2
3
4
5
Time (min)
*
0
20
40
60
Rearing times
*
0
1000
2000
3000
4000
Pathlength (cm)
*
0
20
40
60
80
100
Time (sec)
*
0
10
20
30
Time (sec)
*
G2+Veh2
G2+D2
Iso2+Veh2
Iso2+D2
A
B

41
A. Effects of social isolation and treatment with DHM on anxiety-like behaviors as measured by
the time (min) spent in the open and closed arms of the elevated plus maze. B. Effects of social
isolation and treatment with DHM on locomotor activity, exploratory behaviors as measured by
running distance (total distance of moving), rearing (total number of times of rearings), corner
(the total duration the mouse stayed in the 4 corner 10x10 cm squares), and center time (the total
time duration the mouse stayed in the center 20x20 cm square), in the open field assay. One-way
ANOVA followed by multiple comparison, Holm-Sidak method to the control. For running
length, P < 0.001; For numbers of rearings, P < 0.001. For stay in corners P < 0.001. For stay in
the center, P < 0.001. * = p ≤ 0.05 vs. vehicle group housing control (G2+Veh2). (n = 10-
11/group).

Using a mouse model of social isolation stress, we examined social isolation stress induced
anxiety levels and the effects of DHM with the elevated plus-maze (EPM) and open field (OF)
tests (Dityatev & Bolshakov, 2005; Ieraci et al., 2016; J. Silva, A. S. Shao, et al., 2020). Group-
housed control mice (G2+Veh2) spent 2.31±0.27 (min) of a total 5 min in open arms of the
elevated plus-maze (Fig 1A) and 2.24±0.31 min in the closed arm. Mice socially isolated for 2
weeks, followed by 2 weeks of vehicle treatment (Iso2+Veh2) spent substantially less time
(1.26±0.17 min) in the open arms compared to the closed arms (3.31±0.27 min). Socially
isolated mice treated with DHM (Iso2+D2) resulted in greater time spent in the open arm
(2.07±0.22 min) when compared with untreated socially isolated mice (Table S1). These results
suggest that social isolation increased anxiety levels. Furthermore, DHM administration
ameliorates isolation-induced anxiety behaviors, as observed with increased entry into and
staying in the open arms.

To further examine anxiety-like behavior, locomotor activity, and exploration behavior were
analyzed by measuring the distance traveled of the mice in the open field test (Fig 1B). During
the 10 min observation trial, group-housed mice (G2+Veh2) traveled 2765±161 cm, while
isolated mice (Iso2+Veh2) traveled a shorter (2176±145 cm) pathlength suggesting that social

42
isolation decreased motor activity of these mice. In contrast, isolated mice treated with DHM
(Iso2+D2) resulted in greater running distance (2398±147 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 (46.6±1.5 times vs 28.3±2.1 times) (19.6±0.7 vs
7.6±0.86). Social isolation mice spent more time in the corners compared to group-housed mice
(73±4.2 sec vs 28.2±2.06 sec). Administration of DHM in Iso2+D2 increased the number of
rearing (40.7±1.03 times) and the time in the center (18.3±0.49 sec) while decreasing stay in the
corner duration (38.5±3.5 sec) (Table 1S). Collectively, these results suggest that isolation
decreased exploratory/locomotor activity in adult male C57BL/6J mice and that DHM treatment
ameliorates these behavioral responses in socially isolated mice.

43
Social isolation down regulates gephyrin protein expression, while DHM treatment
improves it

Figure 2. 2 Changes in gephyrin protein expression after social isolation and the effect of DHM
treatment.
Grouped-house (G4+Veh2), single-housed mice (Iso4+Veh2), and single-housed with DHM
treatment (Iso4+D2). β-actin from the same blot was used as a loading control. One-way
ANOVA followed by multiple comparisons, Holm-Sidak’s method; * = p≤ 0.05 vs. group
housing control (G4+Veh2), n=5/group.

Gephyrin is a scaffold protein essential for GABAAR clustering via several mechanisms
(Machado et al., 2011; Parra et al., 2016). The changes in the expression of gephyrin can
partially explain the changes in GABAergic neurotransmission. Gephyrin protein expression was
measured in extracted hippocampi and evaluated by Western blot. Gephyrin expression was 40%
lower in isolated mice compared to group-housed mice (Fig. 2) (Table 2S). DHM treatment up
regulated gephyrin expression at Iso4+D2 group relative to isolated mice without DHM.
Collectively, the behavioral assay and the gephyrin results confirm the phenotype of the social
isolation animal model. The results also suggest that DHM improves gephyrin expression levels
in hippocampi and subsequent GABAAR function.
0.0
0.5
1.0
1.5
b-actin:Gephyrin
*
G4+Veh2
Iso4+Veh2
Iso4+D2

44

Social isolation induces loss and dystrophy in the hippocampus microglia, while DHM
improves it

Figure 2. 3 The effects of social isolation-induced anxiety and DHM treatment on microglia
activation and proliferation in the hippocampal CA area.
A. Representative images showing the effect of social isolation-induced anxiety on the number
of labeled microglia in the hippocampus, Iba-1 (red), DAPI (blue). B. Confocal single-cell
microglia images for the G2+Veh2, Iso+Veh2, and Iso+D2 obtained using a 63X oil-immersion
objective. C. Quantification analysis of the microglia number in CA1 and CA2 area, data
presented as the number of cells per 1mm
2
. One-way ANOVA followed by Sidak multiple
comparisons test was used for statistical analysis. Each point represents cells number in 2-3
individual sections from n=5 mice, values represented as mean± SEM, * = p ≤ 0.05, n=5.

G2+Veh2 Iso2+Veh2 Iso2+D2
G2+Veh2 Iso2+Veh2 Iso2+D2
A
B
C
0
100
200
300
400
500
Cells/mm
2
*
G+Veh2
Iso2+Veh2
Iso2+D2

45

Figure 2. 4 DHM treatment modulates microglia morphology in the CA1 and CA2 area of
hippocampus.
A. Representative photos of the hippocampus CA1 where the white rectangular are regions for
photomicrograph analysis, stained for DAPI (blue, left panel) and Iba-1 (red, right panel) from
the control group. B. Illustration for the box-counting method used for lacunarity calculation. C.
representative images for binary microglial and convex hull (green) and enclosing circle (pink)
that were used to calculate density, perimeter, and maximum span across the hull, and the
corresponding microglial in each group. D. Microglial morphology profile illustrated in
lacunarity, cell perimeter (E), density (cell area/convex hull area) (F), and the maximum span
across the convex hull (G). One-way ANOVA followed by Sidak multiple comparisons test was
used for statistical analysis. Each point represents individual microglia 3-4 cells from n=5 mice,
values represented as mean± SEM, * = p ≤ 0.05.

Microglia number was reduced after short term social isolation in the CA hippocampus brain
region in addition to alteration in morphological characteristics of microglia cells. The changes
appeared due to the activation of cells toward more dystrophic morphology and apoptosis
leading to decrease in microglia number compared to the basal state. Microglia counts in social
isolation group (Iso2+Veh2) was lower (28.50± 1.64 cells/mm
2
) compared to the control group
0.0
0.1
0.2
0.3
0.4
0.5
Lacunarity
*
0
200
400
600
Perimeter ( µm)
*
0.00
0.05
0.10
0.15
0.20
Density
*
0
50
100
150
200
250
Max span ( µm)
*
A
B
C
D E
F
G
G2+Veh2 Iso2+Veh2 Iso2+D2
G+Veh2
Iso2+Veh2
Iso2+D2

46
G2+Veh2 (37.27 ± 2.1) (Fig. 3C). Moreover, microglia cells in Iso2+Veh2 show a significant
decrease in the lacunarity value from 0.353 ± 0.018 to 0.302 ± 0.009 (Fig. 4B) as an evidence of
microglia activation that was restored after DHM treatment. Lacunarity measures cell shape
heterogenicity and changes in the soma (Fernández-Arjona et al., 2017). Moreover, a decrease in
microglial cell area was observed in the social isolation group (Iso2+Veh2) illustrated in the
reduction of the perimeter of the individual cell outline, where social isolation for 2 weeks
followed by 2 weeks of DHM treatment (Iso2+D2) showed an increase in cell perimeter from
278.7±16.76 to 382.9±21.56 µm [F (2, 28) = 17.72, p=<0.0001] (Fig. 4C). Iso2+Veh2 microglia
cells demonstrated a more compact shape illustrated in significantly higher cell density [F (2, 27)
= 3.388, p=0.0387) (Fig. 3D]. In contrast, the DHM isolated group (Iso2+D2) showed higher cell
density which was statistically similar to the control grouped house (G2+Veh2). In addition, we
evaluated the maximum distance between two points across the convex hull and found that DHM
administered social isolated group (Iso2+D2) had an average distance relatively similar to the
control group house (G2+Veh2) (Fig. 4E), while the socially isolated group (Iso2+Veh2) had a
substantially smaller average distance across the convex hull from 172.5±12.04 to 103.2±6.40
µm (F (2, 28) = 14.36, p=<0.0001) (Table 3S). These results suggest a modulation in several
microglia morphological parameters related to the microglia activation after social isolation,
indicating an acute response to social isolation challenges, which were ameliorated by DHM
administration.

47
Social isolation anxiety increases serum corticosterone level, activates NF-κB signaling
pathway, and increases proinflammatory cytokines, while DHM attenuates these changes.

Figure 2. 5 The effect of DHM on the levels of serum corticosterone, p-NF-kB p65 protein
expression, and proinflammatory cytokines expression after social isolation.
A1. Representative Western blots of Phospho-NF-kB p65 (65 kDa), NF-kB p65 (65 kDa), and β
-actin (42 kDa). (A2 and A3) Quantitative analysis ratio for the protein expression of Phospho-
A-1
p-NF- κB p65
NF- κB p65
β-Actin
1
2 3 4
G2+Veh2
G2+D 2
Iso2+Veh2
Iso2+D 2
IFN-y
IL-1 α
TNF- α
IL-2
IL-7
IL-1 β
IL-6
MIP-1 β
CXCL2
CCL17
1
2
0
200
400
600
800
CORT ng/ml
*
0.0
0.5
1.0
1.5
NFkB:b-Actin
0.0
0.5
1.0
1.5
p-NFkB p65:b-Actin
*
A-2 A-3
B C

48
NF-κB p65 and NF-κB p65 with the loading control β-actin F (4,10) = 1.485 p=0.5885. B.
Serum level of corticosterone (ng/ml) after social isolation (Iso2+Veh2) and following DHM
treatment 2 mg/kg. One-way ANOVA followed by multiple comparisons, Holm-Sidak’s method.
C. Heat map of serum cytokines and chemokines level. The color key indicates response level
ranging from the highest (red) to the lowest (pink). Data represented as the inverted mean gray
value expressed in each cytokine/chemokine. Each data point was normalized to the baseline
value (G2+Veh2). (G2+Veh) grouped housed vehicle, (G2+D2) grouped housed DHM,
(Iso2+Veh2) socially isolated vehicle, (Iso2+D2) socially isolated DHM, n = 4-5 per group. * =
p ≤ 0.05 vs. group housing control (G2+Veh2), n = 5/group.

Corticosterone is the primary hormone of the pituitary adrenocortical axis in response to
environmental challenges, and it has an essential function in stress (Kinlein et al., 2019).
Corticosterone level was evaluated after social isolation to illustrate the effect of social isolation
on the hypothalamic–pituitary–adrenal (HPA) axis. Our results revealed a significant increase in
the serum corticosterone level from 285.8±26.65 ng/ml in G2+Veh2 to 466.0±60.51 ng/ml in
Iso2+Veh2 group (F (3, 15) = 7.897), p = 0.002) (Fig. 5A). Administration of DHM at the dose
of 2 mg /kg in the isolation group decreased the corticosterone level to 283.3±27.43 ng/ml closed
to the basal level of G2+Veh2 ((Table 4S). This observation suggests that corticosterone
responds to social isolation-induced stress challenges, and DHM attenuates the stress (anxiety)
following social isolation and reduces corticosterone levels.

To determine the effect of social isolation on inflammation, we examined the protein expression
level of NF-κB p65 in extracted hippocampi utilizing Western blot. NF-κB transcription factor is
an essential inflammatory pathway that plays a crucial role in regulating immune and
inflammatory responses by inducing the expression of cytokines and chemokines genes (T. Liu
et al., 2017). The active phosphorylated NF-κB p65 expression was significantly higher in the
Iso2+Veh2 group compared to the control. DHM treatment group (Iso2+D2) shows a
comparable phospho-NF-κBp65 protein level to the control (G2+Veh2) and treatment control

49
(G2+ D2) (Fig 5B) ((p = 0.574). An increase in the expression of several numbers of
proinflammatory cytokines and chemokines was observed using cytokines profile proteome
assay in the isolated group (Iso2+ Veh2) compared to the control and DHM group-housed
(G2+Veh2) (G2+ D2) (Fig 5C). The DHM treated isolated group (Iso2+D2) showed lower
cytokines and chemokines expression levels compared to (Iso2+Veh2) (Table 2S). These data
suggest that social isolation activates the NF-κB signaling pathway leading to an increase in the
transcription of proinflammatory cytokines while DHM treatment counteract them.

Discussion

The primary goal of this study was to determine the effects of anxiety induced by short-term
social isolation on neuroinflammation and to understand pharmacological mechanisms
underlying the therapeutic effects of DHM. In this study, we found that social isolation induced
anxiety-like behaviors in mice, increased corticosterone level, down regulated gephyrin
expression, enhanced the activation of NF-kB p65 pathway, decrease hippocampal microglial
cell number, and increase the reactive microglia. Importantly, these pathological changes and
behavioral deficits were ameliorated by DHM treatment. Findings from the present work
suggests that short-term social isolation leads to changes in the HPA axis, disrupts GABAergic
neurotransmission, and provokes neuroinflammation. DHM was found to restore these molecular
and cellular changes.

50

Figure 2. 6 Schematic summary of the various pathways involved in social isolation induced
neuroinflammation and anxiety-like behaviors.
Social isolation (SI) induces stress that triggers disruption in the hypothalamus-pituitary-adrenal
axis (HPA), increase in the levels of corticosterone (CORT), and overactivity in the sympathetic
nervous system (SNS), which in turn might trigger NF-κB pathway activation and enhance the
level of pro-inflammatory cytokines. Social isolation induces a reduction in pre-, post-, and/or
extra-synaptic GABAARs, and in gephyrin expression leading to impairment in GABAergic
neurotransmission. GABAARs are also expressed in microglia and astrocytes. Proinflammatory
cytokines and GABAAR disruption provokes microglia and astrocytes activation leading to
further neuroinflammation damages and anxiety-like behaviors. DHM is a positive modulator of
GABAAR that plays a role in counteracting neuroinflammation and contribute to the anxiolytics
effect.

Gephyrin is a key protein that anchors, clusters, and stabilizes GABAergic synapses (Choii &
Ko, 2015). Mounting evidence has indicated the critical role of GABA transmission in the

51
progression of anxiety disorder (Tasan et al., 2011). In our previous study, we found that changes
in gephyrin expression play a role in changes in GABAAR amount and GABAergic function
(Liang, López-Valdés, et al., 2014; J. Silva, A. S. Shao, et al., 2020). Additionally, we found a
reduction in GABAAR-mediated extra-synaptic, pre-, and post-synaptic currents in the social
isolation anxiety model, which was restored after DHM treatment (J. Silva, A. S. Shao, et al.,
2020). To elucidate the mechanism in which DHM modulates GABAARs in the socially isolated
model, gephyrin protein expression was assessed utilizing Western blot. Gephyrin protein
expression was reduced 40% in the isolation group and up regulated after DHM treatment (Fig.
1A). Taking into consideration that GABAAR expression is ubiquitous in the brain and the
primary role of GABA neurotransmitter in anxiety pathogenesis, a reduction in gephyrin protein
level might provide a partial explanation of the behavioral changes shown in (Fig 1B). It is
essential to emphasize the interconnection between GABA and neuroinflammation.
Neuroinflammation decreases GABA synthesis by reducing glutamate acid decarboxylase 67
(GAD67) enzyme, downregulating GABAAR protein expression, and inhibiting GABA current
by decrease GABA neurons density (T. Crowley et al., 2016). Therefore, studies of
neuroinflammation will further clarify the underlining mechanisms of DHM effects on
improving anxiety-like behaviors.

As part of the innate immune system, microglia play a significant role in initiating and mediating
neuroinflammation in the brain (DiSabato et al., 2016). Microglia are not only involved in brain
infection and debris phagocytosis, but also play a crucial role in the physiological development
of the brain by engaging in the shaping process of neuronal circuits and synapse plasticity (Salter
& Beggs, 2014; Wake et al., 2013). Microglia activation occurred after various types of stress

52
(Calcia et al., 2016; Y. L. Wang et al., 2018; Wohleb & Delpech, 2017). We assessed
microgliosis after short-term social isolation using immunohistochemistry staining of Iba1, one
of the most common markers of microglia (Frank et al., 2007; Hinwood et al., 2013; Lehmann et
al., 2019). These data indicate that social isolation-induced anxiety led to a decline in microglia
cell number (Fig. 3C). Several evidence demonstrate a tight connection between the microglial
loss mediated by overactivation and social isolation and other form of stress (Gong et al., 2018;
Kreisel et al., 2014; Tong et al., 2017). Additionally, social isolation triggers microglia
activation, manifested as changes in the cell morphology. High lacunarity value indicates the
heterogeneity of the cells, meaning the single-cell image of microglia has different gap sizes. On
the other hand, a low lacunarity value indicates homogeneity, suggesting the cell shape has less
variance (Fernández-Arjona et al., 2017) . Under the influence of social isolation, the microglial
cell showed a lower lacunarity value compared to the control. This implies the transformation of
the microglial cell to a more homogenous state. This change generally suggests activation of the
microglia from its surveillant state (Karperien et al., 2013). Furthermore, the activated microglia
cells in socially isolated mice showed a reduction in cell size, based on the measurement of the
perimeter of single cell outlines. Reduction of cell perimeter is an illustration of fewer
ramifications, shorter and thicker branches, and a larger soma size of the cell that manifested
during activation (Stein et al., 2017). Microglia density is another morphometric parameter that
was altered after social isolation and DHM treatment. Higher cell density demonstrates the
transformation of the cells to a more compact form during activation (Hinwood et al., 2012;
Tynan et al., 2010). The maximum span across the hull in each microglial cell was significantly
smaller in the social isolation group than the control, reflecting the size of the overall cells and
the length of the processes. Microglial in less ramified form is considered activated or

53
intermediate active, which explains the reduction in the hull span size (Fernández-Arjona et al.,
2019; Karperien et al., 2013). These results are consistent with the findings from several groups
that demonstrated the activated microglia contribute to anxiety induced by social isolation (Du
Preez et al., 2020; Haj-Mirzaian et al., 2020). In addition, several studies showed that
microgliosis mediate synapses loss that is considered the earliest manifestation of AD pathology
(Rajendran & Paolicelli, 2018; Schafer et al., 2012; Yoshiyama et al., 2007).

Accumulated evidence suggests a link between neuroinflammation and the pathology of anxiety
and other psychological disorders (H. Y. Liu et al., 2018; Y. L. Wang et al., 2018).
Neuroinflammation is involved in several pathological changes in the nervous system and the
neuroendocrine system, and it is initiated as a result of the modulation of the HPA axis (Hannibal
& Bishop, 2014). Chronic exposure to stress leads to disruption in HPA axis and an increase in
the cortisol level in humans and corticosterone in rodents (Chrousos, 2000). Animal studies of
the social isolation model have shown that the corticosterone level is higher after exposure to
stress than the group-housed control (Serra et al., 2005). There was an increase in serum
corticosterone level after 4 weeks of social isolation; and our results demonstrate that DHM
treatment at doses of 2 mg/kg reduces corticosterone levels.

The transcription factor NF-κB plays a key function in regulating immune and inflammatory
responses by inducing the expression of cytokines and chemokines genes (T. Liu et al., 2017).
Growing evidence suggests that stress-induced behavioral deficits are mediated by NF-κB
signaling activation, which increases the production of the proinflammatory cytokine and
chemokines leading to microglia activation and ultimately neuroinflammation (Koo et al., 2010;

54
Zlatković, Bernardi, et al., 2014). Our results indicate an increase in the Phospho-NF-kB p65
protein level compared to the nonactive NF-kB p65, which might explain the increase of
proinflammatory cytokines expression, and the microglial activation observed in the social
isolation group. Additionally, DHM treatment attenuates the activation of NF-κB pathway
induced by social isolation, and this finding is consistent with a previous work that demonstrated
a reduction in NF-κB protein expression after DHM treatment (Jing & Li, 2019).

Study limitations:
A previous study from our team showed no difference in GABAARs-mediated neurotransmission
and gephyrin level between 2-wk and 4-wk social isolation (J. Silva, A. S. Shao, et al., 2020).
The current study in neuroinflammation focused only on 2 weeks isolation. However, future
studies might be necessary to include more different time points.
The gender choice in the current study was based on a previous publication from our team (J.
Silva, A. S. Shao, et al., 2020), which was conducted only on males. Future research on the
effects of DHM in socially isolated female mice is necessary to gain insight into the therapeutics
role of DHM in both genders. Despite the limitations, the current study provides important data
for understanding possible mechanisms of actions of DHM in ameliorating anxiety-like
behaviors.
Conclusion:
There has been a growing body of evidence supporting the association of anxiety with cognitive
decline leading researchers to conclude that anxiety symptoms could be predictive for the
progression of Alzheimer’s disease (AD). This work provided insights into the mechanisms that
anxiety induces neuroinflammation and DHM reverses the neuropathology resulted from anxiety

55
as a component of preclinical AD, as well as the utility of DHM as a novel therapy for anxiety.
DHM reduces neuroinflammation, restores GABAergic function by up regulating gephyrin
levels, and decreases serum cortisol levels, therefore, improves the social isolation induced
anxiety and the early onset of AD. DHM could provide an early intervention to manage anxiety
and to reduce the risk of cognitive decline and AD development.

List of abbreviation:
DHM: Dihydromyricetin
NF-κB: Nuclear factor-κB
CNS: Central nervous system
PTSD: Posttraumatic stress disorder
GABAARs: GABAA receptors
PAM: Positive allosteric modulator
HPA: hypothalamic-pituitary-adrenal
MAPK: Mitogen-activated protein kinase
BSA: Bovine serum albumin
PBS: Phosphate buffer saline
TBST: Tris-buffered saline with Tween 20
ELISA: Enzyme Linked Immmunosorbent Assay
ANOVA: Analysis of variance
EPM: Elevated plus-maze
OF: Open field
GAD67: Decarboxylase 67

56
AD: Alzheimer’s disease

Declarations

Ethics approval and consent to participate
All animal experiments were performed according to the protocols approved by the University of
California, Los Angeles (UCLA) and the University of Southern California (USC) Institutional
Animal Care and Use Committees, and all methods were carried out in accordance with relevant
guidelines and regulations.

Consent for publication
Not applicable

Availability of data and materials
All data generated or analyzed during this study are included in this published article and in the
supplementary information files.

Competing interests
The authors declare no conflict of interest and competing interests.

Funding
This continuing research was supported by the National Institute of Health grants AA017991 (to
J.L.), AA022448 (to D.L.D.).

57

Authors' contributions
A.J.A designed and performed experiments, analyzed data, generated figures, and wrote the
manuscript.
A.S.S. established animal model, designed and performed experiments, and wrote the manuscript.
S.W. discussed the design, performed experiments, and wrote the manuscript.
Z.Z. generated images, contributed to methods, and assisted with majority of lab instruments.
C.X. performed experiments and analyzed data.
J.Z. performed experiments and analyzed data.
J.W. selected the methods and mentored technologies of majority of lab instruments.
D.L.D. wrote the manuscript
X.S.M. performed experiments, statistical analyses, discussed the design, and wrote the
manuscript.
J.L. established animal model, designed and performed experiments, wrote the manuscript, and
supervised the project.

Acknowledgements
Carefree Biotechnology Foundation, and Army Health Professions Scholarship Program (HPSP)
(to A.S.S.), Saudi Arabian Cultural Mission (SACM) (to A.J.A).

58

The supplementary file

Table S1: One-way ANOVA and Holm-Sidak tests summary of the behavioral data

One-way ANOVA table

SS DF MS F (DFn, DFd) P value
Open arm Treatment
(between
columns)
6.906 3 2.302
F (3, 39) =
3.721
P=0.0191
Residual (within
columns)
24.13 39 0.6187
Total 31.03 42

Close arm Treatment
(between
columns)
6.104 3 2.035
F (3, 29) =
3.001
P=0.0466
Residual (within
columns)
19.66 29 0.6780
Total 25.76 32

Holm-Sidak test

Mean ± SEM N Mean ± SEM N Mean ± SEM N Mean ± SEM N
Open arm

2.31± 0.27 11 2.3±0.26

11 1.26±0.17 10 2.07±0.22 11
Close arm 2.24±0.31 11 2.269±0.26 11 3.31±0.27 10 2.56±0.28 11
Running 2765±161.2 11 2873±141.2 11 2176±145.9 10 2398±146.9 11
Rearing 46.63±1.52 11 47.25±2.085 11 28.25±2.068 10 40.75±1.03 11
Centre 19.63±0.71 11 21.25±0.64 11 7.63±0.86 10 18.38±0.49 11
Corner 28.25±2.07 11 27.75±2.11 11 73.00±4.31 10 38.50±3.52 11

G2+Veh2 G2+D2 Iso2+Veh2 Iso2+D2

59

Table 2S: Summary data of Western blot

One-way ANOVA table

F Sig. P value

Gephyrin 29.02

* 0.0008

pNF-κB p65 4.401

ns 0.0222

NF-κB p65 0.5559

ns 0.6540

Holm-Sidak test

Mean ±SEM N Mean ± SEM N Mean ± SEM N Mean ± SEM N
Gephyrin 100± 3.522 3 NA

NA 62.20±3.926

3 85.30±3.118 3
pNF-κB p65 0.5421±0.147 4 0.5532±0.1098 4 0.9967±0.06064 5 0.4908±0.1382 5
NF-κB p65 1.13±0.09 4 1.049±0.09 4 0.96±0.13 4 0.99±0.04 4

G2+Veh2 G2+D2 Iso2+Veh2 Iso2+D2

60

Table 3S: summary of microglia analysis data

One-way ANOVA table

F Sig. P value

Lacunarity 3.828 * 0.0412
Perimeter 17.72 * <0.0001
Density 3.388 * 0.0487
Span across hull

14.36 * <0.0001
Microglia cell count 4.871

* 0.0145

Holm-Sidak test

Mean ±SEM N Mean ± SEM N Mean ± SEM N
Lacunarity 0.35± 0.2 7 0.30±0.009

7 0.34±0.01 7
Perimeter 463.5±27.9 10 278.7±16.76 11 382.9±21.56 10
Density 0.120±0.007 10 0.144±0.006 11 0.126±0.006 10
Span across
hull
172.5±12.04 10 103.2±6.40 11 140.0±8.84 10
Microglia cell
count
310.6±17.52 11 237.5±13.74 12 260.6±19.59 11

G2+Veh2 Iso2+Veh2 Iso2+D2

61

Table 4S: summary of corticosterone ELISA data

One-way ANOVA table

F Sig. P value

Corticosterone 7.897

* 0.0022

Holm-Sidak test

Mean± SEM N Mean ± SEM N Mean ± SEM N Mean ±SEM N
Corticosterone 285.8±26.65 5 240.1±19.98 5 466.0±60.51

4 283.3±27.43 5

G2+Veh2 G2+D2 Iso2+Veh2 Iso2+D2

62

CHAPTER 3

Dihydromyricetin improves social isolation-induced cognitive impairments and astrocytic
changes in mice

Saki Watanabe
1†
, Alzahra Al Omran
1†
, Amy S. Shao
2†
, Chen Xue
1†
, Zeyu Zhang
3†
, Jifeng
Zhang
1
, Daryl L. Davies
1
, Xuesi M. Shao
4
,

Junji Watanabe
3
, Jing Liang
1

1. Titus Family Department of Clinical Pharmacy, School of Pharmacy, University of
Southern California, Los Angeles, CA 90033, USA.
2. Homer Stryker M.D. School of Medicine, Western Michigan University, Kalamazoo, MI
49007, USA.
3. Translational Research Laboratory, School of Pharmacy, University of Southern
California, Los Angeles, CA 90033, USA.
4. Neurobiology, David Geffen School of Medicine, University of California Los Angeles,
Los Angeles, CA 90095, USA.
Corresponding author:
Jing Liang, MD, PhD
1985 Zonal Ave, PSC 504
Los Angeles, CA 90089
Tel: 323-442-3118
Email: jliang1@usc.edu
†
These authors contributed equally to this work.

63
Abstract

Social isolation induces stress, anxiety, and mild cognitive impairment that could progress
towards irreversible brain damage. A probable player in the mechanism of social isolation-
induced anxiety is astrocytes, specialized glial cells that support proper brain function. Using a
social isolation mouse model, we observed worsened cognitive and memory abilities with
reductions of Object Recognition Index (ORI) in novel object recognition test and Recognition
Index (RI) in novel context recognition test. Social isolation also increased astrocyte density,
reduced astrocyte size with shorter branches, and reduced morphological complexity in the
hippocampus. Dihydromyricetin, a flavonoid that we previously demonstrated to have anxiolytic
properties, improved memory/cognition and restored astrocyte plasticity in these mice. Our study
indicates astrocytic involvement in social isolation-induced cognitive impairment as well as
anxiety and suggest dihydromyricetin as an early-stage intervention against anxiety, cognitive
impairment, and potential permanent brain damage.

Significance Statement
Social isolation induces stress and anxiety that lead to adverse changes in the brain. Proper brain
function requires numerous essential factors, including specialized cells called astrocytes. In this
paper, we show that mice models of social isolation-induced anxiety exhibit deficits in memory
and cognitive abilities, as well as plasticity changes in hippocampal astrocytes. We also show the
therapeutic activity of dihydromyricetin, an herbal flavonoid component, to restore such abilities
and reshape astrocytes. These findings are critical to demonstrate the impact of social isolation and
stress on the brain and provide a potential preventative measure. Given the significant and

64
continuing rise in anxiety, early and safe intervention is essential to prevent irreversible brain
damage.

Introduction
Anxiety disorders are commonly regarded as a precursor for mild cognitive decline (Porter et al.,
2003). The COVID-19 pandemic and its associated fear and stress have inflicted a great burden on
the mental wellbeing of many individuals, especially as millions have lost their loved ones
(Hossain et al., 2020). Anxiety disorder prevalence is predicted to continue growing in the post-
pandemic period (Hossain et al., 2021). Notably, social isolation has been the norm for many
during the pandemic, and several studies have demonstrated its negative impact on mental
wellbeing, including reports of acute stress disorder (ASD) along with depression, anxiety, and
insomnia (Serafini et al., 2020; Wang et al., 2021; Ye et al., 2020). Its consequences are suggested
to increase the risk of acute or chronic cognitive impairment, which are major signs of dementia
or Alzheimer’s Disease (AD) (de Oliveira, 2021). Under such stress, the brain undergoes a series
of pathophysiological and mental alterations, especially in the hippocampus (Mumtaz et al., 2018).
The hippocampus is a vulnerable and highly plastic region in the brain that plays a major role in
long-term memory, memory consolidation, and emotions like fear, anxiety, and depression
(Bartsch & Wulff, 2015; W. Liu et al., 2017). Its dysfunction is thus suggested to play a key role
in anxiety and cognition.

Synaptic loss is a major correlate of cognitive impairment (Terry et al., 1991). Previously, we
reported impaired cognition and hippocampal GABAergic (gamma-aminobutyric acid) inhibitory
synapses in animal models of transgenic AD and social isolation-induced anxiety (Liang,

65
Lindemeyer, et al., 2014; J Silva et al., 2020). The frequency, amplitude, and size of miniature
inhibitory postsynaptic currents (mIPSCs) were reduced. At the same time, gephyrin, a
postsynaptic GABAAR anchor protein that guides the formation and plasticity of GABAergic
synapses, was reduced by 50%. However, this reduction of gephyrin levels only partially
explains the GABAergic synapse dysfunction.

Glial cells in the brain closely associate with and physically support the structures of neuronal
synapses (Fields & Stevens-Graham, 2002). Astrocytes are specialized glial cells that support the
central nervous system by providing nutrients to neurons, maintaining extracellular environments,
and providing structural support, including that of the synapse (Blackburn et al., 2009; Santello et
al., 2019). Astrocytes not only regulate metabolic supplies via blood vessels and neurons, but they
also perform fine neurotransmission control by tightly enwrapping synapses and supporting
appropriate signaling and insulation (Augusto-Oliveira et al., 2020; Dallerac & Rouach, 2016).
They form complex networks to support synaptic structure (Giaume et al., 2010; Paixão & Klein,
2010), and there is increasing evidence for their involvement in complex behavioral functions
including sleep, depression, and cognitive impairment (Sofroniew, 2014). Therefore, astrocyte
could be a key target to improve cognitive function and mental wellbeing.

Astrocytes respond to stress and other insults in diverse manners to optimize their neuroprotective
abilities. These changes vary with the severity of the damage and, depending on the response, can
be either beneficial or detrimental (Rodríguez-Arellano et al., 2016; Sofroniew, 2014). For
example, mild to moderate reactive astrocytes extend their cytoskeleton, form scars, and surround
damaged tissues to protect healthy ones, while severe changes can cause undesirable upregulation

66
or downregulation of gene expressions, cellular hypertrophy or atrophy, or scattered astrocyte
proliferation (Sofroniew, 2014).

Dihydromyricetin [(2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-
one] (DHM) is a flavonoid component isolated from the herbal plant Ampelopsis grossedentata.
We previously reported that DHM acts as a positive allosteric modulator (PAM) of GABAergic
transmission, and has anxiolytic activity, rescues GABAA receptor (GABAAR) function, and
restores gephyrin expression levels (Liang, Shen, et al., 2014; Shen et al., 2012; J Silva et al.,
2020). Thus, we hypothesize that astrocytes, synaptic supporters, are involved in this pathway. In
this study, we utilized a social isolation-induced anxiety mice model (J Silva et al., 2020) to
examine the effects of social isolation and DHM on cognition and plasticity of astrocytes in the
hippocampus.

Materials and Methods

Animals
All animal experiments were performed according to the protocols approved by the University of
California (UCLA) and University of Southern California (USC) Institutional Animal Care and
Use Committee (IACUC), and all methods were carried out in accordance with relevant guidelines,
regulations, and recommendations, including the ARRIVE guidelines. Six-week-old male
C57BL/6 mice (Charles River Laboratories, Hollister, CA) were housed in the vivarium under a
12 h light/dark cycle with direct bedding and free access to food and water. Animals were
randomly assigned to experimental groups and habituated to the vivarium for 2 d before beginning
experimentation. Two or three mice were allocated in each cage for group housing. For the social
isolation groups, each mouse was separated into single cages wrapped with black plastic bags to

67
prevent social interaction with other mice. We considered the mice to be in absolute social isolation
as we singly housed them, wrapped their cages in opaque black bags, minimally handled them,
and provided no environmental stimuli such as toys. We housed the mice in groups or social
isolation for a period of four weeks. During the last two weeks, we orally administered either
sucrose (vehicle) or DHM (2 mg/kg) daily. Tissue biochemical analyses were conducted at the
University of Southern California (USC).
Groups were randomly separated as follows for a total of 4 weeks before sacrifice:
a. G2+Veh2: 2-week group housing plus 2-week group housing with vehicle (Veh) treatment
b. G2+D2: 2-week group housing plus 2-week group housing with DHM (D) treatment
c. Iso2+Veh2: 2-week social isolation (SoIso) plus 2-week SoIso with vehicle treatment
d. Iso2+D2: 2-week SoIso plus 2-week SoIso with DHM treatment
Treatment preparation
DHM (HPLC purified ≥ 98%, Master Herbs Inc., Pomona, CA) 2 mg/kg was prepared in 3% agar
cubes with 5% sucrose as described previously(J Silva et al., 2020). In short, 3% agar was
dissolved in ~90 °C water, then mixed with DHM + 5% sucrose or 5% sucrose only until cooled
and solidified. Treatment was prepared for the animals by cutting the agar into cubes of 0.5 x 0.5
x 0.5 cm and administering one cube per mouse. Both vehicle and DHM cubes were administrated
orally once a day for the last two weeks during the dark period of the 12-hour light/dark cycle with
minimal disturbance to the mice. Complete consumption of the respective treatment was observed
each day.
Animal behavioral tests
Novel Object Recognition (NOR). We conducted the NOR test according to previous reports
(Liang, Shen, et al., 2014; Martinez-Coria et al., 2010). In short, the object recognition task is

68
based on the cortex-dependent spontaneous tendency of rodents to explore a novel object for a
longer period of time compared to a familiar one. On day one, the animals were familiarized with
the empty open field for 5 mins. On day two, they were subjected to a 5-min exploration session
of two identical, symmetrically placed objects. 24 h later, the animals were subjected to a 3-min
retention session where they were exposed to one familiar object from day two and one novel
object. The times of exploration were recorded, and an object recognition index (ORI%) was
calculated, such that 𝑂𝑅 𝐼 % =
𝑡 𝑛 −𝑡 𝑓 𝑡 𝑛 +𝑡 𝑓 , where tf and tn represent times of exploring the familiar and
novel objects, respectively.

Novel Context Recognition (NCR). We conducted the NCR test, which is dependent on the
hippocampus, as described in previous reports (Liang, Lopez-Valdes, et al., 2014). The
hippocampus plays a role in remembering a particular stimulus or object in a particular place
(Barker & Warburton, 2011). In short, animals were exposed to two identical objects (i.e., two toy
balls) in a round cage for 5 mins and then to another two identical objects (i.e., two small cubes)
in a rectangular cage for 5 mins. After 24 h, 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 a recognition index 𝑅𝐼 % =
(
𝑡 𝑛𝑜𝑣𝑒𝑙 𝑡 𝑛𝑜𝑣𝑒𝑙 +𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 ) × 100 by a blinded scorer.

Immunohistochemistry fluorescent staining, imaging and analysis
Mice were dissected the day following the end day of the 4-week experiments. Left brains were
collected and fixed in 10% formaldehyde (FA, Sigma-Aldrich, USA) overnight at 4°C. Brain

69
tissues were then rinsed with 1X PBS (Sigma) and transferred to 30% sucrose solution (Sigma)
at 4°C for 3 days. Brains were washed in cold PBS and flash frozen in isopentane chilled with
liquid nitrogen. Samples were immediately stored at -80℃. For cryosectioning, frozen brains
were embedded in a mold with O.C.T. compound (Sakura, USA) on dry ice. 30 μm sagittal brain
slices were sectioned at -20°C with Microm HM525 Cryostat (Thermo, Waltham, MA) and
transferred to SuperFrost microscope slides (VWR, USA). Slides were stored in -80°C until
staining.

Antibodies were diluted in 1X PBS-Tween20 (PBS-T) + 10% BSA (w/v). Slides were incubated
in a humidifier in mouse anti-mouse GFAP primary antibody (1:500, Cell Signaling #3670,
RRID:AB_561049) for 2 days at 4°C, and then in goat anti-mouse 550 nm fluorescent antibody
(1:250, DyLight 550 #84540, RRID:AB_10942171) overnight at 4°C, protected from light.
Stained slides were mounted with DAPI mounting medium (Abcam ab104139), coverslipped,
and stored in 4°C in dark until imaging.

All images were acquired on Zeiss LSM 880 confocal microscope (Carl Zeiss Microscopy, White
Plains, NY) using Airyscan fast mode or Airyscan super-resolution mode. Images of the entire DG
were acquired using 20X objective with 0.6X zoom to define the tile scan area and 2.0X zoom to
define the laser settings and perform tile scanning. The following setups were used for the Z-stack
scan: Z-range defined by GFAP (red) channel with 20X objective and 2.0X zoom, 2.5 µm interval,
1024 x 1024 frame size, and average acquisition at 8. After scanning, the images were processed
by Zeiss Zen Black software for Airyscan Processing (3D), stitching, and maximum intensity
projection.

70
Cell morphology was observed using 63X objective in Airyscan super-resolution mode. Single
or near-single cells from similar portion on the DG area were selected using the 20X objective,
snapped with frame size at 1024 x 1024, average acquisition at 1, and then switched to 63X to
perform the Z-stack scan. The following setups were used for the Z-stack scan: Z-range defined
by DAPI (blue) channel with 63X objective and 2.0X zoom, 0.2 µm interval, 1024 x 1024 frame
size, and average acquisition at 1. After scanning, the images were processed by Zeiss Zen Black
software for Airyscan Processing (3D), Gauss, and stack correction. 3D images were generated
using 3D Surface option. All images were unified under same setups of X, Y, Z axis angles,
threshold, light intensity ambient, specular light intensity, and surface shininess.

Quantification and statistical analysis

All statistical analyses were performed using Prism v9.0.2. (GraphPad Software, Inc., La Jolla,
CA, RRID:SCR_002798) or SigmaStat v3.5 (Systat Software, Inc.). Statistical details of the
experiments can be found in the results and figures. Significance was defined as P≤0.05.
Analysis of cell density and size was performed by 3D object counter (FIJI plugin,
RRID:SCR_002285). Cell counts per sample were normalized to the analyzed DG area (in µm
2
)
and then multiplied by 1x10
6
to show the number of astrocytes per 1000 µm
2
. Astrocyte
complexity was analyzed by Sholl analysis (FIJI plugin).

Results

Cognition and memory decline induced by social isolation is ameliorated by DHM
Recognition memory is composed of at least two elements: the familiarity of items and the
contextual information (spatial and/or temporal) in which the items were encountered (Balderas et
al., 2008). In this study, we used two behavioral tests for evaluating both components of

71
recognition memory: novel object recognition (NOR) test for familiarity of items, and novel
context recognition (NCR) for contextual memory. Mice in social isolation exhibited worsened
recognition memory (Fig. 1A). G2+Veh2 mice spent more time exploring the novel objects with
higher Object Recognition Index (ORI = 66.3 ± 4.7 %) than Iso2+Veh2 mice (ORI = 55.3 ± 4.1
%) in NOR test (P = 0.0139, two-way ANOVA with multiple comparisons to the control, Holm-
Sidak method). DHM improved object recognition memory of SoIso mice by roughly 9% (ORI =
64.2 ± 3.8 %). Similarly, Recognition Index (RI) of NCR was calculated in every group of mice
(Fig. 1B). Compared with G2+Veh2, Iso2 +Veh2 mice exhibited reduced RI (51.5 ± 6.5 %, P =
0.002, two-way ANOVA with multiple comparisons to the control, Holm-Sidak method). DHM
administration reversed the RI in Iso2+D2 mice and showed substantial contextual memory
improvement. These results indicate that daily oral administration of DHM restores memory and
cognition in the SoIso mice.

Figure 3. 1 Cognition-related behaviors.
(A) Novel object recognition. ORI = object recognition index. (B) Novel context recognition. RI
= recognition index. G2+Veh2: 2-week grouped plus 2-week grouped with vehicle treatment;
G2+D2: 2-week grouped plus 2-week grouped with DHM treatment; Iso2+Veh2: 2-week SoIso
plus 2-week SoIso with vehicle treatment; Iso2+D2: 2-week SoIso plus 2-week SoIso with DHM

72
administration. Bars represent mean ± SEM. Two-way ANOVA followed by multiple comparisons
with Holm-Sidak method. *, P≤0.05. †, P≤0.05 vs Iso2+D2.

Social isolation and DHM induce changes in astrocytic density and size in the hippocampus
Next, we questioned whether social isolation induces astrocytic changes, as these changes are
commonly observed in individuals with cognitive impairment, dementia, and AD (Dossi et al.,
2018). To determine the effect of social isolation on astrocyte plasticity, we acquired sagittal
mice brain images in the dentate gyrus (DG) using glial fibrillary acidic protein (GFAP) as the
marker. Increased GFAP expression in astrocytes is commonly used as a marker for reactive
astrocytes (Wilhelmsson et al., 2006). Our results show an increase in the number of GFAP-
positive astrocytes in Iso2+Veh2 compared to the control (G2+Veh2) (424.2 vs 277.6 cells/1x10
6

µm, P = 0.0117, two-way ANOVA with multiple comparisons to the control, Holm-Sidak
method), which was reduced by DHM administration (Iso2+D2) back to the control level
(G2+Veh2 [277.6] vs Iso2+D2 [285.5] (Fig. 2A and 2B). Further, social isolation reduced
astrocyte size compared to the control (Fig. 2C) (median 88.246 vs 97.223 µm
2
). Since the
astrocyte size and volume are not normally distributed, we used Kruskal-Wallis one-way
ANOVA with multiple comparisons, Dunn’s method to analyze them. The data are expressed as
medians and percentiles with box plots. On the other hand, astrocyte sizes in DHM-administered
mice (131.739 µm
2
) were larger compared to the control (DHM control (92.15 µm
2
), and SoIso
mice), suggesting possible effects of DHM on mediating anti-inflammatory responses or
recovery of astrocytic activities (Fig. 2C). These differences were mirrored in astrocyte volumes
(Fig. 2D). G2+Veh2 vs G2+D2 showed no significant difference in size and volume.

73

Figure 3. 2 Astrocyte density and size in the dentate gyrus.
(A) 20X Airyscan super resolution images of single or near-single astrocytes in the dentate
gyrus of mice: blue = DAPI, nucleus; red = GFAP, astrocytes. Scale bar = 200 µm. (B) Number
of astrocytes per sample normalized to DG area. Data shown as mean + SEM. Two-way
ANOVA with multiple comparisons, Holm-Sidak method. Astrocyte size in µm
2
(C) and µm
3

(D). Data shown as median + interquartile range (IQR) with all astrocyte size from each group
compiled into a single dataset. Kruskal-Wallis one-way ANOVA on ranks with multiple
comparisons, Dunn’s method. For all graphs, N = 3-4 mice analyzed per group, one section per
mouse. G2+Veh2: 2-week grouped plus 2-week grouped with vehicle treatment; G2+D2: 2-week
grouped plus 2-week grouped with DHM administration; Iso2+Veh2: 2-week SoIso plus 2-week
SoIso with vehicle treatment; Iso2+D2: 2-week SoIso plus 2-week SoIso with DHM
administration. *, P≤0.05; †, P≤0.05 vs Iso2+D2; ‡, P≤0.05 vs G2+D2.

74
Social isolation and DHM induce morphological changes in the astrocytes of the
hippocampus
Astrocytes have dynamic and distinct morphological changes in response to the environment
(Rodríguez-Arellano et al., 2016). Consistent with Fig. 2, Iso2+Veh2 showed more cells per area
with reduction in branches, while DHM administration increased cell size with thicker, longer
branches (Fig. 3). Three-dimensional Z-stack images in comparable areas of the DG for each
group further demonstrate the increased activated astrocyte count in SoIso group (Fig. 3A, B,
center) compared to the control (Fig. 3A, B, left), which was modulated by DHM (Fig. 3A, B,
right). Astrocyte complexity as defined by Sholl analysis showed a decrease in Iso2+Veh2
compared to the control (G2+Veh2) at 10, 15, 20, and 25 µm
2
from the nucleus (P = 0.0462,
<0.0001, 0.0009, and 0.0042, , respectively, two-way ANOVA with multiple comparisons to the
control, Holm-Sidak method) and an increase with DHM administration at the same distances
(Fig. 3C, D).

75

Figure 3. 3 Astrocyte morphology in the dentate gyrus.
(A) Upper: two-dimensional images of the astrocytes analyzed for morphology. Bottom: area of
the DG containing the selected astrocyte. Blue = DAPI, nucleus; red = GFAP, astrocytes. Scale
bar = 20 µm. (B) Three-dimensional images of astrocyte morphology. Scale bar = 20 µm. (C)
Binary representation of astrocytes used for morphological complexity. Scale bar = 20 µm. (D)

76
Quantification of morphological complexity (Sholl analysis). Two-way ANOVA with multiple
comparisons, Holm-Sidak method. G2+Veh2: 2-week grouped plus 2-week grouped with vehicle
treatment; G2+D2: 2-week grouped plus 2-week grouped with DHM administration; Iso2+Veh2:
2-week SoIso plus 2-week SoIso with vehicle treatment; Iso2+D2: 2-week SoIso plus 2-week
SoIso with DHM administration. 5 astrocytes analyzed per mouse. N = 4 mice per group. *,
P≤0.05.

Discussion

Loneliness serves as an early predictor for psychological problems like anxiety disorder that could
further lead to suicidal thoughts and risk of later-life cognitive decline (Evans et al., 2019; Yanguas
et al., 2018). The COVID-19 pandemic and social isolation have amplified anxiety disorder
prevalence that is likely to persist in the post-pandemic period. In this study, we found that social
isolation worsened cognitive and memory abilities as it decreased ORI in NOR and RI in NCR
tests, increased astrocyte density, reduced astrocyte size with shorter branches, and reduced
morphological complexity in the hippocampus. DHM improved memory, cognition, and astrocyte
plasticity in these mice. Our results indicate that social isolation leads to deficits in memory and
cognition, as well as hippocampal astrocyte atrophy in mice models of social isolation-induced
anxiety. Previously, we have shown that four weeks of social isolation considerably elevated
anxious behaviors in mice, while two weeks of DHM administration reduced these behaviors (J
Silva et al., 2020). In this study, we further illustrate the capability of DHM to improve cognitive
loss induced by social isolation. Based on NCR and NOR, memory and cognitive abilities in
socially isolated mice were restored by DHM administration, suggesting that DHM has
neuroprotective properties that result in ameliorating these deficits (Fig. 1). Because the
hippocampus is one of the first regions in the brain to suffer damage in mild cognitive impairment,
including initial stages of AD (Dubois et al., 2016; Frisoni et al., 2010), we focused our imaging
studies on the hippocampus.

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Proper synapse distance and structure is essential for neurotransmission and thus essential for sharp
cognition and memory. Astrocytes support synapse connection and thus are also essential players
in cognition (Augusto-Oliveira et al., 2020; Dallerac & Rouach, 2016). As Buss and colleagues
(2021) have demonstrated, circuit organization breakdown was associated with diminished
cognitive aging and reduction of DG synaptic input onto the CA3 pyramidal neurons (Buss et al.,
2021). Other studies have also found decreased synaptic density with decreased cognitive and
memory abilities in individuals with AD (DeKosky & Scheff, 1990; Kashyap et al., 2019; Terry
et al., 1991). Our present findings show decreased astrocyte complexity and size, in parallel with
diminished cognitive and memory abilities in anxious mice (Fig. 1, Fig. 2C and Fig. 3), suggesting
a reduction in astrocyte capability to support synapses. Further investigation is necessary to
understand the relationship between astrocyte morphology and synaptic density/function,
including studies involving synaptic markers.

The hippocampal astrocytes are mainly protoplasmic (highly branched), highly express GFAP,
and are dynamic in changes related to immune response and synapse elimination (Matias et al.,
2019). Although GFAP expression is generally reduced in aged animals(Rodríguez et al., 2009),
the connection between GFAP expression and astrocyte reactivity in AD is controversial. While
many have found higher hippocampal GFAP levels with AD clinical progression, others have
observed no difference between healthy and afflicted brains in humans (Dossi et al., 2018). The
difference is likely attributed to both brain region (Matias et al., 2019) and disease stage. At early
clinical stages of transgenic AD mice models, astrocytes showed atrophy—reduced volume,
surface area, and morphological complexity—and reduced GFAP expression, while disease

78
progression corresponded to increased GFAP expression (Rodríguez-Arellano et al., 2016). These
observations were mirrored in post-mortem brains of AD patients, in addition to reduced astrocyte
complexity and volume occupied by a single astrocyte (Rodríguez et al., 2009; Rodríguez-Arellano
et al., 2016). Astrocytes in our socially isolated mice displayed increased cell density but reduction
in size and morphological complexity (Fig. 2, 3), suggesting that social isolation can induce
astrocyte atrophy, potentially disrupting synaptic support and neuroprotective abilities. While
Hama et al. (2004) and Oyabu et al. (2020) have demonstrated that increased astrocytic density
increased excitatory synaptic transmission in primary rat and mouse cultures (Hama et al., 2004;
Oyabu et al., 2020), our group previously found reductions in the inhibitory synaptic transmission
(GABAAR) in mice brain slices (J Silva et al., 2020). Thus, in social isolation, increased astrocytic
density may disrupt the balance of synaptic transmission due to increased excitatory transmission
and decreased inhibitory transmission. This imbalance may lead to the nervous, restless, or
aggressive behavior(s) observed in anxiety disorders (Craske et al., 2017). Additional studies
should examine the role of astrocytes in increasing or decreasing synaptic transmission, as well as
upstream mechanisms of astrocytic proliferation and function.

Interestingly, while SoIso alone increased astrocyte density and reduced size, we found that SoIso-
DHM administration did not alter astrocyte density (Fig. 2B) but exhibited larger astrocytes (Fig.
2C, D) compared to control. Furthermore, three-dimensional analysis showed that DHM-
administered mice had longer, thicker astrocyte branches with morphological complexity greater
than those in control, SoIso, and DHM control (Fig. 3). These findings are significant because
after social isolation, DHM not only restored astrocytic density similar to that of control, but it
also altered astrocyte morphology. These changes induced by DHM are similar to those shown in

79
aged mice and rats that were exposed to environmental enrichments like toys, running wheels, and
other mice (Rodríguez-Arellano et al., 2016). These rodents showed increased morphological
complexity and plasticity in astrocytes and improved cognitive abilities (Rodríguez-Arellano et
al., 2016). Our findings mirror these data, as DHM-administered mice exhibited recovery in
cognitive and memory abilities (Fig. 1). Thus, social isolation induces symptoms of early aging in
mice, while DHM recovers such symptoms, potentially by restoring astrocytic ability to support
synapses.

In our previous studies with socially isolated mice, we reported that DHM reversed the reduced
levels of adenine triphosphate (ATP) and gephyrin, a scaffolding protein that supports and
stabilizes the clustering of post-synaptic GABAA receptors(J Silva et al., 2020). We also recently
demonstrated that DHM ameliorated GABAAR-mediated currents, microglia, and
neuroinflammation in the same mice model (Alzahra J. Al Omran et al., 2022; J Silva et al., 2020).
GABAARs are highly expressed in astrocytes and allow them to sense and respond to their
environment (Mederos & Perea, 2019; Verkhratsky & Nedergaard, 2018). Receptor activation
induces membrane depolarization—instead of hyperpolarization as in neurons—and increases
intracellular Ca
2+
, stimulating the release of various signal molecules like ATP (Chung et al.,
2022). Neuroinflammation, however, has been shown to reduce astrocytic GABAAR expression
and neurotransmission (Tadhg Crowley et al., 2016). Collectively, these findings suggest that
social isolation induces improper GABAAR clustering via gephyrin downregulation, subsequently
reducing the ability of astrocytes to respond to their environment. Loss of this ability may induce
astrocytic atrophy, which then leads to loss of homeostatic and/or synaptic functions (Preman et
al., 2021). The increased astrocytic density in the hippocampus could be a result of compensatory

80
mechanisms, in which astrocytes migrate towards areas where synaptic transmission is reduced.
We hypothesize that DHM modulates astrocyte plasticity and reverses/prevents these changes by
promoting proper GABAAR clustering and functioning. Our hypothesis and upstream mechanism
of gephyrin expression will be investigated in future studies.

In this study, we detected changes in memory, cognition, and hippocampal astrocytes in social
isolation-induced anxiety mice models. These substantial changes within a short period of time
suggest that repeated or prolonged anxiety and stress could lead to a greater long-term
consequence, such as permanent brain damage, dementia, and Alzheimer’s Disease (AD).
Furthermore, current anxiolytic medications are often not fully effective nor are readily available
to individuals, so they have minimal ability to prevent these long-term consequences (Craske et
al., 2017). Novel drug development and marketing are timely and costly as well (Hutson et al.,
2017; Insel, 2012; Paul et al., 2010), and thus cannot quickly address the sharp rise in anxiety,
ASD, and declined mental wellbeing due to the pandemic. In contrast, DHM has a high potential
to be developed as a quick and early intervention to anxiety, especially since the extensive
process of developing therapeutics de novo is eliminated (Hutson et al., 2017).

This study is subject to a few limitations. First, anxiety prevalence and pathology has been shown
to differ between sexes (McLean et al., 2011), and our present study only included males. Thus,
mirrored studies in female mice are ongoing. Because the astrocyte is a dense and compact area
with overlapping branches, we could not obtain images for a true, single astrocyte. Morphological
analysis, although threshold-based, may not be entirely accurate. Nonetheless, our findings shed
light on the impact of social isolation on cognition and neuroprotective/astrocytic abilities.

81

Our findings of the adverse cognitive and cellular effects of social isolation suggest that social
isolation can lead to early synaptic loss and aging signs. Further, we demonstrated the
therapeutic activity of DHM to restore these cognitive damages and reshape astrocytes. Our
results suggest astrocyte involvement in social isolation-induced cognitive impairment and
anxiety and demonstrate the potential of astrocytes as a therapeutic target. Further, our study
indicates DHM to be a promising candidate for early intervention against anxiety, cognitive
impairment, and long-term risks of permanent brain damage. Prompt actions will delay brain
aging, cognitive dysfunctions, and potential progression of severe diseases like dementia and
AD. Early intervention is critical to prevent such irreversible outcomes.

AUTHOR CONTRIBUTIONS
A.A.O., A.S.S., and J.L. conceptualized and designed the study. S.W., A.S.S., Z.Z., J.W., and J.L.
contributed to the methodology. S.W., A.A.O., A.S.S., C.X., Z.Z., and J.Z. performed experiments.
A.S.S. created Fig. 1. S.W. and Z.Z. created Fig. 2 and 3. S.W., A.A.O., A.S.S., C.X., Z.Z., X.M.S.,
and J.L. wrote the original draft. All authors contributed to editing and finalizing the manuscript.
D.L.D. and J.L. acquired funds. J.L. supervised the project.

ADDITIONAL INFORMATION
The authors declare no financial interests or conflicts of interest.

82
ACKNOWLEDGEMENTS
This continuing research was supported by the National Institute of Health grants AA17991 (to
J.L.), AA022448 (to D.L.D.), Carefree Biotechnology Foundation, Saudi Arabia Cultural Mission
Scholarship (to A.A.O.), and Army Health Professions Scholarship Program (to A.S.S.). We would
like to thank Dr. Richard W. Olsen, Distinguished Professor at the David Geffen School of
Medicine at UCLA, for his comments.

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CHAPTER 4

Dihydromyricetin ameliorates social isolation-induced anxiety by modulating
mitochondrial function, antioxidant enzymes, and BDNF

Alzahra J. Al Omran
1†
, Saki Watanabe
1
, Ethan C. Hong
1
, Samantha G. Skinner
1
, Mindy
Zhang
1
, Jifeng Zhang
1
, Xuesi M. Shao
2
, Jing Liang
1*

(†) First author
(*) Corresponding author

Authors' information
5. Titus Family Department of Clinical Pharmacy, University of Southern California School
of Pharmacy, Los Angeles, CA, 90033, USA.
6. Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095,
USA.

Abstract:

Stress has been implicated in the etiology of neurological and psychological illnesses. Chronic
social isolation (SI) is a psychological stressor that provokes neurobehavioral changes associated
with psychiatric disorders, including anxiety disorders. Mitochondria dysfunction and oxidative
stress are hallmarks of anxiety pathogenesis. Here we demonstrate the effects of SI-induced
stress on mitochondrial function, antioxidative enzymes, autophagy, and brain derivative

84
neurotrophic factor (BDNF). SI induced a reduction in electron transport chain subunits C-I, C-
II, and C-VI and an increase in hydrogen peroxide. Treatment with Dihydromyricetin (DHM), a
major plant bioactive flavonoid extracted from Ampelopsis grossedentata, counteracted these
changes. A dramatic increase in several primary mitochondrial antioxidative enzymes such as
superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1), peroxiredoxin-3 (PRDX3), and
glutathione peroxidase 4 (GPX4) was observed after SI and a repeated episode of SI. Both SI and
repeated SI induced a reduction in sequestosome 1 (SQSTM1/p62); however, only repeated SI
modulated autophagy primary protein beclin-1 (Bcl-1). In addition, SI and repeated SI modulated
the BDNF-TrkB signaling pathway and the phosphorylation of the downstream extracellular
signal-regulated MAP kinase1/2 (p-Erk p42 and p-Erk p44) cascade. DHM treatment
ameliorated these changes. Collectively, we demonstrated that DHM treatment counteracted the
effects of SI and repeated SI on antioxidative enzymes, autophagy, and the BDNF-TrkB
signaling pathway. These findings highlight the molecular mechanisms that partially explain the
anxiolytic effects of DHM.

Introduction:

In the U.S., anxiety disorders are the most prevalent mental illness. Current estimates suggest
that more than 40 million (18%) adults are suffering from anxiety disorders, which imposes a
great economic burden on public health and accounts for more than $42 billion in costs for
medical evaluation and treatment (Facts and Statistics, 2016). Anxiety disorders are often
comorbid with depression, posttraumatic stress disorder (PSTD), substance use disorders, and
subsequent cognitive impairment (Becker et al., 2018; Kessler et al., 2010). Various
psychological stressors contribute to the pathophysiology of anxiety disorders, which include

85
environmental, socioeconomic, and genetic factors (Hettema et al., 2005; Nugent et al., 2011).
Furthermore, modern society's social and demographic changes have increased loneliness and
social isolation (Beutel et al., 2017; Klinenberg, 2016). This phenomenon is further exacerbated
by the coronavirus disease 2019 (COVID-19) pandemic, which has led to global social isolation
that negatively impacts the public's mental and psychological well-being and creates heightened
anxiety disorders (Loades et al., 2020; Twenge & Joiner, 2020). As a result of the pandemic,
there was an estimated 26% elevation in anxiety disorders rate globally from 2020 to 2021
(Collaborators, 2021). Despite the high prevalence of anxiety, effective and safe therapies are
still considered a growing unmet medical need. Unfortunately, not all patients respond well to
the currently available treatments, and some have treatment resistance, leaving more than 40% of
the patient population lacking effective medications (Roy-Byrne, 2015).

The inhibitory neurotransmitter ɣ-aminobutryic acid (GABA) has long been recognized as the
primary modulator in anxiety disorders (Nuss, 2015). An increasing number of studies have
implied the association of neuroinflammation, mitochondrial dysfunction, and oxidative stress
pathways in the pathogenesis of anxiety (Shao et al., 2015; Zlatković, Bernardi, et al., 2014;
Zlatković, Todorović, et al., 2014). Previous work from our group showed dysregulation in the
hypothalamic-pituitary-adrenal (HPA) axis, manifesting as an increase in serum corticosteroid
levels in the social isolation (SI)-induced anxiety mouse model, which is considered an indicator
of stress (A. J. Al Omran et al., 2022). Furthermore, SI mice showed signs of neuroinflammation
through activation of the NF-kB signaling pathway that triggered microglia morphological
changes (A. J. Al Omran et al., 2022). Additionally, we demonstrated that SI induces an
alteration in mitochondrial energy metabolism represented by a reduction in ATP level (J. Silva,

86
A. S. Shao, et al., 2020). Neuroinflammation negatively impacts mitochondrial viability and
contributes to neuronal excitotoxicity, oxidative stress damage, energy depletion, neurogenesis
impairment, and eventually neuronal death (Banagozar Mohammadi et al., 2019; C. Li et al.,
2017). Therefore, distressed mitochondria also provoke further neuroinflammation, creating a
constant pathological cycle between neuroinflammation, mitochondrial impairment, oxidative
stress, and neurotoxicity. In the present study, we further investigated the effect of psychological
stress on mitochondrial function, the antioxidative defense system, autophagy, and the brain-
derived neurotrophic factor (BDNF) pathway utilizing a SI mouse model. Moreover, we
investigated the impact of repeated SI stress on mitochondrial function, oxidative stress,
autophagy, and the BDNF pathway. These observations were examined in the brain’s prefrontal
cortex (PFC) due to its essential role in regulating fear and anxiety emotions by integrating
inputs from the amygdala and the hippocampus (Burgos-Robles et al., 2009).

Previously, we have shown that dihydromyricetin (DHM) [(2R,3R)-3,5,7-trihydroxy-2-(3,4,5-
trihydroxyphenyl)-2,3-dihydrochromen-4-one], a primary plant bioactive flavonoid extracted
from Ampelopsis grossedentata, is a positive modulator of the GABAA receptor (GABAARs)
(Liang, López-Valdés, et al., 2014; J. Silva, A. S. Shao, et al., 2020). It antagonizes the
detrimental effects of acute and chronic ethanol consumption on GABAARs in animal models
(Liang, López-Valdés, et al., 2014; Shen et al., 2012). Furthermore, we previously demonstrated
that DHM exerts anxiolytic effects in SI mouse model by improving GABAergic
neurotransmission and GABAAR function (J. Silva, A. S. Shao, et al., 2020). Therefore, DHM’s
effect on GABA provides one possible mechanism for its ability to improve anxiety-like
behaviors in SI mice. Notably, GABAergic transmission impairment is a primary factor in

87
neurotoxicity that provokes a chain of events, including mitochondrial dysfunction, changes in
antioxidant enzyme activity, oxidative stress, and neuroplasticity dysfunction. This triggers
several cellular pathways leading to behavioral deficits (Chanana & Kumar, 2016; Zhu et al.,
2019). Therefore, studying the effects of DHM on mitochondria, antioxidative enzyme activity,
and BDNF is essential in elucidating the mechanism of DHM’s anxiolytic action. This study will
help gain insight into the cellular and molecular aspects of anxiety disorders, which is a crucial
step in developing novel and effective therapeutics. In addition, this study will expand our
understanding of DHM’s anxiolytic mechanisms and set the stage for DHM to be a therapy for
anxiety disorders.

Methods:

Animals and treatment
Experimental design:
Eight-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were housed in the
vivarium under controlled temperature, humidity, and 12 h light/dark cycles with free access to
food and water. All animal experiments were performed according to the protocols approved by
the University of Southern California (USC) Institutional Animal Care and Use Committee. Mice
were randomly assigned to either a group-housed group (2-4 mice/cage) or an isolated group. SI
mice were singly housed in opaque-walled cages with reduced bedding, no environmental
enhancers (toys/objects), and minimal human handling with the exception of weekly cage changes.
The treatment groups were organized as described below:

88
A. Social isolation for 4 weeks phase: Animals were either grouped or singly housed for 2 weeks,
followed by 2 weeks of treatment (Fig. 1A).
1. Group-housed, 2-4 mice/cage, and administered vehicle (sucrose) (G+V), n=11
2. Group-housed, 2-4 mice/cage, and administered DHM 2mg/kg (G+D), n=11
3. Isolated, single-housed, and administered vehicle (sucrose) (Iso+V), n=14
4. Isolated, single-housed, and administered DHM 2mg/kg (Iso+D), n=14

After the 4 weeks SI phase, four mice from each group were randomly selected and kept to test
the effect of anxiety withdrawal, followed by a second isolation period. Mice designated for the
isolation group were grouped housed for 4 weeks, followed by another 4 weeks of isolation. The
group housed mice were grouped in the same cage for the rest of the experiment period as shown
in (Fig. 1B).

B. Social isolation withdrawal period followed by repeated social isolation phase:
1. Group-housed, 2-4 mice/cage and administered vehicle (sucrose) (G+V), n=4
2. Group-housed, 2-4 mice/cage and administered DHM 2mg/kg (G+D), n=4
3. Isolated, single-housed, and administered vehicle (sucrose) (R-Iso+V), n=4
4. Isolated, single-housed, and administered DHM 2mg/kg (R-Iso+D), n=4

89

Figure 4. 1 Study design and timeline of the experiments.
(A) Mice were single-housed for 2 weeks followed by 2 weeks of DHM 2mg/kg treatment. (B)
Mice were single-housed for 2 weeks followed by 2 weeks of DHM 2mg/kg treatment. The same
group of mice was group-housed (SI-withdrawal) for 4 weeks, then another 2 weeks repeated SI,
then 2 weeks of DHM 2mg/kg treatment. The group-housed mice (G+V and G+D) remained
group housed for the entire length of the study.

Drug preparations:
DHM (HPLC purified ≥ 98%, Master Herbs Inc., Pomona, CA) was given orally as an agar cube
once per day (2 mg/kg) for 2 weeks (J. Silva, A. S. Shao, et al., 2020). DHM and vehicle agar
cubes were prepared using 3% agar with water and then heated to ~90 °C to dissolve the agar.
Subsequently, DHM + 5% sucrose (for DHM group) or 5% sucrose only (for vehicle group) was
added and mixed until cooled and solidified. Then, the agar was cut into cubes of 0.5 X 0.5 X 0.5
cm each.

90

Drug administration:
During the dark cycle, the mice were given DHM or vehicle by placing the agar cube in a 50 X
50 X 8mm weighing dish and placing it in the cage after removing all food. To give the
treatment for group housed, mice were separated, fed DHM or vehicle, and then returned to the
group housing cage. All animals were observed to ensure complete consumption of agar, which
took 30-90 minutes.

Hydrogen peroxide assay:
Mice were dissected after 4 weeks of social isolation and 12 weeks of repeated social isolation.
The amount of H2O2 in the prefrontal cortex (PFC) tissue was measured using a hydrogen
peroxide assay kit (ab102500, Abcam, Cambridge, MA, USA) according to the manufacturer’s
instructions. The PFC tissues were collected and lysed in ice-cold assay buffer provided by the
kit. The supernatants were collected, deproteinized, and incubated in a working solution
containing OxiRed probe and horseradish peroxidase (HRP). The intensity of the color
developed was measured at 570 nm using a Synergy H1 Hybrid Multi-Mode Reader (BioTek).

Western blot analysis:
Equal protein concentrations of PFC homogenates were separated on 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Bio-Rad
Laboratories, Hercules, CA). Membranes were blocked in 5% skim milk in 1X Tris-buffered
saline with Tween 20 (TBST) for 1 hour at room temperature. Membranes were then incubated
overnight (4 °C) with either mouse anti-mouse OxPhos antibody cocktail (Thermo Fisher 45-

91
8099, 1:200), mouse anti-mouse β-actin (Cell Signaling 4970, 1:1000), rabbit anti-mouse (mAb)
SOD-2 (Cell Signaling 13141, 1:1000), rabbit mAb HO-1 (Cell Signaling 86806, 1:1000), rabbit
mAb PRDX-3 (Abcam ab73349, 1:1000), rabbit mAb GPX-4 (Cell Signaling 52455, 1:1000),
Bcl-1 rabbit mAb Cell Signaling 42406, 1:1000), p62 rabbit mAb Cell Signaling 23214, 1:1000),
BDNF (Cell Signaling 47808, 1:1000) , p-TrkB (Abcam ab229908, 1:1000), p44/42 MAPK
(Erk1/2) (Cell Signaling L34F12, 1:1000) Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling
D13.14.4E, 1:1000). After washing three times with 1X TBST, the membranes were incubated in
a secondary antibody goat anti-rabbit IgG or goat anti-mouse (Bio-Rad 1706515 and 1706516) in
1X TBST for 1 hour. Blots were subsequently developed with an enhanced chemiluminescence
(ECL) system (Bio-Rad 1705061) and visualized using a Chemi-Doc (Bio-Rad) imaging device.

SOD activity:
The colorimetric superoxide dismutase activity assay kit (ab65354, Abcam, Cambridge, MA,
USA) was used to detect the activity of SOD in PFC homogenates. According to the
manufacturer’s instructions, each sample requires three blanks to calculate the inhibition rate in a
96 well plate. Blank-1 contained dH2O+enzyme working solution, blank-2 with sample alone, and
blank-3 with dH2O alone in addition to the fourth well of the sample + enzyme working solution.
The intensity of the developed color was measured at λ = 450 nm using Synergy H1 Hybrid Multi-
Mode Reader (BioTek). The OD values were used to calculate the SOD activity expressed as the
inhibition rate following the equation listed in the instruction.

SOD Activity (inhibition rate %)
=[(blank1−blank3)−(sample−blank2) × 100]/ (blank1−blank3)

92

ELISA analysis:
PFC tissue lysates were homogenized in RIPA lysis buffer (89900, Thermo Scientific, Milford,
MA). The homogenates were then used to quantify BDNF protein concentration using an ELISA
kit and following the manufacturer’s protocol (DBNT00, R&D Systems, Minneapolis, MN,
USA). The intensity of the color developed was measured using a Synergy H1 Hybrid Multi-
Mode Reader (BioTek).

Statistical analysis
All assays were performed at least three times. The data were presented as the mean ± standard
error of the mean using GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA). Two-way
analysis of variance (ANOVA) followed by Holm-Sidak multiple comparison test (SigmaStat,
version 3.5) were performed, and the significance level was set at p < 0.05.

93

Results

The effect of social isolation, repeated social isolation, and DHM treatment on reactive
oxygen species (ROS) formation and mitochondrial complex I, II, and IV protein
expression

Figure 4. 2 SI mice exhibit an increase in H2O2 Levels and a decrease in mitochondrial
complexes I, II, and IV, while DHM normalizes them.
(A) The levels of H2O2 nmol/mg of PFC tissue after 4 weeks of SI (Iso+V), repeated SI (R-
Iso+V), and after DHM treatment (Iso+D, R-Iso+D). (B) Representative Western blots of
OxPhos proteins expression in ETC C-V (50kDa), C-III (48kDa), C-IV (35kDa), C-II (25kDa),
C-I (17kDa), and β-actin (42 kDa). (C, D, E, F, and G) Quantitative analysis ratio of C-I, C-II,
C-III, C-IV, and C-V. Values were normalized by the corresponding β-actin. Data are presented
as mean ± SEM values (n=4-6 for A, n=7 for C, D, E, F, and G ). Two-way ANOVA followed
by multiple comparisons, Holm-Sidak’s method *p<0.05 vs. G+V, #p<0.05 vs. Iso+V or R-
Iso+V.

94
To assess the levels of reactive oxygen species (ROS) production after SI, hydrogen peroxide
(H2O2) activity was measured. H2O2 is a common reactive oxygen metabolic byproduct and a
potent inducer of oxidative stress (X. Hou et al., 2015). The level of H2O2 was substantially
higher in Iso+V (0.134 ± 0.0137 nmol/mg) and repeated SI R-Ios+V (0.143 ± 0.015, p < 0.001)
vs G+V (0.0402 ± 0.0125 nmol/mg, p < 0.001). The detected level of H2O2 was significantly
lower in Iso+D (0.0803 ± 0.0177, p = 0.008) and R-Iso+D (0.083 ± 0.0177, p = 0.018) following
DHM 2mg/kg treatment (Fig. 2A). These results suggest that SI elevates ROS production, while
DHM administration counteracts this effect. Additionally, this observation was consistent even
after a period of SI stress discontinuation followed by repeated SI.

ROS are produced in the mitochondria at various locations on the electron transport chain (ETC),
complex proteins necessary for mitochondrial respiration (Indo et al., 2007). Because stress is
known to play a role in ROS production, protein levels were analyzed in the five mitochondrial
complexes that comprise the ETC (Zlatković & Filipović, 2013). Significant reductions in the
expression of C-I (0.574 ± 0.035), C-II (0.440 ± 0.098), and C-IV (0.503 ± 0.129) were
identified in Iso+V compared to the control G+V C-I (1.116 ± 0.115), C-II (0.851 ± 0.116), and
C-IV (1.052 ± 0.152). DHM treatment restored the protein expressions of C-I (0.983 ± 0.139), C-
II (0.780 ± 0.098), and C-IV (1.036 ± 0.120) in Iso+D group relative to the Iso+V (Fig. 2C, D,
F). SI did not influence the protein expressions of C-III and C-V (Fig. 2E, G). These results
suggest that DHM has pharmacotherapeutic effects on C-I, C-II, and C-IV, which subsequently
improves ROS levels in the PFC.

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Social isolation and repeated social isolation enhance the activities of mitochondrial
antioxidative enzymes, while DHM normalizes them

Figure 4. 3 Modulation of mitochondrial antioxidant enzymes activity and protein levels induced
by SI, repeated SI, and DHM treatment.
(A, B) Representative Western blot of proteins expression of SOD2, HO-1, PRDX-3, GPX4, and
β -actin. (C) Total SOD activity is represented as percent inhibition of H2O2. (D, E, F, and G)
SOD2, HO-1, PRDX-3, and GPX4 antioxidant capacity were detected by measuring the protein

96
expression of each antioxidant enzyme using western blot, and the values were normalized by
the corresponding β-actin. Data are presented as the mean ± SEM. Two-way ANOVA followed
by multiple comparisons, Holm-Sidak’s method *p<0.05 vs. G+V, #p<0.05 vs. Iso+V or R-
Iso+V.

The activity and protein levels of the mitochondrial antioxidant enzymes superoxide dismutase 2
(SOD2), heme oxygenase-1 (HO-1), peroxiredoxin-3 (PRDX3), and glutathione peroxidase 4
(GPX4) were measured to determine the effects of SI and DHM on the mitochondria’s
antioxidant defense system. Superoxide anions (O2
-
) are a byproduct of phosphorylation that can
damage the ETC and other components of the mitochondria. SOD2 converts superoxide radicals
into less reactive H2O2 molecules, preventing further damage (Storz et al., 2005). HO-1 is an
enzyme that has been implicated in the regulation of stress and the cell’s adaptive response to
oxidative injury (Chen et al., 2000). Additionally, PRDX3 prevents ROS from harming the
mitochondria by efficiently removing H2O2 from the mitochondrial matrix (L. Chen et al., 2008).
Moreover, GPX4 is a part of the glutathione redox cycle, which is an important line of defense
against H2O2 and lipid hydroperoxides. GPX4 is a mitochondrial antioxidant enzyme that can
directly reduce these damaging phospholipid hydroperoxides (Imai & Nakagawa, 2003; Ran et
al., 2006). Colorimetric analyses and Western blot were used to determine the activity and
protein ratios, respectively. The activity of total SOD enzyme in Iso+V (106.9 ± 5.93) was
substantially higher than G+V (83.88 ± 5.76). DHM treatment reduced the level of total SOD
relative to the control in Iso+D (75.90 ± 7.66) (Fig. 3C). Additionally, the protein expression of
antioxidant enzymes SOD2 (1.787 ± 0.188), HO-1 (1.584 ± 0.154), and GPX4 (1.754 ± 0.184)
were significantly higher in Iso+V compared to the control (G+V). DHM treatment after SI
(Iso+D) showed protein expression levels similar to the control: SOD2 (1.218 ± 0.163), HO-
1(1.282 ± 0.154), PRDX3 (1.28 ± 0.354), and GPX4 (1.185 ± 0.184) (not statistically different

97
from G+V group). The G+D group showed a significant increase compared to G+V for SOD2
(1.398 ± 0.115), HO-1 (1.316 ± 0.109), and GPX4 (1.409 ± 0.130), except for PRDX3 (1.56 ±
0.26) did not show the difference (Fig. 3D, E, F, and G). These results indicate that SI correlates
with an increase in antioxidant activity protein expression, while DHM has a pharmacologic
effect on normalizing these levels. Furthermore, the level of HO-1 (1.391 ± 0.189), PRDX3
(2.047 ± 0.364), and GPX4 (1.675 ± 0.184) in R-Iso+V was upregulated after repeated SI
compared to the control. While the levels of HO-1 (1.088 ± 0.169), PRDX3 (1.672 ± 0.364), and
GPX4 (1.292 ± 0.184) in R-Iso+D group showed similar protein expression to the control G+V
group (Fig. 3E, F, G). SOD2 expression level did not change among all treatment groups after
repeated SI (Fig. 3D).

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Social isolation and repeated social isolation affect Bcl-1 and p62 protein expression, while
DHM normalizes them

Figure 4. 4 Modulation of Bcl-1 and SQSTM1/p62 after SI, repeated SI, and DHM treatment.
(A, B) Representative blots of Bcl-1 and SQSTM1/p62 after 4 weeks of SI and repeated SI and
2 weeks of DHM treatment, 2mg/kg. (C, D) Quantified relative values of protein expression of
Bcl-1 and SQSTM1/p62 after SI, repeated SI, and 2 weeks of DHM 2mg/kg. β-actin was used as
a loading control. Data are presented as the mean ± SEM (n=4-7). Two-way ANOVA followed
by multiple comparisons, Holm-Sidak’s method *p<0.05 vs. G+V, #p<0.05 vs. Iso+V or R-
Iso+V.

Beclin-1 (Bcl-1) and sequestosome 1 (SQSTM1/p62) are involved in the autophagy
mitochondrial-dependent apoptosis pathway that is activated in response to ROS and oxidative
insults (Shefa et al., 2019). Therefore, we assessed the protein expression of Bcl-1 and p62 after

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SI and repeated SI. The level of Bcl-1 (1.072 ± 0.121) after SI did not show a reduction, yet p62
(0.704 ± 0.083) demonstrates a reduction compared to G+V (0.978 ± 0.083). Furthermore, the
protein expression of Bcl-1 (0.493 ± 0.135) and p62 (0.65 ± 0.083) in R-Iso+V was significantly
decreased compared to the group-housed (G+V). Administration of DHM after repeated SI (R-
Iso+D) restored the level of Bcl-1 (0.901 ± 0.156) and p62 (0.955 ± 0.083) (Fig. 4C, D).
Collectively, these results indicated that autophagy functionality decreased progressively after
multiple SI episodes. Two weeks of DHM treatment counteracted these changes.

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Social isolation and repeated social isolation modulate BDNF-TrkB signaling pathway,
while DHM improves them

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Figure 4. 5 Reduction of BNDF, p-TrkB, p-Erk p42, and p-Erk p44 protein levels after SI and
repeated SI; DHM improves them.
(A, B) Representative blots of BDNF, p-TrkB, t-Erk p42, p-Erk p42, t-Erk p44, and p-Erk p44.
(C) Quantitative measurement of BDNF protein levels after SI and 2mg/kg DHM treatment in
PFC homogenates using ELISA. (D, E, F, G, H, and I) Quantified the relative values of protein
expression of BDNF after repeated SI along with p-TrkB, t-Erk p42, p-Erk p42, t-Erk p44, and p-
Erk p44 values after SI, repeated SI, and 2 weeks of DHM 2mg/kg treatment. β-actin was used as
a loading control. Data are presented as the mean ± SEM (n=4-7). Two-way ANOVA followed
by multiple comparisons, Holm-Sidak’s method *p<0.05 vs. G+V, #p<0.05 vs. Iso+V or R-
Iso+V.

BDNF plays a crucial role in neuroprotection via activation of tropomyosin-related kinase B
(TrkB) receptor (Ji et al., 2016). Once BDNF binds to its receptor, TrkB, several transcriptional
cascades are activated, including the extracellular signal-regulated MAP kinase (Erk1/2)
pathway (Mao et al., 2015). To explore the effect of SI on neuronal survival, the protein levels of
BDNF, TrkB, and Erk1/2 in PFC homogenates were measured using ELISA and Western blot.
The results revealed that SI and repeated SI induced a significant reduction in the BDNF levels
in Iso+V (4.844 ± 0.112 pg/mg) relative to G+V (5.711 ± 0.105 pg/mg) and R-Iso+V (0.657 ±
0.114) vs. its control (set as 1) (Fig. 5C, D). DHM treatment reversed this effect in Iso+D (5.743
± 0.119 pg/mg) and R-Iso+D (1.058 ± 0.242). Additionally, the level of phosphorylated TrkB (p-
TrkB) protein expression reduced after SI (0.777 ± 0.089) and repeated SI (0.607 ± 0.146). DHM
treatment restored the level of p-TrkB in Iso+D (1.128 ± 0.158) and R-Iso+D (1.028 ± 0.127)
(Fig. 5E). SI induced a reduction in phosphorylated Erk p42 (p-Erk p42) (0.663 ± 0.109) and
phosphorylated Erk p44 (p-Erk p44) (0.452 ± 0.146) in Iso+ V, while DHM treatment (Iso+D)
improved p-Erk p42 (1.133 ± 0.102) and p-Erk p44 (1.310 ± 0.146) protein expression (Fig. 5G
and I). The protein expression of p-Erk p42 (0.831 ± 0.116) and p-Erk p44 (0.761 ± 0.164) after
repeated SI (R-Iso+V) were slightly decreased (Fig. 5G and I). Together, the results support the

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hypothesis that SI negatively impacts neurogenesis and neuronal survival balance while DHM
improves them.

Discussion:

Social isolation is considered a common psychological stressor, inducing pathological changes in
the neuroendocrine system, immune system, neurotrophic factors, and mitochondrial functions
(Calcia et al., 2016; Chan et al., 2017; Mumtaz et al., 2018; Murínová et al., 2017; Möller et al.,
2013). In the present study, we demonstrated that short-term SI disrupted mitochondrial activity
through impairment in C-I, C-II, and C-IV in ETC. Additionally, SI increased ROS and several
mitochondrial antioxidative enzymes, including SOD2, HO-1, PRDX-3, and GPX4. Moreover,
disbalance in the autophagy processes and the BDNF-TrkB pathway were also attributed to SI.
We also found that repeated SI induced equivalent effects on ROS production, antioxidative
enzymes, autophagy, and the BDNF-TrkB pathway.

ROS is the downstream product of mitochondrial energy production and plays an essential role
in cellular homeostasis under normal physiological conditions (Diebold & Chandel, 2016;
Schieber & Chandel, 2014). Under stressful conditions, however, ETC subunits undergo
disruption resulting in ROS accumulation that leads to oxidative stress damage (Andreazza et al.,
2010; Xing et al., 2013). In neurons, in particular, oxidative stress causes damage to proteins,
membrane lipids, and DNA that negatively impacts neuronal homeostasis and synaptic viability
(Herbet et al., 2017). Furthermore, ROS accumulation contributes to the prolonged activation of
microglia and astrocytes, causing persistent neuroinflammation, further ROS production, and
additional oxidative damage (S. M. Wang et al., 2018; Wang et al., 2004). Eventually, this series

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of events drive alterations to synaptic plasticity and neurotransmission activities, particularly the
GABAergic system, which eventually result in multiple behavioral deficits and anxiety
disorders. Considering this causative relation between neuroinflammation and mitochondrial
viability, we assessed the level of ROS production after SI and repeated SI. Our results indicated
a significant increase in H2O2 levels in SI and repeated SI (Fig. 2A). Several studies strongly
suggested that the level of ROS production is an indicator of mitochondrial impairment in the SI
anxiety model (Shao et al., 2015; Todorović et al., 2016).

Mitochondria play a predominant role in ATP production, intercellular calcium signaling, and
ROS production and elimination balance. These functions are essential in sustaining and
executing the complex functions of neurotransmission and synaptic plasticity (Billups &
Forsythe, 2002; Chang et al., 2006; Obashi & Okabe, 2013). Mitochondrial protein complexes
that constitute the ETC are vital to mitochondrial energy production through oxidative
phosphorylation (OXPHOS). The viability of the complexes in the ETC is a major indicator of
mitochondrial functionality (Indo et al., 2007). We evaluated the protein expression of
mitochondria complexes after SI and found a reduction in C-I, C-II, and C-IV protein expression
(Fig. 2C, D, F). This finding is consistent with other studies that showed different anxiety animal
models exhibit an impairment in C-I, C-II, and C-IV functions (Gebara et al., 2021; Hollis et al.,
2015; Perić et al., 2018). ETC subunit disruptions contribute to the pathology of neuropsychiatric
and neurodegenerative disorders such as anxiety, depression, PTSD, and dementia (Rammal et
al., 2008; Sarandol et al., 2007; Tezcan et al., 2003).

104
Despite ROS formation, mitochondria also produce several antioxidative enzymes to eliminate
the harmful ROS, such as SOD2, HO-1, PRDX-3, and GPX4 (Filipović et al., 2017; Hollis et al.,
2015). The levels of antioxidative enzymes are a distinct indicator of mitochondrial viability.
Our results indicated increases in total SOD activity and SOD2 protein expression following SI
(Fig. 3C, D). This increase was normalized by DHM treatment to a level relevant to the control.
The SOD2 activity and protein expression in SI was closer to DHM treated group-housed, which
potentially suggests a stress compensation mechanism. Our present data are consistent with
several studies of SI-induced anxiety that showed an increase in SOD activity after SI (Krolow et
al., 2012).

Moreover, considering the antioxidative effect of other mitochondrial enzymes, we studied the
effect of SI on HO-1 activity. HO-1 is an enzyme responsible for catalyzing heme to biliverdin,
iron, and carbon monoxide (CO) (Ryter & Tyrrell, 2000). Biliverdin is eventually converted to
bilirubin, which exhibits antioxidant properties (Ryter & Tyrrell, 2000). HO-1 upregulation can
be neuroprotective or neurotoxic depending on the redox microenvironment condition. A higher
level of CO and iron could exacerbate oxidative stress or initiate an antioxidative role
(Piantadosi, 2008). During stress, HO-1 is upregulated, especially in microglia and astrocytes, as
a protection mechanism against oxidative stress (Nakaso et al., 2000). Our results demonstrated
that SI and repeated SI enhanced the activity of HO-1, while DHM treatment reversed this effect
(Fig. 3E). We suggest that the increase in antioxidant enzymes might be explained as a
compensatory protective mechanism to counteract the effects of SI-induced stress, which DHM
potentially normalizes.

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PRDX-3 is another mitochondrial-specific antioxidant enzyme that is highly reactive and
specific to the H2O2 level, therefore defining the mitochondrial ETC functionality (Cunniff et al.,
2014). In SI and repeated SI, the level of PRDX-3 was higher than the control and relatively
close to the DHM positive control (Fig. 3F). PRDX-3 plays a critical role in excessive H2O2
elimination. Interestingly enough, neurons have the ability to overexpress PRDX-3 during
cellular oxidative stress injury (Bettegazzi et al., 2019; W. Hu et al., 2018). Hence, it is
speculated that PRDX-3 elevation is a protection mechanism counteracting the impact of SI. The
increase in PRDX-3 could work as a compensatory protective mechanism to enhance
neuroprotection pathways, as reported in several studies (Bettegazzi et al., 2019; Ogłodek & Just,
2018; Wen et al., 2014). The level of antioxidant enzymes in SI mice treated with DHM is
relatively closer to the control. This suggests that DHM treatment reduces stress via attenuating
the ROS effect and maintaining mitochondrial function.

Another antioxidative enzyme that is associated with stress-related disorders is GPX4. GPX4 is a
primary antioxidant enzyme with a unique enzymatic property in protecting against H2O2 in
lipoproteins; therefore, it preserves the integrity of the mitochondrial membrane during stress
(Jelinek et al., 2018; Wigner et al., 2020). In the current study, SI and repeated SI increased
GPX4 expression in the PFC (Fig. 3G). The elevated level of GPX4 was normalized by DHM
treatment. This could be due to the fact that overexpression of GPX4 could potentially play a
protective role in maintaining ATP production and preventing the loss of mitochondrial
membrane potential during oxidative stress (Liang et al., 2007). Studies showed that
overproduction of GPX4 after brain injury provides neuronal protection against oxidative
damage (Liang et al., 2007; Ran et al., 2006; Savaskan et al., 2007).

106

Autophagy is a vital process of selective degradation of cellular components. In the central
nervous system (CNS), autophagy sustains neuronal integrity and maintains neuroplasticity via
regulating the clustering of synaptic vesicles and facilitating synaptic pruning (Nikoletopoulou et
al., 2017). Therefore, autophagy dysregulation has been proposed to be associated with various
neuropsychological disorders (Pierone et al., 2020). At the cellular level, proper mitophagy, a
process essential for mitochondria quality control in the CNS, is essential to prevent ROS
accumulation by eliminating the cytosolic mitochondrial DNA (Nakahira et al., 2011).
Downregulation of autophagy-related proteins such as Bcl-1 and SQSTM1/p62 has been
associated with impairment of autophagy/mitophagy and, ultimately, development of behavioral
deficits (Geng et al., 2019). In the current study, social isolation for four weeks did not show a
significant difference in Bcl-1, but a reduction in p62 was observed. However, repeated SI
induced downregulation of both Bcl-1 and p62 (Fig. 4). DHM treatment alleviates the reduction
of Bcl-1 and p62 protein expression and restores autophagy. Taken together, disruption in Bcl-1
and p62 contributes to insufficient mitophagy, resulting in increased oxidative stress and,
ultimately, behavioral deficits. This finding is consistent with multiple studies that showed a
reduction in Bcl-1 and p62 in stress-related models (Einat et al., 2005; Geng et al., 2019; M.
Wang et al., 2019; Yang et al., 2017; Zheng et al., 2017).

BDNF is the most abundant neurotrophin in the brain, and it is essential in neurogenesis and
synaptic regulation (Jakawich et al., 2010). The BDNF-TrkB pathway and the downstream
transcriptional factor Erk1/2 are highly correlated with neurogenesis and synaptic plasticity
(Silva-Peña et al., 2019). Notably, neuronal atrophy associated with BDNF downregulation in

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various stress-related brain regions, particularly the hippocampus and PFC, was strongly
associated with anxiety (Duman & Monteggia, 2006). Several preclinical and clinical studies
have demonstrated a causal relationship between BNDF dysfunction and anxiety, depression,
and cognitive behaviors (Ieraci et al., 2016; Suliman et al., 2013). Anxiety and stress-related
studies demonstrated a reduction in BNDF, p-TrkB, and p-Erk1/2 that ultimately led to
neurogenesis impairment (Chiba et al., 2012; Evans et al., 2012; Tong et al., 2021). The present
study demonstrated a reduction in the PFC BDNF-TrkB pathway and p-Erk1/2 expression
following SI and repeated SI (Fig. 5). This reduction was improved by DHM administration.
Therefore, the increase in BDNF expression following DHM treatment might indicate that DHM
could work as a potential stimulator for neurogenesis. Consistent with our findings, several
studies revealed an enhancement in BDNF levels after DHM treatment in different disease
models (Ge et al., 2019; Ren et al., 2018). The data suggest that DHM potentially has a
therapeutic effect by maintaining and restoring neurogenesis and synapse function in the PFC.

Overall, the current results suggest that DHM produces its anxiolytic effects by modulating the
function of mitochondria, reducing oxidative stress, maintaining antioxidant levels, normalizing
autophagy, and enhancing BDNF. Additionally, since DHM counteracts the impact of repeated
social isolation stress, our findings suggest that DHM can provide long-term protection against
stress-induced CNS impairment and behavioral consequences. One limitation of the current
study is that the number of mice in repeated SI groups was low. Therefore, despite the trend of
reduction in antioxidative enzymes and p-Erk1/2 that was observed, the power was not deemed
high enough to promote statistical significance. Further studies looking at other anxiety-related

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brain regions, such as the hippocampus and amygdala, are necessary to analyze the connections
between these regions and further verify the therapeutic effect of DHM.

Funding and Disclosure:
Carefree Biotechnology Foundation, and Saudi Arabian Cultural Mission (to A.J.A). The authors
have nothing to disclose.

Acknowledgments
We thank N. Kirmiz for manuscript editing. We also would like to thank D. L. Davies, C. Folk,
C. Xue, and L. Qi for their help during this project.

Author contributions
A.J.A designed and performed experiments, analyzed data, generated figures, and wrote the
manuscript.
S.W. discussed the design, performed experiments, and wrote the manuscript.
E.C.H. performed experiments, analyzed data, and wrote the manuscript.
S.G.S. performed experiments, analyzed data, and wrote the manuscript.
M.Z. performed experiments, analyzed data, and wrote the manuscript.
J.Z. performed experiments and analyzed data.
X. M. S. discussed the design of experiments, statistical analyses, and wrote the manuscript.
J.L. established animal model, designed experiments, statistical analyses, wrote the manuscript
and supervised the project.

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CHAPTER 5

CONCLUSIONS

Anxiety disorders are the most frequent among all mental diseases. Anxiety symptoms are
ubiquitous across all other psychiatric and neurodegenerative diseases. Anxiety disorders are
commonly considered as a precursor for cognitive decline and Alzheimer’s disease (AD).
Despite the high prevalence rate of anxiety disorders, only a small percentage of these
individuals have access to effective treatment. The increased undertreated population is
continuously imposing public health issue that escalated during COVID-19. The currently
available anti-anxiety medications are suboptimal in terms of safety, tolerability, and efficacy,
leaving a high percentage of anxiety patients not responding to treatment and with a high
potential to relapse. Therefore, my dissertation aimed to develop DHM as a novel anxiolytic to
mitigate anxiety symptoms and enhance cognition. Altogether, these studies support my
dissertation hypothesis that DHM alleviates anxiety behaviors by modulating
neuroinflammation, oxidative stress, autophagy, and neurotrophic factors following social
isolation-induced stress and anxiety behaviors. My studies demonstrated that DHM attenuates
neuroinflammation-mediated stress by inhibiting the activation of NFκB, microglia, and
astrocytes. I also identified that DHM protects mitochondrial redox signaling and maintains
antioxidative enzyme defense system, thereby reducing the impact of oxidative stress.
Furthermore, I demonstrated that DHM significantly ameliorates anxiety-related behaviors in
long-term studies (12 weeks) after sporadic intervals of stress.

110
To investigate the mechanism of DHM in reducing neuroinflammation after social isolation-
induced stress and anxiety model in C57J6/B mice, I investigated the key factors that potentially
initiate neuroinflammation, which are peripheral corticosterone (rodent version of cortisol) and
proinflammatory cytokines. I found that DHM treatment (2 mg/kg) normalizes serum
corticosterone levels to stress-free mice. Also, DHM decreases the expression of several
proinflammatory cytokines, which was also demonstrated by inhibiting NFκB signaling pathway
activation. Afterward, I examined the effects of social isolation on the CNS inflammation by
studying hippocampal microglia and astrocytes. I identified that DHM ameliorates the impact of
stress on microglia overactivation illustrated as modulation of morphological changes, including
decreases in lacunarity, perimeter, cell size, and increases in cell density. Additionally, I
demonstrated that DHM counteracts the impact of stress on astrocyte overactivation and restores
their density, size, and branch length. The primary effect of neuroinflammation appears in
microglia and astrocyte plasticity, which are known to support GABAergic synapses and
maintain their functions. Furthermore, I demonstrated that DHM treatment modulates the level of
gephyrin, which is consider an indicator of the GABAARs functionality. Therefore, DHM's role
in modulating neuroinflammation modifies GABAergic neurotransmitters, GABAARs clustering,
and functioning, thereby ameliorating behavioral deficits and reducing anxiety.

Beside neuroinflammation, neuronal plasticity along with the proper synapse distance and
structure, are vital in maintaining neurotransmission and thus essential for sharp cognition and
memory (Augusto-Oliveira et al., 2020). Thus, any disturbances in the synapse are considered a
major factor in cognitive and memory decline. Astrocytes and microglia, as mentioned above,
are essential in supporting the homeostasis of the overall neuronal synapse, particularly

111
GABAergic synapses (Matejuk & Ransohoff, 2020). Therefore, further investigations that focus
on restoring stress-induced abnormalities in astrocytes and microglia are critical to elucidating
our understanding of DHM’s role in improving cognitive function cognitive improvement. I
found that DHM counteracts the impact of stress on astrogliosis and microgliosis after social
isolation-induced stress and restores their density and vital morphology.

Due to chronic neuroinflammation contributes to producing a massive amount of ROS and the
subsequent detrimental oxidative stress-induced damage, I investigated the role of DHM in
overcoming the ROS-mediated effects. My study demonstrated that DHM reduces the level of
H2O2; this is not only due to DHM’s antioxidant characteristics but also its anti-
neuroinflammation properties. Moreover, anxiety disorders are correlated with alteration in
mitochondrial energy metabolism, as evidenced in both animal and human studies (Anglin et al.,
2012; Hollis et al., 2015). In the social-isolation model, a reduction of mitochondrial ATP level
was previously observed (J Silva et al., 2020). Since dysfunctional ETC produce less ATP, I
investigated ETC alterations after stress. I identified that daily DHM administration counteracts
the impact of stress-induced anxiety in ETC activity by measuring protein expression levels,
particularly C-I, C-II, and C-IV. Thus, by protecting the integrity of ETC after DHM treatment
enhances the mitochondrial ATP output and reduces oxidative stress. Given that neurons
consume the highest amount of energy in synaptic transmission (Harris et al., 2012), DHM’s
bioenergetic enhancement ultimately attenuates anxiety phenotypes and improves cognitive
abilities. I also identified that social isolation-induced stress is associated with dramatic increases
in several mitochondrial antioxidative enzymes, including SOD2, HO-1, PRDX3, and GPX4.
Increasing the levels of various antioxidant enzymes as mentioned above, as coping and

112
protective mechanisms during stress, is in agreement with several previous stress-related studies
(Bettegazzi et al., 2019; Krolow et al., 2012; Liang et al., 2007; Nakaso et al., 2000). I found that
DHM treatment modulates the level of antioxidative enzymes to a level comparable to the
control (stress-free mice), therefore, improves mitochondrial function and reduces ROS. Given
the fundamental function of mitochondria in neurotransmitters production, especially the
excitatory glutamate and inhibitory GABA, promotes neurotransmitters balance and
neuroprotection to mitigate anxiety and cognitive reduction.

Up- or down-regulation of autophagy is linked to psychological and neurodegenerative
disorders, including anxiety disorders, dementia, and AD. In fact, autophagy is crucial for
protecting the integrity of the synaptic structure and regulating neurotransmitter release; thus,
autophagy impairment leads to neurotoxicity and synaptic morphological defects. My findings
demonstrated that DHM enhances autophagy performance by reversing reduction in beclin 1 and
p62 protein expression, thereby improving the autophagic degradation and recycling of excess
neurotransmitters and damaged synaptic proteins. Collectively, this led to the modulation the
GABAergic neurotransmission, improving pre- and post-synaptic remodeling, regulating
synaptic vesicles, increasing surface GABAAR presentation, and enhancing neuronal plasticity,
thus, attenuating anxiety and promoting cognitive/memory abilities. Furthermore, BDNF has a
crucial role in neurogenesis and synaptic regulation, BDNF downregulation is strongly
associated with psychiatric and neurodegenerative diseases and linked to the pathological
process of excitotoxicity, oxidative stress, and neuroinflammation. I identified that DHM
enhances BNDF-TrkB signaling activity and the downstream MAPK/ERK cascade. Thereby

113
promoting neuronal protection and synapse recovery after social isolation-induced stress that
eventually modulates behavioral deficits and improves cognition.

In an effort to study the influence of DHM treatment on anxiety behaviors, our model was
designed to mimic circumstances that relate to human life events, such as intermittent stress.
This was achieved by inducing stress on socially isolated mice in sporadic intervals. I found that
repeated social isolation can sensitize the mice to stress-induced damage, notably by
investigating pathways involved in oxidative stress, autophagy, and BDNF activity and
expression. This body of work demonstrates that long-term DHM treatment can modulate the
impact imposed by chronic stress. Collectively, this set of experiments are essential as “a proof
of concept” for the potential use of DHM for long-term neuroprotection that reduces stress and
anxiety while safeguarding memory and cognition.

In summary, I identified that neuroinflammation, oxidative stress, autophagy impairment, and
BNDF dysfunction, tightly contribute to the pathophysiological responses to social isolation
induced stress/anxiety. The anxiety symptoms could be predictive for the progression of
dementia and AD (Becker et al., 2018). Despite this severe upward trend of both AD and
anxiety, the mechanistic linkage between the two has not yet been carefully investigated. To
date, there are no cures for AD and the recently approved AD medication—aducanumab—is
questionable in terms of therapeutic efficacy. In addition, current treatments that aim to reduce
anxiety symptoms—which include psychotherapy, medications, or a combination of both—are
not always effective. This work clarified that social isolation-induced anxiety could cause
behavioral deficits and decreases cognition and memory in consequence of neuroinflammation,

114
oxidative stress, autophagy impairment, and BNDF dysfunction. These pathological pathways
are also implicated in AD pathogenesis. This work provided clear evidence for the linkage
between anxiety and AD through neuroinflammation and the subsequent pathologies.
Additionally, DHM reverses these pathological events, attenuates anxiety, and enhances
cognition and memory. The ultimate goal of my dissertation is to set the stage for DHM to be
used clinically as a novel therapy for anxiety disorders and as early intervention against
cognitive/memory impairment to intervene the early onset of AD.

115

BIBLIOGRAPHY

Agarwal, S. D., & Landon, B. E. (2019). Patterns in Outpatient Benzodiazepine Prescribing in
the United States. JAMA Netw Open, 2(1), e187399.
https://doi.org/10.1001/jamanetworkopen.2018.7399
Akimova, E., Lanzenberger, R., & Kasper, S. (2009). The serotonin-1A receptor in anxiety
disorders. Biol Psychiatry, 66(7), 627-635.
https://doi.org/10.1016/j.biopsych.2009.03.012
Al Omran, A. J., Shao, A. S., Watanabe, S., Zhang, Z., Zhang, J., Xue, C., . . . Liang, J. (2022).
Social isolation induces neuroinflammation and microglia overactivation, while
dihydromyricetin prevents and improves them. Journal of Neuroinflammation, 19(1), 2.
https://doi.org/10.1186/s12974-021-02368-9
Al Omran, A. J., Shao, A. S., Watanabe, S., Zhang, Z., Zhang, J., Xue, C., . . . Liang, J. (2022).
Social isolation induces neuroinflammation and microglia overactivation, while
dihydromyricetin prevents and improves them. J Neuroinflammation, 19(1), 2.
https://doi.org/10.1186/s12974-021-02368-9
Alvarez-Castelao, B., & Schuman, E. M. (2015). The Regulation of Synaptic Protein Turnover. J
Biol Chem, 290(48), 28623-28630. https://doi.org/10.1074/jbc.R115.657130
Andreazza, A. C., Andersen, M. L., Alvarenga, T. A., de-Oliveira, M. R., Armani, F., Ruiz, F.
S., . . . Tufik, S. (2010). Impairment of the mitochondrial electron transport chain due to
sleep deprivation in mice. J Psychiatr Res, 44(12), 775-780.
https://doi.org/10.1016/j.jpsychires.2010.01.015

116
Andrew H. Rogers, S. T. W. s. a.-h. t. c. u.-i. f. f. h. r. i. w. u. x. h. (2019). Anxiety
Comorbidities: Mood Disorders, Substance Use Disorders, and Chronic Medical Illness.
In Current Clinical Psychiatry book series.
Andreyev, A. Y., Kushnareva, Y. E., & Starkov, A. A. (2005). Mitochondrial metabolism of
reactive oxygen species. Biochemistry (Mosc), 70(2), 200-214.
https://doi.org/10.1007/s10541-005-0102-7
Anglin, R. E., Garside, S. L., Tarnopolsky, M. A., Mazurek, M. F., & Rosebush, P. I. (2012).
The psychiatric manifestations of mitochondrial disorders: a case and review of the
literature. J Clin Psychiatry, 73(4), 506-512. https://doi.org/10.4088/JCP.11r07237
Ariosa, A. R., & Klionsky, D. J. (2016). Autophagy core machinery: overcoming spatial barriers
in neurons. J Mol Med (Berl), 94(11), 1217-1227. https://doi.org/10.1007/s00109-016-
1461-9
Assmann, N., Schramm, E., Kriston, L., Hautzinger, M., Härter, M., Schweiger, U., & Klein, J.
P. (2018). Moderating effect of comorbid anxiety disorders on treatment outcome in a
randomized controlled psychotherapy trial in early-onset persistently depressed
outpatients. Depress Anxiety, 35(10), 1001-1008. https://doi.org/10.1002/da.22839
Augusto-Oliveira, M., Arrifano, G., Takeda, P., Lopes-Araújo, A., Santos-Sacramento, L.,
Anthony, D., . . . Crespo-Lopez, M. (2020). Astroglia-specific contributions to the
regulation of synapses, cognition and behaviour. Neuroscience & Biobehavioral Reviews
118. DOI:10.1016/j.neubiorev.2020.07.039.
Baandrup, L., Ebdrup, B. H., Rasmussen, J., Lindschou, J., Gluud, C., & Glenthøj, B. Y. (2018).
Pharmacological interventions for benzodiazepine discontinuation in chronic

117
benzodiazepine users. Cochrane Database Syst Rev, 3, CD011481.
https://doi.org/10.1002/14651858.CD011481.pub2
Bachmanov, A. A., Reed, D. R., Beauchamp, G. K., & Tordoff, M. G. (2002). Food intake, water
intake, and drinking spout side preference of 28 mouse strains. Behav Genet, 32(6), 435-
443. https://doi.org/10.1023/a:1020884312053
Balderas, I., Rodriguez-Ortiz, C. J., Salgado-Tonda, P., Chavez-Hurtado, J., McGaugh, J. L., &
Bermudez-Rattoni, F. (2008). The consolidation of object and context recognition
memory involve different regions of the temporal lobe. Learn Mem, 15(9), 618-624.
https://doi.org/10.1101/lm.1028008
Banagozar Mohammadi, A., Torbati, M., Farajdokht, F., Sadigh-Eteghad, S., Fazljou, S. M. B.,
Vatandoust, S. M., . . . Mahmoudi, J. (2019). Sericin alleviates restraint stress induced
depressive- and anxiety-like behaviors via modulation of oxidative stress,
neuroinflammation and apoptosis in the prefrontal cortex and hippocampus. Brain Res,
1715, 47-56. https://doi.org/10.1016/j.brainres.2019.03.020
Bandelow, B., & Michaelis, S. (2015). Epidemiology of anxiety disorders in the 21st century.
Dialogues Clin Neurosci, 17(3), 327-335.
Bandelow, B., Michaelis, S., & Wedekind, D. (2017). Treatment of anxiety disorders. Dialogues
Clin Neurosci, 19(2), 93-107.
Barker, G. R. I., & Warburton, E. C. (2011). When Is the Hippocampus Involved in Recognition
Memory? The Journal of Neuroscience, 31(29), 10721.
https://doi.org/10.1523/JNEUROSCI.6413-10.2011

118
Bartsch, T., & Wulff, P. (2015). The hippocampus in aging and disease: From plasticity to
vulnerability. Neuroscience, 309, 1-16.
https://doi.org/10.1016/j.neuroscience.2015.07.084
Baumann, S. W., Baur, R., & Sigel, E. (2002). Forced subunit assembly in alpha1beta2gamma2
GABAA receptors. Insight into the absolute arrangement. J Biol Chem, 277(48), 46020-
46025. https://doi.org/10.1074/jbc.M207663200
Baur, R., Minier, F., & Sigel, E. (2006). A GABA(A) receptor of defined subunit composition
and positioning: concatenation of five subunits. FEBS Lett, 580(6), 1616-1620.
https://doi.org/10.1016/j.febslet.2006.02.002
Baxter, A. J., Vos, T., Scott, K. M., Norman, R. E., Flaxman, A. D., Blore, J., & Whiteford, H.
A. (2014). The regional distribution of anxiety disorders: implications for the Global
Burden of Disease Study, 2010. Int J Methods Psychiatr Res, 23(4), 422-438.
https://doi.org/10.1002/mpr.1444
Becker, E., Orellana Rios, C. L., Lahmann, C., Rücker, G., Bauer, J., & Boeker, M. (2018).
Anxiety as a risk factor of Alzheimer's disease and vascular dementia. Br J Psychiatry,
213(5), 654-660. https://doi.org/10.1192/bjp.2018.173
Belzer, K., & Schneier, F. R. (2004). Comorbidity of anxiety and depressive disorders: issues in
conceptualization, assessment, and treatment. J Psychiatr Pract, 10(5), 296-306.
https://doi.org/10.1097/00131746-200409000-00003
Bettegazzi, B., Pelizzoni, I., Salerno Scarzella, F., Restelli, L. M., Zacchetti, D., Maltecca, F., . . .
Codazzi, F. (2019). Upregulation of Peroxiredoxin 3 Protects. Oxid Med Cell Longev,
2019, 4721950. https://doi.org/10.1155/2019/4721950

119
Beutel, M. E., Klein, E. M., Brähler, E., Reiner, I., Jünger, C., Michal, M., . . . Tibubos, A. N.
(2017). Loneliness in the general population: prevalence, determinants and relations to
mental health. BMC Psychiatry, 17(1), 97. https://doi.org/10.1186/s12888-017-1262-x
Bhat, R., Axtell, R., Mitra, A., Miranda, M., Lock, C., Tsien, R. W., & Steinman, L. (2010).
Inhibitory role for GABA in autoimmune inflammation. Proc Natl Acad Sci U S A,
107(6), 2580-2585. https://doi.org/10.1073/pnas.0915139107
Billups, B., & Forsythe, I. D. (2002). Presynaptic mitochondrial calcium sequestration influences
transmission at mammalian central synapses. J Neurosci, 22(14), 5840-5847.
https://doi.org/20026597
Blackburn, D., Sargsyan, S., Monk, P. N., & Shaw, P. J. (2009). Astrocyte function and role in
motor neuron disease: a future therapeutic target? Glia, 57(12), 1251-1264.
https://doi.org/10.1002/glia.20848
Bluthé, R. M., Walter, V., Parnet, P., Layé, S., Lestage, J., Verrier, D., . . . Dantzer, R. (1994).
Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism. C
R Acad Sci III, 317(6), 499-503.
Bramham, C. R., & Messaoudi, E. (2005). BDNF function in adult synaptic plasticity: the
synaptic consolidation hypothesis. Prog Neurobiol, 76(2), 99-125.
https://doi.org/10.1016/j.pneurobio.2005.06.003
Brigelius-Flohé, R., & Maiorino, M. (2013). Glutathione peroxidases. Biochim Biophys Acta,
1830(5), 3289-3303. https://doi.org/10.1016/j.bbagen.2012.11.020
Bruce, S. E., Yonkers, K. A., Otto, M. W., Eisen, J. L., Weisberg, R. B., Pagano, M., . . . Keller,
M. B. (2005). Influence of psychiatric comorbidity on recovery and recurrence in

120
generalized anxiety disorder, social phobia, and panic disorder: a 12-year prospective
study. Am J Psychiatry, 162(6), 1179-1187. https://doi.org/10.1176/appi.ajp.162.6.1179
Bulfin, L. J., Clarke, M. A., Buller, K. M., & Spencer, S. J. (2011). Anxiety and hypothalamic-
pituitary-adrenal axis responses to psychological stress are attenuated in male rats made
lean by large litter rearing. Psychoneuroendocrinology, 36(7), 1080-1091.
https://doi.org/10.1016/j.psyneuen.2011.01.006
Burgos-Robles, A., Vidal-Gonzalez, I., & Quirk, G. J. (2009). Sustained conditioned responses
in prelimbic prefrontal neurons are correlated with fear expression and extinction failure.
J Neurosci, 29(26), 8474-8482. https://doi.org/10.1523/JNEUROSCI.0378-09.2009
Buss, E. W., Corbett, N. J., Roberts, J. G., Ybarra, N., Musial, T. F., Simkin, D., . . . Nicholson,
D. A. (2021). Cognitive aging is associated with redistribution of synaptic weights in the
hippocampus. Proceedings of the National Academy of Sciences, 118(8), e1921481118.
https://doi.org/10.1073/pnas.1921481118
Calcia, M. A., Bonsall, D. R., Bloomfield, P. S., Selvaraj, S., Barichello, T., & Howes, O. D.
(2016). Stress and neuroinflammation: a systematic review of the effects of stress on
microglia and the implications for mental illness. Psychopharmacology (Berl), 233(9),
1637-1650. https://doi.org/10.1007/s00213-016-4218-9
CDC. (2020). Mental health: Household Pulse Survey. In.
Chan, J. N., Lee, J. C., Lee, S. S., Hui, K. K., Chan, A. H., Fung, T. K., . . . Ngai, S. P. (2017).
Interaction Effect of Social Isolation and High Dose Corticosteroid on Neurogenesis and
Emotional Behavior. Front Behav Neurosci, 11, 18.
https://doi.org/10.3389/fnbeh.2017.00018

121
Chanana, P., & Kumar, A. (2016). GABA-BZD Receptor Modulating Mechanism of Panax
quinquefolius against 72-h Sleep Deprivation Induced Anxiety like Behavior: Possible
Roles of Oxidative Stress, Mitochondrial Dysfunction and Neuroinflammation. Front
Neurosci, 10, 84. https://doi.org/10.3389/fnins.2016.00084
Chang, D. T., Honick, A. S., & Reynolds, I. J. (2006). Mitochondrial trafficking to synapses in
cultured primary cortical neurons. J Neurosci, 26(26), 7035-7045.
https://doi.org/10.1523/JNEUROSCI.1012-06.2006
Chang, T. S., Cho, C. S., Park, S., Yu, S., Kang, S. W., & Rhee, S. G. (2004). Peroxiredoxin III,
a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria. J
Biol Chem, 279(40), 41975-41984. https://doi.org/10.1074/jbc.M407707200
Chantarujikapong, S. I., Scherrer, J. F., Xian, H., Eisen, S. A., Lyons, M. J., Goldberg, J., . . .
True, W. R. (2001). A twin study of generalized anxiety disorder symptoms, panic
disorder symptoms and post-traumatic stress disorder in men. Psychiatry Res, 103(2-3),
133-145. https://doi.org/10.1016/s0165-1781(01)00285-2
Chen, C. T., Green, J. T., Orr, S. K., & Bazinet, R. P. (2008). Regulation of brain
polyunsaturated fatty acid uptake and turnover. Prostaglandins Leukot Essent Fatty
Acids, 79(3-5), 85-91. https://doi.org/10.1016/j.plefa.2008.09.003
Chen, F., Zhou, L., Bai, Y., Zhou, R., & Chen, L. (2015). Hypothalamic-pituitary-adrenal axis
hyperactivity accounts for anxiety- and depression-like behaviors in rats perinatally
exposed to bisphenol A. J Biomed Res, 29(3), 250-258.
https://doi.org/10.7555/JBR.29.20140058

122
Chen, K., Gunter, K., & Maines, M. D. (2000). Neurons overexpressing heme oxygenase-1 resist
oxidative stress-mediated cell death. J Neurochem, 75(1), 304-313.
https://doi.org/10.1046/j.1471-4159.2000.0750304.x
Chen, K., Holschneider, D. P., Wu, W., Rebrin, I., & Shih, J. C. (2004). A spontaneous point
mutation produces monoamine oxidase A/B knock-out mice with greatly elevated
monoamines and anxiety-like behavior. J Biol Chem, 279(38), 39645-39652.
https://doi.org/10.1074/jbc.M405550200
Chen, L., Na, R., Gu, M., Salmon, A. B., Liu, Y., Liang, H., . . . Ran, Q. (2008). Reduction of
mitochondrial H2O2 by overexpressing peroxiredoxin 3 improves glucose tolerance in
mice. Aging Cell, 7(6), 866-878. https://doi.org/10.1111/j.1474-9726.2008.00432.x
Chen, S., Jiang, H., Liu, Y., Hou, Z., Yue, Y., Zhang, Y., . . . Yuan, Y. (2017). Combined serum
levels of multiple proteins in tPA-BDNF pathway may aid the diagnosis of five mental
disorders. Sci Rep, 7(1), 6871. https://doi.org/10.1038/s41598-017-06832-6
Chiba, S., Numakawa, T., Ninomiya, M., Richards, M. C., Wakabayashi, C., & Kunugi, H.
(2012). Chronic restraint stress causes anxiety- and depression-like behaviors,
downregulates glucocorticoid receptor expression, and attenuates glutamate release
induced by brain-derived neurotrophic factor in the prefrontal cortex. Prog
Neuropsychopharmacol Biol Psychiatry, 39(1), 112-119.
https://doi.org/10.1016/j.pnpbp.2012.05.018
Choii, G., & Ko, J. (2015). Gephyrin: a central GABAergic synapse organizer. Exp Mol Med, 47,
e158. https://doi.org/10.1038/emm.2015.5

123
Christoffel, D. J., Golden, S. A., & Russo, S. J. (2011). Structural and synaptic plasticity in
stress-related disorders. Rev Neurosci, 22(5), 535-549.
https://doi.org/10.1515/RNS.2011.044
Christopherson, K. S., Ullian, E. M., Stokes, C. C., Mullowney, C. E., Hell, J. W., Agah, A., . . .
Barres, B. A. (2005). Thrombospondins are astrocyte-secreted proteins that promote CNS
synaptogenesis. Cell, 120(3), 421-433. https://doi.org/10.1016/j.cell.2004.12.020
Chrousos, G. P. (2000). The role of stress and the hypothalamic-pituitary-adrenal axis in the
pathogenesis of the metabolic syndrome: neuro-endocrine and target tissue-related
causes. Int J Obes Relat Metab Disord, 24 Suppl 2, S50-55.
https://doi.org/10.1038/sj.ijo.0801278
Chung, W., Wang, D.-S., Khodaei, S., Pinguelo, A., & Orser, B. A. (2022). GABAA Receptors
in Astrocytes Are Targets for Commonly Used Intravenous and Inhalational General
Anesthetic Drugs [Original Research]. Frontiers in Aging Neuroscience, 13.
Collaborators, C.-M. D. (2021). Global prevalence and burden of depressive and anxiety
disorders in 204 countries and territories in 2020 due to the COVID-19 pandemic.
Lancet, 398(10312), 1700-1712. https://doi.org/10.1016/S0140-6736(21)02143-7
Collaborators, G. D. a. H. (2018). Global, regional, and national disability-adjusted life-years
(DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195
countries and territories, 1990-2017: a systematic analysis for the Global Burden of
Disease Study 2017. Lancet, 392(10159), 1859-1922. https://doi.org/10.1016/S0140-
6736(18)32335-3
Coppen, A. (1967). The biochemistry of affective disorders. Br J Psychiatry, 113(504), 1237-
1264. https://doi.org/10.1192/bjp.113.504.1237

124
Costello, E. J., Egger, H. L., & Angold, A. (2005). The developmental epidemiology of anxiety
disorders: phenomenology, prevalence, and comorbidity. Child Adolesc Psychiatr Clin N
Am, 14(4), 631-648, vii. https://doi.org/10.1016/j.chc.2005.06.003
CPNP, T. C. o. P. a. N. P. (2022). Treatment Guidelines: Anxiety and Related Disorders
Craske, M. G., Stein, M. B., Eley, T. C., Milad, M. R., Holmes, A., Rapee, R. M., & Wittchen,
H.-U. (2017). Anxiety disorders. Nature Reviews Disease Primers, 3(1), 17024.
https://doi.org/10.1038/nrdp.2017.24
Crowley, T., Cryan, J. F., Downer, E. J., & O'Leary, O. F. (2016). Inhibiting neuroinflammation:
The role and therapeutic potential of GABA in neuro-immune interactions. Brain Behav
Immun, 54, 260-277. https://doi.org/10.1016/j.bbi.2016.02.001
Crowley, T., Cryan, J. F., Downer, E. J., & O’Leary, O. F. (2016). Inhibiting neuroinflammation:
The role and therapeutic potential of GABA in neuro-immune interactions. Brain,
Behavior, and Immunity, 54, 260-277.
https://doi.org/https://doi.org/10.1016/j.bbi.2016.02.001
Crowley, T., Fitzpatrick, J. M., Kuijper, T., Cryan, J. F., O'Toole, O., O'Leary, O. F., & Downer,
E. J. (2015). Modulation of TLR3/TLR4 inflammatory signaling by the GABAB receptor
agonist baclofen in glia and immune cells: relevance to therapeutic effects in multiple
sclerosis. Front Cell Neurosci, 9, 284. https://doi.org/10.3389/fncel.2015.00284
Cryan, J. F., & Sweeney, F. F. (2011). The age of anxiety: role of animal models of anxiolytic
action in drug discovery. Br J Pharmacol, 164(4), 1129-1161.
https://doi.org/10.1111/j.1476-5381.2011.01362.x

125
Csabai, D., Wiborg, O., & Czéh, B. (2018). Reduced Synapse and Axon Numbers in the
Prefrontal Cortex of Rats Subjected to a Chronic Stress Model for Depression. Front Cell
Neurosci, 12, 24. https://doi.org/10.3389/fncel.2018.00024
Cunniff, B., Wozniak, A. N., Sweeney, P., DeCosta, K., & Heintz, N. H. (2014). Peroxiredoxin 3
levels regulate a mitochondrial redox setpoint in malignant mesothelioma cells. Redox
Biol, 3, 79-87. https://doi.org/10.1016/j.redox.2014.11.003
Cutler, D. M., & Summers, L. H. (2020). The COVID-19 Pandemic and the $16 Trillion Virus.
JAMA, 324(15), 1495-1496. https://doi.org/10.1001/jama.2020.19759
Daffre, C. (2020). Neurocircuitry of Anxiety Disorders. In E. F. P.-S. Katelyn I. Oliver (Ed.).
Current Clinical Psychiatry.: Humana, Cham.
Dallerac, G., & Rouach, N. (2016). Astrocytes as New Targets to Improve Cognitive Functions.
Progress in Neurobiology 144, DOI:10.1016/j.pneurobio.2016.01.003.
Davalos, D., Grutzendler, J., Yang, G., Kim, J. V., Zuo, Y., Jung, S., . . . Gan, W. B. (2005).
ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci, 8(6),
752-758. https://doi.org/10.1038/nn1472
Davies, A. M. (1994). The role of neurotrophins in the developing nervous system. J Neurobiol,
25(11), 1334-1348. https://doi.org/10.1002/neu.480251103
De Luca, L. A., Menani, J. V., & Johnson, A. K. (2014). Neurobiology of Body Fluid
Homeostasis: Transduction and Integration. In. https://doi.org/NBK200964
de Oliveira, G. C. (2021). Dementia or psychosis precipitated by social isolation? A brief case
report in COVID-19 pandemic times. Alzheimers Dement (Amst), 13(1), e12166.
https://doi.org/10.1002/dad2.12166

126
DeKosky, S. T., & Scheff, S. W. (1990). Synapse loss in frontal cortex biopsies in Alzheimer's
disease: Correlation with cognitive severity. Annals of Neurology, 27(5), 457-464.
https://doi.org/https://doi.org/10.1002/ana.410270502
Del Arco, A., & Mora, F. (2009). Neurotransmitters and prefrontal cortex-limbic system
interactions: implications for plasticity and psychiatric disorders. J Neural Transm
(Vienna), 116(8), 941-952. https://doi.org/10.1007/s00702-009-0243-8
DePierro, J., Lowe, S., & Katz, C. (2020). Lessons learned from 9/11: Mental health perspectives
on the COVID-19 pandemic. Psychiatry Res, 288, 113024.
https://doi.org/10.1016/j.psychres.2020.113024
Devane, C. L., Chiao, E., Franklin, M., & Kruep, E. J. (2005). Anxiety disorders in the 21st
century: status, challenges, opportunities, and comorbidity with depression. Am J Manag
Care, 11(12 Suppl), S344-353.
Diebold, L., & Chandel, N. S. (2016). Mitochondrial ROS regulation of proliferating cells. Free
Radic Biol Med, 100, 86-93. https://doi.org/10.1016/j.freeradbiomed.2016.04.198
DiSabato, D. J., Quan, N., & Godbout, J. P. (2016). Neuroinflammation: the devil is in the
details. J Neurochem, 139 Suppl 2, 136-153. https://doi.org/10.1111/jnc.13607
Dityatev, A. E., & Bolshakov, V. Y. (2005). Amygdala, long-term potentiation, and fear
conditioning. Neuroscientist, 11(1), 75-88. https://doi.org/10.1177/1073858404270857
Dossi, E., Vasile, F., & Rouach, N. (2018). Human astrocytes in the diseased brain. Brain Res
Bull, 136, 139-156. https://doi.org/10.1016/j.brainresbull.2017.02.001
DSM-5. (2013). Diagnostic And Statistical Manual Of Mental Disorders, Fifth
EditionInternational Statistical Classification of Diseases and Related Health Problemsof

127
Diseases and Related Health Problems.
https://dsm.psychiatryonline.org/doi/book/10.1176/appi.books.9780890425596
Du Preez, A., Law, T., Onorato, D., Lim, Y. M., Eiben, P., Musaelyan, K., . . . Fernandes, C.
(2020). The type of stress matters: repeated injection and permanent social isolation
stress in male mice have a differential effect on anxiety- and depressive-like behaviours,
and associated biological alterations. Transl Psychiatry, 10(1), 325.
https://doi.org/10.1038/s41398-020-01000-3
Dubois, B., Hampel, H., Feldman, H. H., Scheltens, P., Aisen, P., Andrieu, S., . . . Washington
Dc, U. S. A. (2016). Preclinical Alzheimer's disease: Definition, natural history, and
diagnostic criteria [https://doi.org/10.1016/j.jalz.2016.02.002]. Alzheimer's & Dementia,
12(3), 292-323. https://doi.org/https://doi.org/10.1016/j.jalz.2016.02.002
Duman, R. S., & Monteggia, L. M. (2006). A neurotrophic model for stress-related mood
disorders. Biol Psychiatry, 59(12), 1116-1127.
https://doi.org/10.1016/j.biopsych.2006.02.013
Duval, E. R., Javanbakht, A., & Liberzon, I. (2015). Neural circuits in anxiety and stress
disorders: a focused review. Ther Clin Risk Manag, 11, 115-126.
https://doi.org/10.2147/TCRM.S48528
Einat, H., Yuan, P., & Manji, H. K. (2005). Increased anxiety-like behaviors and mitochondrial
dysfunction in mice with targeted mutation of the Bcl-2 gene: further support for the
involvement of mitochondrial function in anxiety disorders. Behav Brain Res, 165(2),
172-180. https://doi.org/10.1016/j.bbr.2005.06.012

128
Ericsson, A., Kovács, K. J., & Sawchenko, P. E. (1994). A functional anatomical analysis of
central pathways subserving the effects of interleukin-1 on stress-related neuroendocrine
neurons. J Neurosci, 14(2), 897-913.
Evans, I. E. M., Martyr, A., Collins, R., Brayne, C., & Clare, L. (2019). Social Isolation and
Cognitive Function in Later Life: A Systematic Review and Meta-Analysis. J Alzheimers
Dis, 70(s1), S119-S144. https://doi.org/10.3233/JAD-180501
Evans, J., Sun, Y., McGregor, A., & Connor, B. (2012). Allopregnanolone regulates
neurogenesis and depressive/anxiety-like behaviour in a social isolation rodent model of
chronic stress. Neuropharmacology, 63(8), 1315-1326.
https://doi.org/10.1016/j.neuropharm.2012.08.012
Facts and Statistics. (2016).
Fedoce, A. D. G., Ferreira, F., Bota, R. G., Bonet-Costa, V., Sun, P. Y., & Davies, K. J. A.
(2018). The role of oxidative stress in anxiety disorder: cause or consequence? Free
Radic Res, 52(7), 737-750. https://doi.org/10.1080/10715762.2018.1475733
Fergusson, D. M., Horwood, L. J., & Lynskey, M. T. (1996). Childhood sexual abuse and
psychiatric disorder in young adulthood: II. Psychiatric outcomes of childhood sexual
abuse. J Am Acad Child Adolesc Psychiatry, 35(10), 1365-1374.
https://doi.org/10.1097/00004583-199610000-00024
Fernández-Arjona, M. D. M., Grondona, J. M., Fernández-Llebrez, P., & López-Ávalos, M. D.
(2019). Microglial Morphometric Parameters Correlate With the Expression Level of IL-
1β, and Allow Identifying Different Activated Morphotypes. Front Cell Neurosci, 13,
472. https://doi.org/10.3389/fncel.2019.00472

129
Fernández-Arjona, M. D. M., Grondona, J. M., Granados-Durán, P., Fernández-Llebrez, P., &
López-Ávalos, M. D. (2017). Microglia Morphological Categorization in a Rat Model of
Neuroinflammation by Hierarchical Cluster and Principal Components Analysis. Front
Cell Neurosci, 11, 235. https://doi.org/10.3389/fncel.2017.00235
Fields, R., & Stevens-Graham, B. (2002). New Insights into Neuron-Glia Communication.
Science. 298(5593): 556 –562. doi: 10.1126/science.298.5593.556.
Filipović, D., Todorović, N., Bernardi, R. E., & Gass, P. (2017). Oxidative and nitrosative stress
pathways in the brain of socially isolated adult male rats demonstrating depressive- and
anxiety-like symptoms. Brain Struct Funct, 222(1), 1-20. https://doi.org/10.1007/s00429-
016-1218-9
Frank, M. G., Baratta, M. V., Sprunger, D. B., Watkins, L. R., & Maier, S. F. (2007). Microglia
serve as a neuroimmune substrate for stress-induced potentiation of CNS pro-
inflammatory cytokine responses. Brain Behav Immun, 21(1), 47-59.
https://doi.org/10.1016/j.bbi.2006.03.005
Frisoni, G. B., Fox, N. C., Jack, C. R., Scheltens, P., & Thompson, P. M. (2010). The clinical use
of structural MRI in Alzheimer disease. Nature Reviews Neurology, 6(2), 67-77.
https://doi.org/10.1038/nrneurol.2009.215
Garakani, A., Murrough, J. W., Freire, R. C., Thom, R. P., Larkin, K., Buono, F. D., & Iosifescu,
D. V. (2020). Pharmacotherapy of Anxiety Disorders: Current and Emerging Treatment
Options. Front Psychiatry, 11, 595584. https://doi.org/10.3389/fpsyt.2020.595584
Garcia-Segura, L. M., & Melcangi, R. C. (2006). Steroids and glial cell function. Glia, 54(6),
485-498. https://doi.org/10.1002/glia.20404

130
Ge, H., Guan, S., Shen, Y., Sun, M., Hao, Y., He, L., . . . Gao, Y. (2019). Dihydromyricetin
affects BDNF levels in the nervous system in rats with comorbid diabetic neuropathic
pain and depression. Sci Rep, 9(1), 14619. https://doi.org/10.1038/s41598-019-51124-w
Gebara, E., Zanoletti, O., Ghosal, S., Grosse, J., Schneider, B. L., Knott, G., . . . Sandi, C.
(2021). Mitofusin-2 in the Nucleus Accumbens Regulates Anxiety and Depression-like
Behaviors Through Mitochondrial and Neuronal Actions. Biol Psychiatry, 89(11), 1033-
1044. https://doi.org/10.1016/j.biopsych.2020.12.003
Geng, J., Liu, J., Yuan, X., Liu, W., & Guo, W. (2019). Andrographolide triggers autophagy-
mediated inflammation inhibition and attenuates chronic unpredictable mild stress
(CUMS)-induced depressive-like behavior in mice. Toxicol Appl Pharmacol, 379,
114688. https://doi.org/10.1016/j.taap.2019.114688
Giaume, C., Koulakoff, A., Roux, L., Holcman, D., & Rouach, N. (2010). Astroglial networks: a
step further in neuroglial and gliovascular interactions. Nature Reviews Neuroscience,
11(2), 87-99. https://doi.org/10.1038/nrn2757
Gilman, S. E., Ni, M. Y., Dunn, E. C., Breslau, J., McLaughlin, K. A., Smoller, J. W., & Perlis,
R. H. (2015). Contributions of the social environment to first-onset and recurrent mania.
Mol Psychiatry, 20(3), 329-336. https://doi.org/10.1038/mp.2014.36
Goddard, A. W., Ball, S. G., Martinez, J., Robinson, M. J., Yang, C. R., Russell, J. M., &
Shekhar, A. (2010). Current perspectives of the roles of the central norepinephrine
system in anxiety and depression. Depress Anxiety, 27(4), 339-350.
https://doi.org/10.1002/da.20642
Goddard, A. W., Mason, G. F., Appel, M., Rothman, D. L., Gueorguieva, R., Behar, K. L., &
Krystal, J. H. (2004). Impaired GABA neuronal response to acute benzodiazepine

131
administration in panic disorder. Am J Psychiatry, 161(12), 2186-2193.
https://doi.org/10.1176/appi.ajp.161.12.2186
Gomez, A. F., Barthel, A. L., & Hofmann, S. G. (2018). Comparing the efficacy of
benzodiazepines and serotonergic anti-depressants for adults with generalized anxiety
disorder: a meta-analytic review. Expert Opin Pharmacother, 19(8), 883-894.
https://doi.org/10.1080/14656566.2018.1472767
Gong, Y., Tong, L., Yang, R., Hu, W., Xu, X., Wang, W., . . . Huang, C. (2018). Dynamic
changes in hippocampal microglia contribute to depressive-like behavior induced by
early social isolation. Neuropharmacology, 135, 223-233.
https://doi.org/10.1016/j.neuropharm.2018.03.023
Guo, Z., Chen, X., Huang, Z., Chen, D., Yu, B., He, J., . . . Chen, H. (2021). Effect of dietary
dihydromyricetin supplementation on lipid metabolism, antioxidant capacity and skeletal
muscle fiber type transformation in mice. Anim Biotechnol, 1-8.
https://doi.org/10.1080/10495398.2021.2006204
Gyoneva, S., Davalos, D., Biswas, D., Swanger, S. A., Garnier-Amblard, E., Loth, F., . . .
Traynelis, S. F. (2014). Systemic inflammation regulates microglial responses to tissue
damage in vivo. Glia, 62(8), 1345-1360. https://doi.org/10.1002/glia.22686
Haj-Mirzaian, A., Khosravi, A., Rahbar, A., Ramezanzadeh, K., Nikbakhsh, R., Pirri, F., . . .
Dehpour, A. R. (2020). The potential role of very small embryonic-like stem cells in the
neuroinflammation induced by social isolation stress: Introduction of a new paradigm.
Brain Res Bull, 163, 21-30. https://doi.org/10.1016/j.brainresbull.2020.07.006
Ham, B. J., Sung, Y., Kim, N., Kim, S. J., Kim, J. E., Kim, D. J., . . . Lyoo, I. K. (2007).
Decreased GABA levels in anterior cingulate and basal ganglia in medicated subjects

132
with panic disorder: a proton magnetic resonance spectroscopy (1H-MRS) study. Prog
Neuropsychopharmacol Biol Psychiatry, 31(2), 403-411.
https://doi.org/10.1016/j.pnpbp.2006.10.011
Hama, H., Hara, C., Yamaguchi, K., & Miyawaki, A. (2004). PKC Signaling Mediates Global
Enhancement of Excitatory Synaptogenesis in Neurons Triggered by Local Contact with
Astrocytes. Neuron, 41(3), 405-415. https://doi.org/https://doi.org/10.1016/S0896-
6273(04)00007-8
Hamon, M., & Blier, P. (2013). Monoamine neurocircuitry in depression and strategies for new
treatments. Prog Neuropsychopharmacol Biol Psychiatry, 45, 54-63.
https://doi.org/10.1016/j.pnpbp.2013.04.009
Han, J. M., Lim, H. N., & Jung, H. J. (2017). Hovenia dulcis Thunb. and its active compound
ampelopsin inhibit angiogenesis through suppression of VEGFR2 signaling and HIF-1α
expression. Oncol Rep, 38(6), 3430-3438. https://doi.org/10.3892/or.2017.6021
Hannibal, K. E., & Bishop, M. D. (2014). Chronic stress, cortisol dysfunction, and pain: a
psychoneuroendocrine rationale for stress management in pain rehabilitation. Phys Ther,
94(12), 1816-1825. https://doi.org/10.2522/ptj.20130597
Harris, J. J., Jolivet, R., & Attwell, D. (2012). Synaptic energy use and supply. Neuron, 75(5),
762-777. https://doi.org/10.1016/j.neuron.2012.08.019
Haxhibeqiri, S., Haxhibeqiri, V., Agani, F., Goci Uka, A., Hoxha, B., Dzubur Kulenovic, A., . . .
Deckert, J. (2019). Association of Neuropeptide S Receptor 1 and Glutamate
Decarboxylase 1 Gene Polymorphisms with Posttraumatic Stress Disorder. Psychiatr
Danub, 31(2), 249-255. https://doi.org/10.24869/psyd.2019.249

133
Herbet, M., Korga, A., Gawrońska-Grzywacz, M., Izdebska, M., Piątkowska-Chmiel, I.,
Poleszak, E., . . . Dudka, J. (2017). Chronic Variable Stress Is Responsible for Lipid and
DNA Oxidative Disorders and Activation of Oxidative Stress Response Genes in the
Brain of Rats. Oxid Med Cell Longev, 2017, 7313090.
https://doi.org/10.1155/2017/7313090
Hermida-Ameijeiras, A., Méndez-Alvarez, E., Sánchez-Iglesias, S., Sanmartín-Suárez, C., &
Soto-Otero, R. (2004). Autoxidation and MAO-mediated metabolism of dopamine as a
potential cause of oxidative stress: role of ferrous and ferric ions. Neurochem Int, 45(1),
103-116. https://doi.org/10.1016/j.neuint.2003.11.018
Hershenberg, R., Gros, D. F., & Brawman-Mintzer, O. (2014). Role of atypical antipsychotics in
the treatment of generalized anxiety disorder. CNS Drugs, 28(6), 519-533.
https://doi.org/10.1007/s40263-014-0162-6
Hettema, J. M., An, S. S., Neale, M. C., Bukszar, J., van den Oord, E. J., Kendler, K. S., & Chen,
X. (2006). Association between glutamic acid decarboxylase genes and anxiety disorders,
major depression, and neuroticism. Mol Psychiatry, 11(8), 752-762.
https://doi.org/10.1038/sj.mp.4001845
Hettema, J. M., Neale, M. C., & Kendler, K. S. (2001). A review and meta-analysis of the
genetic epidemiology of anxiety disorders. Am J Psychiatry, 158(10), 1568-1578.
https://doi.org/10.1176/appi.ajp.158.10.1568
Hettema, J. M., Prescott, C. A., Myers, J. M., Neale, M. C., & Kendler, K. S. (2005). The
structure of genetic and environmental risk factors for anxiety disorders in men and
women. Arch Gen Psychiatry, 62(2), 182-189. https://doi.org/10.1001/archpsyc.62.2.182

134
Hinwood, M., Morandini, J., Day, T. A., & Walker, F. R. (2012). Evidence that microglia
mediate the neurobiological effects of chronic psychological stress on the medial
prefrontal cortex. Cereb Cortex, 22(6), 1442-1454. https://doi.org/10.1093/cercor/bhr229
Hinwood, M., Tynan, R. J., Charnley, J. L., Beynon, S. B., Day, T. A., & Walker, F. R. (2013).
Chronic stress induced remodeling of the prefrontal cortex: structural re-organization of
microglia and the inhibitory effect of minocycline. Cereb Cortex, 23(8), 1784-1797.
https://doi.org/10.1093/cercor/bhs151
Hoehn, T., Braune, S., Scheibe, G., & Albus, M. (1997). Physiological, biochemical and
subjective parameters in anxiety patients with panic disorder during stress exposure as
compared with healthy controls. Eur Arch Psychiatry Clin Neurosci, 247(5), 264-274.
https://doi.org/10.1007/BF02900305
Hoge, E. A., Brandstetter, K., Moshier, S., Pollack, M. H., Wong, K. K., & Simon, N. M. (2009).
Broad spectrum of cytokine abnormalities in panic disorder and posttraumatic stress
disorder. Depress Anxiety, 26(5), 447-455. https://doi.org/10.1002/da.20564
Hollis, F., van der Kooij, M. A., Zanoletti, O., Lozano, L., Cantó, C., & Sandi, C. (2015).
Mitochondrial function in the brain links anxiety with social subordination. Proc Natl
Acad Sci U S A, 112(50), 15486-15491. https://doi.org/10.1073/pnas.1512653112
Hossain, M. M., Rahman, M., Trisha, N. F., Tasnim, S., Nuzhath, T., Hasan, N. T., . . . Ma, P.
(2021). Prevalence of anxiety and depression in South Asia during COVID-19: A
systematic review and meta-analysis. Heliyon, 7(4), e06677.
https://doi.org/10.1016/j.heliyon.2021.e06677

135
Hossain, M. M., Sultana, A., & Purohit, N. (2020). Mental health outcomes of quarantine and
isolation for infection prevention: a systematic umbrella review of the global evidence.
Epidemiol Health, 42, e2020038. https://doi.org/10.4178/epih.e2020038
Hou, R., Garner, M., Holmes, C., Osmond, C., Teeling, J., Lau, L., & Baldwin, D. S. (2017).
Peripheral inflammatory cytokines and immune balance in Generalised Anxiety Disorder:
Case-controlled study. Brain Behav Immun, 62, 212-218.
https://doi.org/10.1016/j.bbi.2017.01.021
Hou, X., Tong, Q., Wang, W., Xiong, W., Shi, C., & Fang, J. (2015). Dihydromyricetin protects
endothelial cells from hydrogen peroxide-induced oxidative stress damage by regulating
mitochondrial pathways. Life Sci, 130, 38-46. https://doi.org/10.1016/j.lfs.2015.03.007
Hou, X. L., Tong, Q., Wang, W. Q., Shi, C. Y., Xiong, W., Chen, J., . . . Fang, J. G. (2015).
Suppression of Inflammatory Responses by Dihydromyricetin, a Flavonoid from
Ampelopsis grossedentata, via Inhibiting the Activation of NF-κB and MAPK Signaling
Pathways. J Nat Prod, 78(7), 1689-1696. https://doi.org/10.1021/acs.jnatprod.5b00275
Hu, Q., Zhang, T., Yi, L., Zhou, X., & Mi, M. (2018). Dihydromyricetin inhibits NLRP3
inflammasome-dependent pyroptosis by activating the Nrf2 signaling pathway in
vascular endothelial cells. Biofactors, 44(2), 123-136. https://doi.org/10.1002/biof.1395
Hu, W., Dang, X. B., Wang, G., Li, S., & Zhang, Y. L. (2018). Peroxiredoxin-3 attenuates
traumatic neuronal injury through preservation of mitochondrial function. Neurochem Int,
114, 120-126. https://doi.org/10.1016/j.neuint.2018.02.004
Hu, Z., Yang, B., Mo, X., & Xiao, H. (2015). Mechanism and Regulation of Autophagy and Its
Role in Neuronal Diseases. Mol Neurobiol, 52(3), 1190-1209.
https://doi.org/10.1007/s12035-014-8921-4

136
Huang, G., Mei, X., & Hu, J. (2017). The Antioxidant Activities of Natural Polysaccharides.
Curr Drug Targets, 18(11), 1296-1300.
https://doi.org/10.2174/1389450118666170123145357
Hutson, P. H., Clark, J. A., & Cross, A. J. (2017). CNS Target Identification and Validation:
Avoiding the Valley of Death or Naive Optimism? Annual Review of Pharmacology and
Toxicology, 57(1), 171-187. https://doi.org/10.1146/annurev-pharmtox-010716-104624
Hyun, T. K., Eom, S. H., Yu, C. Y., & Roitsch, T. (2010). Hovenia dulcis--an Asian traditional
herb. Planta Med, 76(10), 943-949. https://doi.org/10.1055/s-0030-1249776
ICD-11. (2022). International Statistical Classification of Diseases and Related Health
Problemsof Diseases and Related Health Problems.
https://www.who.int/standards/classifications/classification-of-diseases
Ieraci, A., Mallei, A., & Popoli, M. (2016). Social Isolation Stress Induces Anxious-Depressive-
Like Behavior and Alterations of Neuroplasticity-Related Genes in Adult Male Mice.
Neural Plast, 2016, 6212983. https://doi.org/10.1155/2016/6212983
Imai, H., & Nakagawa, Y. (2003). Biological significance of phospholipid hydroperoxide
glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radic Biol Med, 34(2),
145-169. https://doi.org/10.1016/s0891-5849(02)01197-8
Indo, H. P., Davidson, M., Yen, H. C., Suenaga, S., Tomita, K., Nishii, T., . . . Majima, H. J.
(2007). Evidence of ROS generation by mitochondria in cells with impaired electron
transport chain and mitochondrial DNA damage. Mitochondrion, 7(1-2), 106-118.
https://doi.org/10.1016/j.mito.2006.11.026
Insel, T. R. (2012). Next-Generation Treatments for Mental Disorders. Science Translational
Medicine, 4(155), 155ps119-155ps119. https://doi.org/10.1126/scitranslmed.3004873

137
Jakawich, S. K., Nasser, H. B., Strong, M. J., McCartney, A. J., Perez, A. S., Rakesh, N., . . .
Sutton, M. A. (2010). Local presynaptic activity gates homeostatic changes in
presynaptic function driven by dendritic BDNF synthesis. Neuron, 68(6), 1143-1158.
https://doi.org/10.1016/j.neuron.2010.11.034
Jelinek, A., Heyder, L., Daude, M., Plessner, M., Krippner, S., Grosse, R., . . . Culmsee, C.
(2018). Mitochondrial rescue prevents glutathione peroxidase-dependent ferroptosis.
Free Radic Biol Med, 117, 45-57. https://doi.org/10.1016/j.freeradbiomed.2018.01.019
Jha, M. K., Jo, M., Kim, J. H., & Suk, K. (2019). Microglia-Astrocyte Crosstalk: An Intimate
Molecular Conversation. Neuroscientist, 25(3), 227-240.
https://doi.org/10.1177/1073858418783959
Ji, L. L., Peng, J. B., Fu, C. H., Cao, D., Li, D., Tong, L., & Wang, Z. Y. (2016). Activation of
Sigma-1 receptor ameliorates anxiety-like behavior and cognitive impairments in a rat
model of post-traumatic stress disorder. Behav Brain Res, 311, 408-415.
https://doi.org/10.1016/j.bbr.2016.05.056
Jin, X. T., Galvan, A., Wichmann, T., & Smith, Y. (2011). Localization and Function of GABA
Transporters GAT-1 and GAT-3 in the Basal Ganglia. Front Syst Neurosci, 5, 63.
https://doi.org/10.3389/fnsys.2011.00063
Jing, N., & Li, X. (2019). Dihydromyricetin Attenuates Inflammation through TLR4/NF-kappaB
Pathway. Open Med (Wars), 14, 719-725. https://doi.org/10.1515/med-2019-0083
Jurga, A. M., Paleczna, M., & Kuter, K. Z. (2020). Overview of General and Discriminating
Markers of Differential Microglia Phenotypes. Front Cell Neurosci, 14, 198.
https://doi.org/10.3389/fncel.2020.00198

138
Kalisch, R., Schubert, M., Jacob, W., Kessler, M. S., Hemauer, R., Wigger, A., . . . Auer, D. P.
(2006). Anxiety and hippocampus volume in the rat. Neuropsychopharmacology, 31(5),
925-932. https://doi.org/10.1038/sj.npp.1300910
Kalueff, A. V., & Nutt, D. J. (2007). Role of GABA in anxiety and depression. Depress Anxiety,
24(7), 495-517. https://doi.org/10.1002/da.20262
Karperien, A., Ahammer, H., & Jelinek, H. F. (2013). Quantitating the subtleties of microglial
morphology with fractal analysis. Front Cell Neurosci, 7, 3.
https://doi.org/10.3389/fncel.2013.00003
Kashyap, G., Bapat, D., Das, D., Gowaikar, R., Amritkar, R. E., Rangarajan, G., . . . Ambika, G.
(2019). Synapse loss and progress of Alzheimer’s disease -A network model. Scientific
Reports, 9(1), 6555. https://doi.org/10.1038/s41598-019-43076-y
Kasote, D. M., Hegde, M. V., & Katyare, S. S. (2013). Mitochondrial dysfunction in psychiatric
and neurological diseases: cause(s), consequence(s), and implications of antioxidant
therapy. Biofactors, 39(4), 392-406. https://doi.org/10.1002/biof.1093
Kessler, R. C., Berglund, P., Demler, O., Jin, R., Merikangas, K. R., & Walters, E. E. (2005).
Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the National
Comorbidity Survey Replication. Arch Gen Psychiatry, 62(6), 593-602.
https://doi.org/10.1001/archpsyc.62.6.593
Kessler, R. C., Birnbaum, H. G., Shahly, V., Bromet, E., Hwang, I., McLaughlin, K. A., . . .
Stein, D. J. (2010). Age differences in the prevalence and co-morbidity of DSM-IV major
depressive episodes: results from the WHO World Mental Health Survey Initiative.
Depress Anxiety, 27(4), 351-364. https://doi.org/10.1002/da.20634

139
Kinlein, S. A., Phillips, D. J., Keller, C. R., & Karatsoreos, I. N. (2019). Role of corticosterone in
altered neurobehavioral responses to acute stress in a model of compromised
hypothalamic-pituitary-adrenal axis function. Psychoneuroendocrinology, 102, 248-255.
https://doi.org/10.1016/j.psyneuen.2018.12.010
Kjelstrup, K. G., Tuvnes, F. A., Steffenach, H. A., Murison, R., Moser, E. I., & Moser, M. B.
(2002). Reduced fear expression after lesions of the ventral hippocampus. Proc Natl Acad
Sci U S A, 99(16), 10825-10830. https://doi.org/10.1073/pnas.152112399
Klausberger, T., & Somogyi, P. (2008). Neuronal diversity and temporal dynamics: the unity of
hippocampal circuit operations. Science, 321(5885), 53-57.
https://doi.org/10.1126/science.1149381
Klinenberg, E. (2016). Social Isolation, Loneliness, and Living Alone: Identifying the Risks for
Public Health. Am J Public Health, 106(5), 786-787.
https://doi.org/10.2105/AJPH.2016.303166
Ko, Y. H., Kim, S. K., Lee, S. Y., & Jang, C. G. (2020). Flavonoids as therapeutic candidates for
emotional disorders such as anxiety and depression. Arch Pharm Res, 43(11), 1128-1143.
https://doi.org/10.1007/s12272-020-01292-5
Konnopka, A., Leichsenring, F., Leibing, E., & König, H. H. (2009). Cost-of-illness studies and
cost-effectiveness analyses in anxiety disorders: a systematic review. J Affect Disord,
114(1-3), 14-31. https://doi.org/10.1016/j.jad.2008.07.014
Koo, J. W., Russo, S. J., Ferguson, D., Nestler, E. J., & Duman, R. S. (2010). Nuclear factor-
kappaB is a critical mediator of stress-impaired neurogenesis and depressive behavior.
Proc Natl Acad Sci U S A, 107(6), 2669-2674. https://doi.org/10.1073/pnas.0910658107

140
Kou, X., Liu, X., Chen, X., Li, J., Yang, X., Fan, J., . . . Chen, N. (2016). Ampelopsin attenuates
brain aging of D-gal-induced rats through miR-34a-mediated SIRT1/mTOR signal
pathway. Oncotarget, 7(46), 74484-74495. https://doi.org/10.18632/oncotarget.12811
Kreisel, T., Frank, M. G., Licht, T., Reshef, R., Ben-Menachem-Zidon, O., Baratta, M. V., . . .
Yirmiya, R. (2014). Dynamic microglial alterations underlie stress-induced depressive-
like behavior and suppressed neurogenesis. Mol Psychiatry, 19(6), 699-709.
https://doi.org/10.1038/mp.2013.155
Krolow, R., Noschang, C., Weis, S. N., Pettenuzzo, L. F., Huffell, A. P., Arcego, D. M., . . .
Dalmaz, C. (2012). Isolation stress during the prepubertal period in rats induces long-
lasting neurochemical changes in the prefrontal cortex. Neurochem Res, 37(5), 1063-
1073. https://doi.org/10.1007/s11064-012-0709-1
Lacerda-Pinheiro, S. F., Pinheiro Junior, R. F., Pereira de Lima, M. A., Lima da Silva, C. G.,
Vieira dos Santos, M. o. S., Teixeira Júnior, A. G., . . . Bianco, B. A. (2014). Are there
depression and anxiety genetic markers and mutations? A systematic review. J Affect
Disord, 168, 387-398. https://doi.org/10.1016/j.jad.2014.07.016
Lamark, T., Svenning, S., & Johansen, T. (2017). Regulation of selective autophagy: the
p62/SQSTM1 paradigm. Essays Biochem, 61(6), 609-624.
https://doi.org/10.1042/EBC20170035
Lanzenberger, R. R., Mitterhauser, M., Spindelegger, C., Wadsak, W., Klein, N., Mien, L.
K., . . . Tauscher, J. (2007). Reduced serotonin-1A receptor binding in social anxiety
disorder. Biol Psychiatry, 61(9), 1081-1089.
https://doi.org/10.1016/j.biopsych.2006.05.022

141
Lehmann, M. L., Weigel, T. K., Poffenberger, C. N., & Herkenham, M. (2019). The Behavioral
Sequelae of Social Defeat Require Microglia and Are Driven by Oxidative Stress in
Mice. J Neurosci, 39(28), 5594-5605. https://doi.org/10.1523/JNEUROSCI.0184-
19.2019
Lenze, E. J. (2003). Comorbidity of depression and anxiety in the elderly. Curr Psychiatry Rep,
5(1), 62-67. https://doi.org/10.1007/s11920-003-0011-7
Lessmann, V., Gottmann, K., & Malcangio, M. (2003). Neurotrophin secretion: current facts and
future prospects. Prog Neurobiol, 69(5), 341-374. https://doi.org/10.1016/s0301-
0082(03)00019-4
Li, C., Li, M., Yu, H., Shen, X., Wang, J., Sun, X., . . . Wang, C. (2017). Neuropeptide VGF C-
Terminal Peptide TLQP-62 Alleviates Lipopolysaccharide-Induced Memory Deficits and
Anxiety-like and Depression-like Behaviors in Mice: The Role of BDNF/TrkB Signaling.
ACS Chem Neurosci, 8(9), 2005-2018. https://doi.org/10.1021/acschemneuro.7b00154
Li, H., Li, Q., Liu, Z., Yang, K., Chen, Z., Cheng, Q., & Wu, L. (2017). The Versatile Effects of
Dihydromyricetin in Health. Evid Based Complement Alternat Med, 2017, 1053617.
https://doi.org/10.1155/2017/1053617
Li, X., Liu, J., Lin, J., Wang, T., Huang, J., Lin, Y., & Chen, D. (2016). Protective Effects of
Dihydromyricetin against •OH-Induced Mesenchymal Stem Cells Damage and
Mechanistic Chemistry. Molecules, 21(5). https://doi.org/10.3390/molecules21050604
Liang, H., Van Remmen, H., Frohlich, V., Lechleiter, J., Richardson, A., & Ran, Q. (2007).
Gpx4 protects mitochondrial ATP generation against oxidative damage. Biochem Biophys
Res Commun, 356(4), 893-898. https://doi.org/10.1016/j.bbrc.2007.03.045

142
Liang, J., Lindemeyer, A., Shen, Y., Lopez-Valdes, H., Martinez-Coria, H., Shao, X., & Olsen,
R. (2014). Dihydromyricetin ameliorates behavioral deficits and reverses neuropathology
of transgenic mouse models of Alzheimer’s disease. Neurochem Res. (6):1171-81. .
Liang, J., Lopez-Valdes, H. E., Martinez-Coria, H., Lindemeyer, A. K., Shen, Y., Shao, X. M., &
Olsen, R. W. (2014). Dihydromyricetin ameliorates behavioral deficits and reverses
neuropathology of transgenic mouse models of Alzheimer's disease. Neurochem Res,
39(6), 1171-1181. https://doi.org/10.1007/s11064-014-1304-4
Liang, J., López-Valdés, H. E., Martínez-Coria, H., Lindemeyer, A. K., Shen, Y., Shao, X. M., &
Olsen, R. W. (2014). Dihydromyricetin ameliorates behavioral deficits and reverses
neuropathology of transgenic mouse models of Alzheimer's disease. Neurochem Res,
39(6), 1171-1181. https://doi.org/10.1007/s11064-014-1304-4
Liang, J., Shen, Y., Shao, X. M., Scott, M. B., Ly, E., Wong, S., . . . Spigelman, I. (2014).
Dihydromyricetin prevents fetal alcohol exposure-induced behavioral and physiological
deficits: the roles of GABAA receptors in adolescence. Neurochem Res, 39(6), 1147-
1161. https://doi.org/10.1007/s11064-014-1291-5
Lichtenberg, D., & Pinchuk, I. (2015). Oxidative stress, the term and the concept. Biochem
Biophys Res Commun, 461(3), 441-444. https://doi.org/10.1016/j.bbrc.2015.04.062
Licznerski, P., & Duman, R. S. (2013). Remodeling of axo-spinous synapses in the
pathophysiology and treatment of depression. Neuroscience, 251, 33-50.
https://doi.org/10.1016/j.neuroscience.2012.09.057
Licznerski, P., & Jonas, E. A. (2018). BDNF signaling: Harnessing stress to battle mood
disorder. Proc Natl Acad Sci U S A, 115(15), 3742-3744.
https://doi.org/10.1073/pnas.1803645115

143
Lieb, R., Isensee, B., Höfler, M., Pfister, H., & Wittchen, H. U. (2002). Parental major
depression and the risk of depression and other mental disorders in offspring: a
prospective-longitudinal community study. Arch Gen Psychiatry, 59(4), 365-374.
https://doi.org/10.1001/archpsyc.59.4.365
Lin, M. T., & Beal, M. F. (2006). Mitochondrial dysfunction and oxidative stress in
neurodegenerative diseases. Nature, 443(7113), 787-795.
https://doi.org/10.1038/nature05292
Liu, H. Y., Yue, J., Hu, L. N., Cheng, L. F., Wang, X. S., Wang, X. J., & Feng, B. (2018).
Chronic minocycline treatment reduces the anxiety-like behaviors induced by repeated
restraint stress through modulating neuroinflammation. Brain Res Bull, 143, 19-26.
https://doi.org/10.1016/j.brainresbull.2018.08.015
Liu, L., Wan, J., Lang, H., Si, M., Zhu, J., Zhou, Y., & Mi, M. (2017). Dihydromyricetin delays
the onset of hyperglycemia and ameliorates insulin resistance without excessive weight
gain in Zucker diabetic fatty rats. Mol Cell Endocrinol, 439, 105-115.
https://doi.org/10.1016/j.mce.2016.10.028
Liu, P., Zou, D., Chen, K., Zhou, Q., Gao, Y., Huang, Y., . . . Mi, M. (2016). Dihydromyricetin
Improves Hypobaric Hypoxia-Induced Memory Impairment via Modulation of SIRT3
Signaling. Mol Neurobiol, 53(10), 7200-7212. https://doi.org/10.1007/s12035-015-9627-
y
Liu, T., Zhang, L., Joo, D., & Sun, S. C. (2017). NF-κB signaling in inflammation. Signal
Transduct Target Ther, 2. https://doi.org/10.1038/sigtrans.2017.23

144
Liu, T. T., Zeng, Y., Tang, K., Chen, X., Zhang, W., & Xu, X. L. (2017). Dihydromyricetin
ameliorates atherosclerosis in LDL receptor deficient mice. Atherosclerosis, 262, 39-50.
https://doi.org/10.1016/j.atherosclerosis.2017.05.003
Liu, W., Ge, T., Leng, Y., Pan, Z., Fan, J., Yang, W., & Cui, R. (2017). The Role of Neural
Plasticity in Depression: From Hippocampus to Prefrontal Cortex. Neural Plast, 2017,
6871089. https://doi.org/10.1155/2017/6871089
Liu, W. Z., Zhang, W. H., Zheng, Z. H., Zou, J. X., Liu, X. X., Huang, S. H., . . . Pan, B. X.
(2020). Identification of a prefrontal cortex-to-amygdala pathway for chronic stress-
induced anxiety. Nat Commun, 11(1), 2221. https://doi.org/10.1038/s41467-020-15920-7
Liu, Y., Zhao, J., & Guo, W. (2018). Emotional Roles of Mono-Aminergic Neurotransmitters in
Major Depressive Disorder and Anxiety Disorders. Front Psychol, 9, 2201.
https://doi.org/10.3389/fpsyg.2018.02201
Loades, M. E., Chatburn, E., Higson-Sweeney, N., Reynolds, S., Shafran, R., Brigden, A., . . .
Crawley, E. (2020). Rapid Systematic Review: The Impact of Social Isolation and
Loneliness on the Mental Health of Children and Adolescents in the Context of COVID-
19. J Am Acad Child Adolesc Psychiatry, 59(11), 1218-1239.e1213.
https://doi.org/10.1016/j.jaac.2020.05.009
Lähdepuro, A., Savolainen, K., Lahti-Pulkkinen, M., Eriksson, J. G., Lahti, J., Tuovinen, S., . . .
Räikkönen, K. (2019). The Impact of Early Life Stress on Anxiety Symptoms in Late
Adulthood. Sci Rep, 9(1), 4395. https://doi.org/10.1038/s41598-019-40698-0
Machado, P., Rostaing, P., Guigonis, J. M., Renner, M., Dumoulin, A., Samson, M., . . . Triller,
A. (2011). Heat shock cognate protein 70 regulates gephyrin clustering. J Neurosci,
31(1), 3-14. https://doi.org/10.1523/JNEUROSCI.2533-10.2011

145
Maday, S., Wallace, K. E., & Holzbaur, E. L. (2012). Autophagosomes initiate distally and
mature during transport toward the cell soma in primary neurons. J Cell Biol, 196(4),
407-417. https://doi.org/10.1083/jcb.201106120
Magariños, A. M., Verdugo, J. M., & McEwen, B. S. (1997). Chronic stress alters synaptic
terminal structure in hippocampus. Proc Natl Acad Sci U S A, 94(25), 14002-14008.
https://doi.org/10.1073/pnas.94.25.14002
Mahmoud, S., Gharagozloo, M., Simard, C., & Gris, D. (2019). Astrocytes Maintain Glutamate
Homeostasis in the CNS by Controlling the Balance between Glutamate Uptake and
Release. Cells, 8(2). https://doi.org/10.3390/cells8020184
Mailly, F., Marin, P., Israël, M., Glowinski, J., & Prémont, J. (1999). Increase in external
glutamate and NMDA receptor activation contribute to H2O2-induced neuronal
apoptosis. J Neurochem, 73(3), 1181-1188. https://doi.org/10.1046/j.1471-
4159.1999.0731181.x
Mao, X. Y., Cao, Y. G., Ji, Z., Zhou, H. H., Liu, Z. Q., & Sun, H. L. (2015). Topiramate protects
against glutamate excitotoxicity via activating BDNF/TrkB-dependent ERK pathway in
rodent hippocampal neurons. Prog Neuropsychopharmacol Biol Psychiatry, 60, 11-17.
https://doi.org/10.1016/j.pnpbp.2015.01.015
Martin, E. I., Ressler, K. J., Binder, E., & Nemeroff, C. B. (2009). The neurobiology of anxiety
disorders: brain imaging, genetics, and psychoneuroendocrinology. Psychiatr Clin North
Am, 32(3), 549-575. https://doi.org/10.1016/j.psc.2009.05.004
Martinez-Coria, H., Green, K. N., Billings, L. M., Kitazawa, M., Albrecht, M., Rammes, G., . . .
LaFerla, F. M. (2010). Memantine improves cognition and reduces Alzheimer's-like

146
neuropathology in transgenic mice. Am J Pathol, 176(2), 870-880.
https://doi.org/10.2353/ajpath.2010.090452
Matejuk, A., & Ransohoff, R. M. (2020). Crosstalk Between Astrocytes and Microglia: An
Overview. Front Immunol, 11, 1416. https://doi.org/10.3389/fimmu.2020.01416
Matheson, S. L., Shepherd, A. M., Pinchbeck, R. M., Laurens, K. R., & Carr, V. J. (2013).
Childhood adversity in schizophrenia: a systematic meta-analysis. Psychol Med, 43(2),
225-238. https://doi.org/10.1017/S0033291712000785
Matias, I., Morgado, J., & Gomes, F. C. A. (2019). Astrocyte Heterogeneity: Impact to Brain
Aging and Disease [Review]. Frontiers in Aging Neuroscience, 11(59).
https://doi.org/10.3389/fnagi.2019.00059
Mazure, N. M., & Pouysségur, J. (2010). Hypoxia-induced autophagy: cell death or cell
survival? Curr Opin Cell Biol, 22(2), 177-180. https://doi.org/10.1016/j.ceb.2009.11.015
McEwen, B. S., & Wingfield, J. C. (2003). The concept of allostasis in biology and biomedicine.
Horm Behav, 43(1), 2-15. https://doi.org/10.1016/s0018-506x(02)00024-7
McKlveen, J. M., Myers, B., Flak, J. N., Bundzikova, J., Solomon, M. B., Seroogy, K. B., &
Herman, J. P. (2013). Role of prefrontal cortex glucocorticoid receptors in stress and
emotion. Biol Psychiatry, 74(9), 672-679. https://doi.org/10.1016/j.biopsych.2013.03.024
McLean, C. P., Asnaani, A., Litz, B. T., & Hofmann, S. G. (2011). Gender differences in anxiety
disorders: Prevalence, course of illness, comorbidity and burden of illness. Journal of
Psychiatric Research, 45(8), 1027-1035.
https://doi.org/https://doi.org/10.1016/j.jpsychires.2011.03.006
Mederos, S., & Perea, G. (2019). GABAergic ‐astrocyte signaling: a refinement of inhibitory
brain networks. Glia, 67(10), 1842-1851.

147
Miao, Z., Wang, Y., & Sun, Z. (2020). The Relationships Between Stress, Mental Disorders, and
Epigenetic Regulation of BDNF. Int J Mol Sci, 21(4).
https://doi.org/10.3390/ijms21041375
Mitra, R., Jadhav, S., McEwen, B. S., Vyas, A., & Chattarji, S. (2005). Stress duration modulates
the spatiotemporal patterns of spine formation in the basolateral amygdala. Proc Natl
Acad Sci U S A, 102(26), 9371-9376. https://doi.org/10.1073/pnas.0504011102
Mizushima, N. (2018). A brief history of autophagy from cell biology to physiology and disease.
Nat Cell Biol, 20(5), 521-527. https://doi.org/10.1038/s41556-018-0092-5
Mody, I., & Maguire, J. (2011). The reciprocal regulation of stress hormones and GABA(A)
receptors. Front Cell Neurosci, 6, 4. https://doi.org/10.3389/fncel.2012.00004
Mumtaz, F., Khan, M. I., Zubair, M., & Dehpour, A. R. (2018). Neurobiology and consequences
of social isolation stress in animal model-A comprehensive review. Biomed
Pharmacother, 105, 1205-1222. https://doi.org/10.1016/j.biopha.2018.05.086
Murínová, J., Hlaváčová, N., Chmelová, M., & Riečanský, I. (2017). The Evidence for Altered
BDNF Expression in the Brain of Rats Reared or Housed in Social Isolation: A
Systematic Review. Front Behav Neurosci, 11, 101.
https://doi.org/10.3389/fnbeh.2017.00101
Möller, M., Du Preez, J. L., Viljoen, F. P., Berk, M., Emsley, R., & Harvey, B. H. (2013). Social
isolation rearing induces mitochondrial, immunological, neurochemical and behavioural
deficits in rats, and is reversed by clozapine or N-acetyl cysteine. Brain Behav Immun,
30, 156-167. https://doi.org/10.1016/j.bbi.2012.12.011
Nakahira, K., Haspel, J. A., Rathinam, V. A., Lee, S. J., Dolinay, T., Lam, H. C., . . . Choi, A. M.
(2011). Autophagy proteins regulate innate immune responses by inhibiting the release of

148
mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol, 12(3), 222-
230. https://doi.org/10.1038/ni.1980
Nakaso, K., Kitayama, M., Mizuta, E., Fukuda, H., Ishii, T., Nakashima, K., & Yamada, K.
(2000). Co-induction of heme oxygenase-1 and peroxiredoxin I in astrocytes and
microglia around hemorrhagic region in the rat brain. Neurosci Lett, 293(1), 49-52.
https://doi.org/10.1016/s0304-3940(00)01491-9
Nash, J. R., Sargent, P. A., Rabiner, E. A., Hood, S. D., Argyropoulos, S. V., Potokar, J. P., . . .
Nutt, D. J. (2008). Serotonin 5-HT1A receptor binding in people with panic disorder:
positron emission tomography study. Br J Psychiatry, 193(3), 229-234.
https://doi.org/10.1192/bjp.bp.107.041186
Neis, V. B., Rosa, P. B., Moretti, M., & Rodrigues, A. L. S. (2018). Involvement of Heme
Oxygenase-1 in Neuropsychiatric and Neurodegenerative Diseases. Curr Pharm Des,
24(20), 2283-2302. https://doi.org/10.2174/1381612824666180717160623
Nikoletopoulou, V., Sidiropoulou, K., Kallergi, E., Dalezios, Y., & Tavernarakis, N. (2017).
Modulation of Autophagy by BDNF Underlies Synaptic Plasticity. Cell Metab, 26(1),
230-242.e235. https://doi.org/10.1016/j.cmet.2017.06.005
Nimmerjahn, A., Kirchhoff, F., & Helmchen, F. (2005). Resting microglial cells are highly
dynamic surveillants of brain parenchyma in vivo. Science, 308(5726), 1314-1318.
https://doi.org/10.1126/science.1110647
Nugent, N. R., Tyrka, A. R., Carpenter, L. L., & Price, L. H. (2011). Gene-environment
interactions: early life stress and risk for depressive and anxiety disorders.
Psychopharmacology (Berl), 214(1), 175-196. https://doi.org/10.1007/s00213-010-2151-
x

149
Nuss, P. (2015). Anxiety disorders and GABA neurotransmission: a disturbance of modulation.
Neuropsychiatr Dis Treat, 11, 165-175. https://doi.org/10.2147/NDT.S58841
Obashi, K., & Okabe, S. (2013). Regulation of mitochondrial dynamics and distribution by
synapse position and neuronal activity in the axon. Eur J Neurosci, 38(3), 2350-2363.
https://doi.org/10.1111/ejn.12263
Ogłodek, E. A., & Just, M. J. (2018). The association between inflammatory markers (iNOS,
HO-1, IL-33, MIP-1β) and depression with and without posttraumatic stress disorder.
Pharmacol Rep, 70(6), 1065-1072. https://doi.org/10.1016/j.pharep.2018.06.001
Olfson, M., Mojtabai, R., Merikangas, K. R., Compton, W. M., Wang, S., Grant, B. F., &
Blanco, C. (2017). Reexamining associations between mania, depression, anxiety and
substance use disorders: results from a prospective national cohort. Mol Psychiatry,
22(2), 235-241. https://doi.org/10.1038/mp.2016.64
Olsen, R. W., Yang, J., King, R. G., Dilber, A., Stauber, G. B., & Ransom, R. W. (1986).
Barbiturate and benzodiazepine modulation of GABA receptor binding and function. Life
Sci, 39(21), 1969-1976. https://doi.org/10.1016/0024-3205(86)90320-6
Oyabu, K., Takeda, K., Kawano, H., Kubota, K., Watanabe, T., Harata, N. C., . . . Iwasaki, K.
(2020). Presynaptically silent synapses are modulated by the density of surrounding
astrocytes. Journal of Pharmacological Sciences, 144(2), 76-82.
https://doi.org/https://doi.org/10.1016/j.jphs.2020.07.009
Paixão, S., & Klein, R. (2010). Neuron-astrocyte communication and synaptic plasticity. Curr
Opin Neurobiol, 20(4), 466-473. https://doi.org/10.1016/j.conb.2010.04.008

150
Paolicelli, R. C., Bolasco, G., Pagani, F., Maggi, L., Scianni, M., Panzanelli, P., . . . Gross, C. T.
(2011). Synaptic pruning by microglia is necessary for normal brain development.
Science, 333(6048), 1456-1458. https://doi.org/10.1126/science.1202529
Parkhurst, C. N., Yang, G., Ninan, I., Savas, J. N., Yates, J. R., Lafaille, J. J., . . . Gan, W. B.
(2013). Microglia promote learning-dependent synapse formation through brain-derived
neurotrophic factor. Cell, 155(7), 1596-1609. https://doi.org/10.1016/j.cell.2013.11.030
Parra, L. A., Baust, T. B., Smith, A. D., Jaumotte, J. D., Zigmond, M. J., Torres, S., . . . Torres,
G. E. (2016). The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to
Regulate Enzyme Activity and Synaptic Vesicle Localization. J Biol Chem, 291(34),
17510-17522. https://doi.org/10.1074/jbc.M116.728782
Patil, P., Thakur, A., Sharma, A., & Flora, S. J. S. (2020). Natural products and their derivatives
as multifunctional ligands against Alzheimer's disease. Drug Dev Res, 81(2), 165-183.
https://doi.org/10.1002/ddr.21587
Paul, S. M., Mytelka, D. S., Dunwiddie, C. T., Persinger, C. C., Munos, B. H., Lindborg, S. R.,
& Schacht, A. L. (2010). How to improve R&D productivity: the pharmaceutical
industry's grand challenge. Nature Reviews Drug Discovery, 9(3), 203-214.
https://doi.org/10.1038/nrd3078
Peirson, S. N., Brown, L. A., Pothecary, C. A., Benson, L. A., & Fisk, A. S. (2018). Light and
the laboratory mouse. J Neurosci Methods, 300, 26-36.
https://doi.org/10.1016/j.jneumeth.2017.04.007
Perea, G., Navarrete, M., & Araque, A. (2009). Tripartite synapses: astrocytes process and
control synaptic information. Trends Neurosci, 32(8), 421-431.
https://doi.org/10.1016/j.tins.2009.05.001

151
Perić, I., Costina, V., Stanisavljević, A., Findeisen, P., & Filipović, D. (2018). Proteomic
characterization of hippocampus of chronically socially isolated rats treated with
fluoxetine: Depression-like behaviour and fluoxetine mechanism of action.
Neuropharmacology, 135, 268-283. https://doi.org/10.1016/j.neuropharm.2018.03.034
Pham, X., Sun, C., Chen, X., van den Oord, E. J., Neale, M. C., Kendler, K. S., & Hettema, J. M.
(2009). Association study between GABA receptor genes and anxiety spectrum disorders.
Depress Anxiety, 26(11), 998-1003. https://doi.org/10.1002/da.20628
Phelps, E. A., & LeDoux, J. E. (2005). Contributions of the amygdala to emotion processing:
from animal models to human behavior. Neuron, 48(2), 175-187.
https://doi.org/10.1016/j.neuron.2005.09.025
Phillips, C. (2017). Brain-Derived Neurotrophic Factor, Depression, and Physical Activity:
Making the Neuroplastic Connection. Neural Plast, 2017, 7260130.
https://doi.org/10.1155/2017/7260130
Piantadosi, C. A. (2008). Carbon monoxide, reactive oxygen signaling, and oxidative stress. Free
Radic Biol Med, 45(5), 562-569. https://doi.org/10.1016/j.freeradbiomed.2008.05.013
Pierone, B. C., Pereira, C. A., Garcez, M. L., & Kaster, M. P. (2020). Stress and signaling
pathways regulating autophagy: From behavioral models to psychiatric disorders. Exp
Neurol, 334, 113485. https://doi.org/10.1016/j.expneurol.2020.113485
Piirainen, S., Youssef, A., Song, C., Kalueff, A. V., Landreth, G. E., Malm, T., & Tian, L.
(2017). Psychosocial stress on neuroinflammation and cognitive dysfunctions in
Alzheimer's disease: the emerging role for microglia? Neurosci Biobehav Rev, 77, 148-
164. https://doi.org/10.1016/j.neubiorev.2017.01.046

152
Pinna, G., Agis-Balboa, R. C., Zhubi, A., Matsumoto, K., Grayson, D. R., Costa, E., & Guidotti,
A. (2006). Imidazenil and diazepam increase locomotor activity in mice exposed to
protracted social isolation. Proc Natl Acad Sci U S A, 103(11), 4275-4280.
https://doi.org/10.1073/pnas.0600329103
Pinna, G., Costa, E., & Guidotti, A. (2004). Fluoxetine and norfluoxetine stereospecifically
facilitate pentobarbital sedation by increasing neurosteroids. Proc Natl Acad Sci U S A,
101(16), 6222-6225. https://doi.org/10.1073/pnas.0401479101
Porter, V. R., Buxton, W. G., Fairbanks, L. A., Strickland, T., O'Connor, S. M., Rosenberg-
Thompson, S., & Cummings, J. L. (2003). Frequency and characteristics of anxiety
among patients with Alzheimer's disease and related dementias. J Neuropsychiatry Clin
Neurosci, 15(2), 180-186. https://doi.org/10.1176/jnp.15.2.180
Preman, P., Alfonso-Triguero, M., Alberdi, E., Verkhratsky, A., & Arranz, A. M. (2021).
Astrocytes in Alzheimer’s Disease: Pathological Significance and Molecular Pathways.
Cells, 10(3). https://doi.org/10.3390/cells10030540
Prinz, M., & Priller, J. (2014). Microglia and brain macrophages in the molecular age: from
origin to neuropsychiatric disease. Nat Rev Neurosci, 15(5), 300-312.
https://doi.org/10.1038/nrn3722
Qi, S., Xin, Y., Guo, Y., Diao, Y., Kou, X., Luo, L., & Yin, Z. (2012). Ampelopsin reduces
endotoxic inflammation via repressing ROS-mediated activation of PI3K/Akt/NF-κB
signaling pathways. Int Immunopharmacol, 12(1), 278-287.
https://doi.org/10.1016/j.intimp.2011.12.001
Quan, N., & Banks, W. A. (2007). Brain-immune communication pathways. Brain Behav
Immun, 21(6), 727-735. https://doi.org/10.1016/j.bbi.2007.05.005

153
Rajendran, L., & Paolicelli, R. C. (2018). Microglia-Mediated Synapse Loss in Alzheimer's
Disease. J Neurosci, 38(12), 2911-2919. https://doi.org/10.1523/JNEUROSCI.1136-
17.2017
Rammal, H., Bouayed, J., Younos, C., & Soulimani, R. (2008). Evidence that oxidative stress is
linked to anxiety-related behaviour in mice. Brain Behav Immun, 22(8), 1156-1159.
https://doi.org/10.1016/j.bbi.2008.06.005
Ran, Q., Gu, M., Van Remmen, H., Strong, R., Roberts, J. L., & Richardson, A. (2006).
Glutathione peroxidase 4 protects cortical neurons from oxidative injury and amyloid
toxicity. J Neurosci Res, 84(1), 202-208. https://doi.org/10.1002/jnr.20868
Ravindran, S., Jahir Hussain, S., Boovarahan, S. R., & Kurian, G. A. (2017). Sodium thiosulfate
post-conditioning protects rat hearts against ischemia reperfusion injury via reduction of
apoptosis and oxidative stress. Chem Biol Interact, 274, 24-34.
https://doi.org/10.1016/j.cbi.2017.07.002
Reed, G. M., First, M. B., Kogan, C. S., Hyman, S. E., Gureje, O., Gaebel, W., . . . Saxena, S.
(2019). Innovations and changes in the ICD-11 classification of mental, behavioural and
neurodevelopmental disorders. World Psychiatry, 18(1), 3-19.
https://doi.org/10.1002/wps.20611
Ren, Z., Yan, P., Zhu, L., Yang, H., Zhao, Y., Kirby, B. P., . . . Zhen, X. (2018).
Dihydromyricetin exerts a rapid antidepressant-like effect in association with
enhancement of BDNF expression and inhibition of neuroinflammation.
Psychopharmacology (Berl), 235(1), 233-244. https://doi.org/10.1007/s00213-017-4761-
z

154
Revicki, D. A., Travers, K., Wyrwich, K. W., Svedsäter, H., Locklear, J., Mattera, M. S., . . .
Montgomery, S. (2012). Humanistic and economic burden of generalized anxiety
disorder in North America and Europe. J Affect Disord, 140(2), 103-112.
https://doi.org/10.1016/j.jad.2011.11.014
Rhie, S. J., Jung, E. Y., & Shim, I. (2020). The role of neuroinflammation on pathogenesis of
affective disorders. J Exerc Rehabil, 16(1), 2-9. https://doi.org/10.12965/jer.2040016.008
Rodríguez, J. J., Olabarria, M., Chvatal, A., & Verkhratsky, A. (2009). Astroglia in dementia and
Alzheimer's disease. Cell Death & Differentiation, 16(3), 378-385.
https://doi.org/10.1038/cdd.2008.172
Rodríguez-Arellano, J. J., Parpura, V., Zorec, R., & Verkhratsky, A. (2016). Astrocytes in
physiological aging and Alzheimer’s disease. Neuroscience, 323, 170-182.
https://doi.org/https://doi.org/10.1016/j.neuroscience.2015.01.007
Rosenfeld, R. D., Zeni, L., Haniu, M., Talvenheimo, J., Radka, S. F., Bennett, L., . . . Welcher,
A. A. (1995). Purification and identification of brain-derived neurotrophic factor from
human serum. Protein Expr Purif, 6(4), 465-471. https://doi.org/10.1006/prep.1995.1062
Roy-Byrne, P. (2015). Treatment-refractory anxiety; definition, risk factors, and treatment
challenges. Dialogues Clin Neurosci, 17(2), 191-206.
Rudolph, U., & Knoflach, F. (2011). Beyond classical benzodiazepines: novel therapeutic
potential of GABAA receptor subtypes. Nat Rev Drug Discov, 10(9), 685-697.
https://doi.org/10.1038/nrd3502
Ruszkiewicz, J., & Albrecht, J. (2015). Changes in the mitochondrial antioxidant systems in
neurodegenerative diseases and acute brain disorders. Neurochem Int, 88, 66-72.
https://doi.org/10.1016/j.neuint.2014.12.012

155
Ryter, S. W., & Tyrrell, R. M. (2000). The heme synthesis and degradation pathways: role in
oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties. Free Radic
Biol Med, 28(2), 289-309. https://doi.org/10.1016/s0891-5849(99)00223-3
Salim, S., Chugh, G., & Asghar, M. (2012). Inflammation in anxiety. Adv Protein Chem Struct
Biol, 88, 1-25. https://doi.org/10.1016/B978-0-12-398314-5.00001-5
Saloni Dattani, H. R. a. M. R. (2021). Mental Health. In.
Salter, M. W., & Beggs, S. (2014). Sublime microglia: expanding roles for the guardians of the
CNS. Cell, 158(1), 15-24. https://doi.org/10.1016/j.cell.2014.06.008
Santello, M., Toni, N., & Volterra, A. (2019). Astrocyte function from information processing to
cognition and cognitive impairment. Nature Neuroscience 22: 154 –166.
Santos, P., Herrmann, A. P., Elisabetsky, E., & Piato, A. (2019). Anxiolytic properties of
compounds that counteract oxidative stress, neuroinflammation, and glutamatergic
dysfunction: a review. Braz J Psychiatry, 41(2), 168-178. https://doi.org/10.1590/1516-
4446-2018-0005
Sarandol, A., Sarandol, E., Eker, S. S., Erdinc, S., Vatansever, E., & Kirli, S. (2007). Major
depressive disorder is accompanied with oxidative stress: short-term antidepressant
treatment does not alter oxidative-antioxidative systems. Hum Psychopharmacol, 22(2),
67-73. https://doi.org/10.1002/hup.829
Sarter, M., Bruno, J. P., & Parikh, V. (2007). Abnormal neurotransmitter release underlying
behavioral and cognitive disorders: toward concepts of dynamic and function-specific
dysregulation. Neuropsychopharmacology, 32(7), 1452-1461.
https://doi.org/10.1038/sj.npp.1301285

156
Sartori, S. B., & Singewald, N. (2019). Novel pharmacological targets in drug development for
the treatment of anxiety and anxiety-related disorders. Pharmacol Ther, 204, 107402.
https://doi.org/10.1016/j.pharmthera.2019.107402
Sarup, A., Larsson, O. M., & Schousboe, A. (2003). GABA transporters and GABA-
transaminase as drug targets. Curr Drug Targets CNS Neurol Disord, 2(4), 269-277.
https://doi.org/10.2174/1568007033482788
Savaskan, N. E., Borchert, A., Bräuer, A. U., & Kuhn, H. (2007). Role for glutathione
peroxidase-4 in brain development and neuronal apoptosis: specific induction of enzyme
expression in reactive astrocytes following brain injury. Free Radic Biol Med, 43(2), 191-
201. https://doi.org/10.1016/j.freeradbiomed.2007.03.033
Schafer, D. P., Lehrman, E. K., Kautzman, A. G., Koyama, R., Mardinly, A. R., Yamasaki,
R., . . . Stevens, B. (2012). Microglia sculpt postnatal neural circuits in an activity and
complement-dependent manner. Neuron, 74(4), 691-705.
https://doi.org/10.1016/j.neuron.2012.03.026
Schieber, M., & Chandel, N. S. (2014). ROS function in redox signaling and oxidative stress.
Curr Biol, 24(10), R453-462. https://doi.org/10.1016/j.cub.2014.03.034
Schipper, H. M., Song, W., Tavitian, A., & Cressatti, M. (2019). The sinister face of heme
oxygenase-1 in brain aging and disease. Prog Neurobiol, 172, 40-70.
https://doi.org/10.1016/j.pneurobio.2018.06.008
Schleimer, S. B., Hinton, T., Dixon, G., & Johnston, G. A. (2004). GABA transporters GAT-1
and GAT-3 in the human dorsolateral prefrontal cortex in schizophrenia.
Neuropsychobiology, 50(3), 226-230. https://doi.org/10.1159/000079975

157
Schneider, J. L., & Cuervo, A. M. (2014). Autophagy and human disease: emerging themes.
Curr Opin Genet Dev, 26, 16-23. https://doi.org/10.1016/j.gde.2014.04.003
Schneier, F. R., Liebowitz, M. R., Abi-Dargham, A., Zea-Ponce, Y., Lin, S. H., & Laruelle, M.
(2000). Low dopamine D(2) receptor binding potential in social phobia. Am J Psychiatry,
157(3), 457-459. https://doi.org/10.1176/appi.ajp.157.3.457
Schousboe, A., Scafidi, S., Bak, L. K., Waagepetersen, H. S., & McKenna, M. C. (2014).
Glutamate metabolism in the brain focusing on astrocytes. Adv Neurobiol, 11, 13-30.
https://doi.org/10.1007/978-3-319-08894-5_2
Sen, S., Duman, R., & Sanacora, G. (2008). Serum brain-derived neurotrophic factor, depression,
and antidepressant medications: meta-analyses and implications. Biol Psychiatry, 64(6),
527-532. https://doi.org/10.1016/j.biopsych.2008.05.005
Serafini, G., Parmigiani, B., Amerio, A., Aguglia, A., Sher, L., & Amore, M. (2020). The
psychological impact of COVID-19 on the mental health in the general population. QJM:
An International Journal of Medicine, 113(8), 531-537.
https://doi.org/10.1093/qjmed/hcaa201
Serra, M., Pisu, M. G., Floris, I., & Biggio, G. (2005). Social isolation-induced changes in the
hypothalamic-pituitary-adrenal axis in the rat. Stress, 8(4), 259-264.
https://doi.org/10.1080/10253890500495244
Sferrazza, G., Brusotti, G., Zonfrillo, M., Temporini, C., Tengattini, S., Bononi, M., . . .
Pierimarchi, P. (2021). Thumberg: Phytochemistry, Pharmacology, Toxicology and
Regulatory Framework for Its Use in the European Union. Molecules, 26(4).
https://doi.org/10.3390/molecules26040903

158
Shao, Y., Yan, G., Xuan, Y., Peng, H., Huang, Q. J., Wu, R., & Xu, H. (2015). Chronic social
isolation decreases glutamate and glutamine levels and induces oxidative stress in the rat
hippocampus. Behav Brain Res, 282, 201-208. https://doi.org/10.1016/j.bbr.2015.01.005
Shefa, U., Jeong, N. Y., Song, I. O., Chung, H. J., Kim, D., Jung, J., & Huh, Y. (2019).
Mitophagy links oxidative stress conditions and neurodegenerative diseases. Neural
Regen Res, 14(5), 749-756. https://doi.org/10.4103/1673-5374.249218
Shekhar, A., Hingtgen, J. N., & DiMicco, J. A. (1990). GABA receptors in the posterior
hypothalamus regulate experimental anxiety in rats. Brain Res, 512(1), 81-88.
https://doi.org/10.1016/0006-8993(90)91173-e
Shen, Q., Lal, R., Luellen, B. A., Earnheart, J. C., Andrews, A. M., & Luscher, B. (2010).
gamma-Aminobutyric acid-type A receptor deficits cause hypothalamic-pituitary-adrenal
axis hyperactivity and antidepressant drug sensitivity reminiscent of melancholic forms
of depression. Biol Psychiatry, 68(6), 512-520.
https://doi.org/10.1016/j.biopsych.2010.04.024
Shen, Y., Lindemeyer, A. K., Gonzalez, C., Shao, X. M., Spigelman, I., Olsen, R. W., & Liang,
J. (2012). Dihydromyricetin as a novel anti-alcohol intoxication medication. J Neurosci,
32(1), 390-401. https://doi.org/10.1523/JNEUROSCI.4639-11.2012
Silva, J., Shao, A., Shen, Y., Davies, D., Olsen, R., Holschneider, D., . . . and Liang, J. (2020).
Modulation of Hippocampal GABAergic Neurotransmission and Gephyrin Levels by
Dihydromyricetin Improves Anxiety. Front. Pharmacol.,
https://doi.org/10.3389/fphar.2020.01008.
Silva, J., Shao, A. S., Shen, Y., Davies, D. L., Olsen, R. W., Holschneider, D. P., . . . Liang, J.
(2020). Modulation of Hippocampal GABAergic Neurotransmission and Gephyrin

159
Levels by Dihydromyricetin Improves Anxiety. Front Pharmacol, 11, 1008.
https://doi.org/10.3389/fphar.2020.01008
Silva, J., Yu, X., Moradian, R., Folk, C., Spatz, M. H., Kim, P., . . . Liang, J. (2020).
Dihydromyricetin Protects the Liver via Changes in Lipid Metabolism and Enhanced
Ethanol Metabolism. Alcohol Clin Exp Res, 44(5), 1046-1060.
https://doi.org/10.1111/acer.14326
Silva-Peña, D., Rivera, P., Alén, F., Vargas, A., Rubio, L., García-Marchena, N., . . . Suárez, J.
(2019). Oleoylethanolamide Modulates BDNF-ERK Signaling and Neurogenesis in the
Hippocampi of Rats Exposed to Δ. Front Mol Neurosci, 12, 96.
https://doi.org/10.3389/fnmol.2019.00096
Smoller, J. W. (2016). The Genetics of Stress-Related Disorders: PTSD, Depression, and
Anxiety Disorders. Neuropsychopharmacology, 41(1), 297-319.
https://doi.org/10.1038/npp.2015.266
Sofroniew, M. V. (2009). Molecular dissection of reactive astrogliosis and glial scar formation.
Trends Neurosci, 32(12), 638-647. https://doi.org/10.1016/j.tins.2009.08.002
Sofroniew, M. V. (2014). Astrogliosis. Cold Spring Harb Perspect Biol, 7(2), a020420.
https://doi.org/10.1101/cshperspect.a020420
Sofroniew, M. V., & Vinters, H. V. (2010). Astrocytes: biology and pathology. Acta
Neuropathol, 119(1), 7-35. https://doi.org/10.1007/s00401-009-0619-8
Song, Q., Liu, L., Yu, J., Zhang, J., Xu, M., Sun, L., . . . Meng, G. (2017). Dihydromyricetin
attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of
oxidative stress. Eur J Pharmacol, 807, 159-167.
https://doi.org/10.1016/j.ejphar.2017.04.014

160
Spampinato, S. F., Bortolotto, V., Canonico, P. L., Sortino, M. A., & Grilli, M. (2019).
Astrocyte-Derived Paracrine Signals: Relevance for Neurogenic Niche Regulation and
Blood-Brain Barrier Integrity. Front Pharmacol, 10, 1346.
https://doi.org/10.3389/fphar.2019.01346
Stein, D. J., Vasconcelos, M. F., Albrechet-Souza, L., Ceresér, K. M. M., & de Almeida, R. M.
M. (2017). Microglial Over-Activation by Social Defeat Stress Contributes to Anxiety-
and Depressive-Like Behaviors. Front Behav Neurosci, 11, 207.
https://doi.org/10.3389/fnbeh.2017.00207
Storz, P., Döppler, H., & Toker, A. (2005). Protein kinase D mediates mitochondrion-to-nucleus
signaling and detoxification from mitochondrial reactive oxygen species. Mol Cell Biol,
25(19), 8520-8530. https://doi.org/10.1128/MCB.25.19.8520-8530.2005
Ströhle, A., Gensichen, J., & Domschke, K. (2018). The Diagnosis and Treatment of Anxiety
Disorders. Dtsch Arztebl Int, 155(37), 611-620. https://doi.org/10.3238/arztebl.2018.0611
Stübner, S., Grohmann, R., Greil, W., Zhang, X., Müller-Oerlinghausen, B., Bleich, S., . . .
Neyazi, A. (2018). Suicidal Ideation and Suicidal Behavior as Rare Adverse Events of
Antidepressant Medication: Current Report from the AMSP Multicenter Drug Safety
Surveillance Project. Int J Neuropsychopharmacol, 21(9), 814-821.
https://doi.org/10.1093/ijnp/pyy048
Suliman, S., Hemmings, S. M., & Seedat, S. (2013). Brain-Derived Neurotrophic Factor (BDNF)
protein levels in anxiety disorders: systematic review and meta-regression analysis. Front
Integr Neurosci, 7, 55. https://doi.org/10.3389/fnint.2013.00055

161
Szepesi, Z., Manouchehrian, O., Bachiller, S., & Deierborg, T. (2018). Bidirectional Microglia-
Neuron Communication in Health and Disease. Front Cell Neurosci, 12, 323.
https://doi.org/10.3389/fncel.2018.00323
Tang, N., Ma, J., Wang, K. S., Mi, C., Lv, Y., Piao, L. X., . . . Jin, X. (2016). Dihydromyricetin
suppresses TNF-α-induced NF-κB activation and target gene expression. Mol Cell
Biochem, 422(1-2), 11-20. https://doi.org/10.1007/s11010-016-2799-6
Tasan, R. O., Bukovac, A., Peterschmitt, Y. N., Sartori, S. B., Landgraf, R., Singewald, N., &
Sperk, G. (2011). Altered GABA transmission in a mouse model of increased trait
anxiety. Neuroscience, 183, 71-80. https://doi.org/10.1016/j.neuroscience.2011.03.051
Taylor, S., Abramowitz, J. S., & McKay, D. (2012). Non-adherence and non-response in the
treatment of anxiety disorders. J Anxiety Disord, 26(5), 583-589.
https://doi.org/10.1016/j.janxdis.2012.02.010
Terry, R., Masliah, E., Salmon, D., Butters, N., DeTeresa, R., Hill, R., . . . Katzman, R. (1991).
Physical Basis of Cognitive Alterations in Alzheimer's Disease: Synapse loss is the major
correlate of cognitive impairment. Ann Neurol. 30:572-580.
Tezcan, E., Atmaca, M., Kuloglu, M., & Ustundag, B. (2003). Free radicals in patients with post-
traumatic stress disorder. Eur Arch Psychiatry Clin Neurosci, 253(2), 89-91.
https://doi.org/10.1007/s00406-003-0413-x
Thompson, C. (2002). Onset of action of antidepressants: results of different analyses. Hum
Psychopharmacol, 17 Suppl 1, S27-32. https://doi.org/10.1002/hup.386
Todorović, N., Tomanović, N., Gass, P., & Filipović, D. (2016). Olanzapine modulation of
hepatic oxidative stress and inflammation in socially isolated rats. Eur J Pharm Sci, 81,
94-102. https://doi.org/10.1016/j.ejps.2015.10.010

162
Tomoda, T., Yang, K., & Sawa, A. (2020). Neuronal Autophagy in Synaptic Functions and
Psychiatric Disorders. Biol Psychiatry, 87(9), 787-796.
https://doi.org/10.1016/j.biopsych.2019.07.018
Tong, L., Gong, Y., Wang, P., Hu, W., Wang, J., Chen, Z., . . . Huang, C. (2017). Microglia Loss
Contributes to the Development of Major Depression Induced by Different Types of
Chronic Stresses. Neurochem Res, 42(10), 2698-2711. https://doi.org/10.1007/s11064-
017-2270-4
Tong, L., Li, M. D., Nie, P. Y., Chen, Y., Chen, Y. L., & Ji, L. L. (2021). miR-132
downregulation alleviates behavioral impairment of rats exposed to single prolonged
stress, reduces the level of apoptosis in PFC, and upregulates the expression of MeCP2
and BDNF. Neurobiol Stress, 14, 100311. https://doi.org/10.1016/j.ynstr.2021.100311
Torres-Bondia, F., de Batlle, J., Galván, L., Buti, M., Barbé, F., & Piñol-Ripoll, G. (2020).
Trends in the consumption rates of benzodiazepines and benzodiazepine-related drugs in
the health region of Lleida from 2002 to 2015. BMC Public Health, 20(1), 818.
https://doi.org/10.1186/s12889-020-08984-z
Twenge, J. M., & Joiner, T. E. (2020). U.S. Census Bureau-assessed prevalence of anxiety and
depressive symptoms in 2019 and during the 2020 COVID-19 pandemic. Depress
Anxiety. https://doi.org/10.1002/da.23077
Tye, K. M., Mirzabekov, J. J., Warden, M. R., Ferenczi, E. A., Tsai, H. C., Finkelstein, J., . . .
Deisseroth, K. (2013). Dopamine neurons modulate neural encoding and expression of
depression-related behaviour. Nature, 493(7433), 537-541.
https://doi.org/10.1038/nature11740

163
Tynan, R. J., Naicker, S., Hinwood, M., Nalivaiko, E., Buller, K. M., Pow, D. V., . . . Walker, F.
R. (2010). Chronic stress alters the density and morphology of microglia in a subset of
stress-responsive brain regions. Brain Behav Immun, 24(7), 1058-1068.
https://doi.org/10.1016/j.bbi.2010.02.001
Verkhratsky, A., & Nedergaard, M. (2018). Physiology of astroglia. Physiological reviews,
98(1), 239-389.
Vieira, M. M., Ferreira, T. B., Pacheco, P. A., Barros, P. O., Almeida, C. R., Araújo-Lima, C.
F., . . . Bento, C. A. (2010). Enhanced Th17 phenotype in individuals with generalized
anxiety disorder. J Neuroimmunol, 229(1-2), 212-218.
https://doi.org/10.1016/j.jneuroim.2010.07.018
Vogelzangs, N., Beekman, A. T., de Jonge, P., & Penninx, B. W. (2013). Anxiety disorders and
inflammation in a large adult cohort. Transl Psychiatry, 3, e249.
https://doi.org/10.1038/tp.2013.27
Volterra, A., & Meldolesi, J. (2005). Astrocytes, from brain glue to communication elements: the
revolution continues. Nat Rev Neurosci, 6(8), 626-640. https://doi.org/10.1038/nrn1722
von Bernhardi, R., Heredia, F., Salgado, N., & Muñoz, P. (2016). Microglia Function in the
Normal Brain. Adv Exp Med Biol, 949, 67-92. https://doi.org/10.1007/978-3-319-40764-
7_4
Wake, H., Moorhouse, A. J., Miyamoto, A., & Nabekura, J. (2013). Microglia: actively
surveying and shaping neuronal circuit structure and function. Trends Neurosci, 36(4),
209-217. https://doi.org/10.1016/j.tins.2012.11.007

164
Walker, K. A., Ficek, B. N., & Westbrook, R. (2019). Understanding the Role of Systemic
Inflammation in Alzheimer's Disease. ACS Chem Neurosci, 10(8), 3340-3342.
https://doi.org/10.1021/acschemneuro.9b00333
Walter, H. J., Bukstein, O. G., Abright, A. R., Keable, H., Ramtekkar, U., Ripperger-Suhler, J.,
& Rockhill, C. (2020). Clinical Practice Guideline for the Assessment and Treatment of
Children and Adolescents With Anxiety Disorders. J Am Acad Child Adolesc Psychiatry,
59(10), 1107-1124. https://doi.org/10.1016/j.jaac.2020.05.005
Wang, M., Bi, Y., Zeng, S., Liu, Y., Shao, M., Liu, K., . . . Lv, Z. (2019). Modified Xiaoyao San
ameliorates depressive-like behaviors by triggering autophagosome formation to alleviate
neuronal apoptosis. Biomed Pharmacother, 111, 1057-1065.
https://doi.org/10.1016/j.biopha.2018.12.141
Wang, S. M., Lim, S. W., Wang, Y. H., Lin, H. Y., Lai, M. D., Ko, C. Y., & Wang, J. M. (2018).
Astrocytic CCAAT/Enhancer-binding protein delta contributes to reactive oxygen species
formation in neuroinflammation. Redox Biol, 16, 104-112.
https://doi.org/10.1016/j.redox.2018.02.011
Wang, T., Qin, L., Liu, B., Liu, Y., Wilson, B., Eling, T. E., . . . Hong, J. S. (2004). Role of
reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia. J
Neurochem, 88(4), 939-947. https://doi.org/10.1046/j.1471-4159.2003.02242.x
Wang, Y., Shi, L., Que, J., Lu, Q., Liu, L., Lu, Z., . . . Shi, J. (2021). The impact of quarantine on
mental health status among general population in China during the COVID-19 pandemic.
Molecular Psychiatry. https://doi.org/10.1038/s41380-021-01019-y
Wang, Y., Wang, W., & Qiu, E. (2017). Protection of oxidative stress induced apoptosis in
osteosarcoma cells by dihydromyricetin through down-regulation of caspase activation

165
and up-regulation of BcL-2. Saudi J Biol Sci, 24(4), 837-842.
https://doi.org/10.1016/j.sjbs.2016.12.004
Wang, Y. L., Han, Q. Q., Gong, W. Q., Pan, D. H., Wang, L. Z., Hu, W., . . . Liu, Q. (2018).
Microglial activation mediates chronic mild stress-induced depressive- and anxiety-like
behavior in adult rats. J Neuroinflammation, 15(1), 21. https://doi.org/10.1186/s12974-
018-1054-3
Wang, Z. H., Xiang, J., Liu, X., Yu, S. P., Manfredsson, F. P., Sandoval, I. M., . . . Ye, K.
(2019). Deficiency in BDNF/TrkB Neurotrophic Activity Stimulates δ-Secretase by
Upregulating C/EBPβ in Alzheimer's Disease. Cell Rep, 28(3), 655-669.e655.
https://doi.org/10.1016/j.celrep.2019.06.054
Wellman, C. L. (2001). Dendritic reorganization in pyramidal neurons in medial prefrontal
cortex after chronic corticosterone administration. J Neurobiol, 49(3), 245-253.
https://doi.org/10.1002/neu.1079
Wen, L., Jin, Y., Li, L., Sun, S., Cheng, S., Zhang, S., . . . Svenningsson, P. (2014). Exercise
prevents raphe nucleus mitochondrial overactivity in a rat depression model. Physiol
Behav, 132, 57-65. https://doi.org/10.1016/j.physbeh.2014.04.050
Wigner, P., Synowiec, E., Czarny, P., Bijak, M., Jóźwiak, P., Szemraj, J., . . . Śliwiński, T.
(2020). Effects of venlafaxine on the expression level and methylation status of genes
involved in oxidative stress in rats exposed to a chronic mild stress. J Cell Mol Med,
24(10), 5675-5694. https://doi.org/10.1111/jcmm.15231
Wilhelmsson, U., Bushong, E. A., Price, D. L., Smarr, B. L., Phung, V., Terada, M., . . . Pekny,
M. (2006). Redefining the concept of reactive astrocytes as cells that remain within their

166
unique domains upon reaction to injury. Proc Natl Acad Sci U S A, 103(46), 17513-
17518. https://doi.org/10.1073/pnas.0602841103
Wilkinson, B. L., & Landreth, G. E. (2006). The microglial NADPH oxidase complex as a
source of oxidative stress in Alzheimer's disease. J Neuroinflammation, 3, 30.
https://doi.org/10.1186/1742-2094-3-30
Wohleb, E. S., & Delpech, J. C. (2017). Dynamic cross-talk between microglia and peripheral
monocytes underlies stress-induced neuroinflammation and behavioral consequences.
Prog Neuropsychopharmacol Biol Psychiatry, 79(Pt A), 40-48.
https://doi.org/10.1016/j.pnpbp.2016.04.013
Won, E., & Kim, Y. K. (2020). Neuroinflammation-Associated Alterations of the Brain as
Potential Neural Biomarkers in Anxiety Disorders. Int J Mol Sci, 21(18).
https://doi.org/10.3390/ijms21186546
Wu, B., Lin, J., Luo, J., Han, D., Fan, M., Guo, T., . . . Yi, F. (2017). Dihydromyricetin Protects
against Diabetic Cardiomyopathy in Streptozotocin-Induced Diabetic Mice. Biomed Res
Int, 2017, 3764370. https://doi.org/10.1155/2017/3764370
Wu, F., Li, Y., Song, H., Zhang, Y., Jiang, M., Wang, F., . . . Tang, D. (2016). Preventive Effect
of Dihydromyricetin against Cisplatin-Induced Nephrotoxicity In Vitro and In Vivo. Evid
Based Complement Alternat Med, 2016, 7937385. https://doi.org/10.1155/2016/7937385
Xiang, S., Pan, W., & Kastin, A. J. (2005). Strategies to create a regenerating environment for
the injured spinal cord. Curr Pharm Des, 11(10), 1267-1277.
https://doi.org/10.2174/1381612053507431
Xie, C., Chen, Z., Zhang, C., Xu, X., Jin, J., Zhan, W., . . . Wang, J. (2016). Dihydromyricetin
ameliorates oleic acid-induced lipid accumulation in L02 and HepG2 cells by inhibiting

167
lipogenesis and oxidative stress. Life Sci, 157, 131-139.
https://doi.org/10.1016/j.lfs.2016.06.001
Xing, G., Barry, E. S., Benford, B., Grunberg, N. E., Li, H., Watson, W. D., & Sharma, P.
(2013). Impact of repeated stress on traumatic brain injury-induced mitochondrial
electron transport chain expression and behavioral responses in rats. Front Neurol, 4,
196. https://doi.org/10.3389/fneur.2013.00196
Xu, B., Huang, S., Wang, C., Zhang, H., Fang, S., & Zhang, Y. (2017). Anti‑inflammatory
effects of dihydromyricetin in a mouse model of asthma. Mol Med Rep, 15(6), 3674-
3680. https://doi.org/10.3892/mmr.2017.6428
Yang, Y., & Herrup, K. (2007). Cell division in the CNS: protective response or lethal event in
post-mitotic neurons? Biochim Biophys Acta, 1772(4), 457-466.
https://doi.org/10.1016/j.bbadis.2006.10.002
Yang, Y., Hu, Z., Du, X., Davies, H., Huo, X., & Fang, M. (2017). miR-16 and Fluoxetine Both
Reverse Autophagic and Apoptotic Change in Chronic Unpredictable Mild Stress Model
Rats. Front Neurosci, 11, 428. https://doi.org/10.3389/fnins.2017.00428
Yanguas, J., Pinazo-Henandis, S., & Tarazona-Santabalbina, F. J. (2018). The complexity of
loneliness. Acta Biomed, 89(2), 302-314. https://doi.org/10.23750/abm.v89i2.7404
Ye, Z., Yang, X., Zeng, C., Wang, Y., Shen, Z., Li, X., & Lin, D. (2020). Resilience, Social
Support, and Coping as Mediators between COVID-19-related Stressful Experiences and
Acute Stress Disorder among College Students in China. Applied Psychology: Health and
Well-Being, 12(4), 1074-1094. https://doi.org/https://doi.org/10.1111/aphw.12211

168
Yoshiyama, Y., Higuchi, M., Zhang, B., Huang, S. M., Iwata, N., Saido, T. C., . . . Lee, V. M.
(2007). Synapse loss and microglial activation precede tangles in a P301S tauopathy
mouse model. Neuron, 53(3), 337-351. https://doi.org/10.1016/j.neuron.2007.01.010
Youdim, K. A., Dobbie, M. S., Kuhnle, G., Proteggente, A. R., Abbott, N. J., & Rice-Evans, C.
(2003). Interaction between flavonoids and the blood-brain barrier: in vitro studies. J
Neurochem, 85(1), 180-192. https://doi.org/10.1046/j.1471-4159.2003.01652.x
Young, K., & Morrison, H. (2018). Quantifying Microglia Morphology from Photomicrographs
of Immunohistochemistry Prepared Tissue Using ImageJ. J Vis Exp(136).
https://doi.org/10.3791/57648
Yuan, K., Gong, Y. M., Liu, L., Sun, Y. K., Tian, S. S., Wang, Y. J., . . . Lu, L. (2021).
Prevalence of posttraumatic stress disorder after infectious disease pandemics in the
twenty-first century, including COVID-19: a meta-analysis and systematic review. Mol
Psychiatry. https://doi.org/10.1038/s41380-021-01036-x
Zhang, Y. S., Ning, Z. X., Yang, S. Z., & Wu, H. (2003). Antioxidation properties and
mechanism of action of dihydromyricetin from Ampelopsis grossedentata. Yao Xue Xue
Bao, 38(4), 241-244.
Zheng, S., Han, F., Shi, Y., Wen, L., & Han, D. (2017). Single-Prolonged-Stress-Induced
Changes in Autophagy-Related Proteins Beclin-1, LC3, and p62 in the Medial Prefrontal
Cortex of Rats with Post-traumatic Stress Disorder. J Mol Neurosci, 62(1), 43-54.
https://doi.org/10.1007/s12031-017-0909-x
Zhou, Q., Gu, Y., Lang, H., Wang, X., Chen, K., Gong, X., . . . Mi, M. (2017). Dihydromyricetin
prevents obesity-induced slow-twitch-fiber reduction partially via FLCN/FNIP1/AMPK

169
pathway. Biochim Biophys Acta Mol Basis Dis, 1863(6), 1282-1291.
https://doi.org/10.1016/j.bbadis.2017.03.019
Zhu, G., Sun, X., Yang, Y., Du, Y., Lin, Y., Xiang, J., & Zhou, N. (2019). Reduction of BDNF
results in GABAergic neuroplasticity dysfunction and contributes to late-life anxiety
disorder. Behav Neurosci, 133(2), 212-224. https://doi.org/10.1037/bne0000301
Zlatković, J., Bernardi, R. E., & Filipović, D. (2014). Protective effect of Hsp70i against chronic
social isolation stress in the rat hippocampus. J Neural Transm (Vienna), 121(1), 3-14.
https://doi.org/10.1007/s00702-013-1066-1
Zlatković, J., & Filipović, D. (2013). Chronic social isolation induces NF-κB activation and
upregulation of iNOS protein expression in rat prefrontal cortex. Neurochem Int, 63(3),
172-179. https://doi.org/10.1016/j.neuint.2013.06.002
Zlatković, J., Todorović, N., Bošković, M., Pajović, S. B., Demajo, M., & Filipović, D. (2014).
Different susceptibility of prefrontal cortex and hippocampus to oxidative stress
following chronic social isolation stress. Mol Cell Biochem, 393(1-2), 43-57.
https://doi.org/10.1007/s11010-014-2045-z

Abstract (if available)

Abstract Anxiety disorders affect over 20% of the US population and 3.8% worldwide. Anxiety disorders are considered the most prevalent mental disease and the sixth leading cause of disability. They dramatically impact an individual’s quality of life, work performance, and well-being. Despite the availability of several anxiolytic medications, many patients are struggling with treatment resistance, disease relapse, or debilitating adverse effects. Regardless of the distinctive difference in clinical presentation among the various types of anxiety disorders, they share various pathogenesis pathways, including disruption in GABAergic neurotransmission, neuroinflammation, oxidative stress, autophagy impairment, and BDNF reduction. Dihydromyricetin (DHM), the major bioactive flavonoid extracted from Hovenia dulcis, exhibits anti-anxiety effects and enhances cognitive and memory properties. Even though DHM's effect in modulating anxiety behaviors and enhancing cognition was previously reported in multiple disease models, a lack of thorough understanding of DHM's anxiolytics and enhancing cognition/memory, the mechanisms remain to be elucidated. Using social isolation-induced stress in C57BL/6J mouse model, I found that oral administration of DHM (2 mg/kg) restores GABAergic function by up-regulating gephyrin levels. DHM ameliorates neuroinflammation by inhibiting the nuclear factor kappa B (NFκB) pathway activation, thereby reducing the production of proinflammatory cytokines and the overactivation of microglia and astrocytes. Moreover, DHM modulates alterations in mitochondrial function by enhancing the performance of multiple mitochondrial antioxidative enzymes, including SOD2, HO-1, PRDX3, and GPX4, thereby reducing oxidative stress. DHM improves the homeostasis of synaptic plasticity not only by providing a neuroprotective effect and maintaining the viability of microglia and astrocytes but also by reversing the stress impact on neuronal autophagy and the BNDF-TRkB signaling pathway. My dissertation work collectively supports DHM's use as a novel therapy to treat anxiety disorders and improve cognition and memory.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Al Omran, Alzahra (author)

Core Title The therapeutic effects of dihydromyricetin (DHM) on anxiety disorders

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Clinical and Experimental Therapeutics

Degree Conferral Date 2022-08

Publication Date 07/09/2022

Defense Date 06/06/2022

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

Tag anxiety disorders,DHM,dihydromyricetin,mitochondria,neuroinflammation,OAI-PMH Harvest,oxidative stress

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Liang , Jing (committee chair), Cadenas, Enrique (committee member), Davies , Daryl L (committee member)

Creator Email aalomran@usc.edu,ajalomran@outlook.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111371068

Unique identifier UC111371068

Legacy Identifier etd-AlOmranAlz-10822

Document Type Dissertation

Format application/pdf (imt)

Rights Al Omran, Alzahra

Type texts

Source 20220713-usctheses-batch-952 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu