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Memory abnormalities in Alzheimer's disease and anxiety models
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Memory abnormalities in Alzheimer's disease and anxiety models
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Content
MEMORY ABNORMALITIES IN ALZHEIMER‟S DISEASE AND ANXIETY
MODELS
by
Chanpreet Singh
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 PHILOSOPHY
(NEUROSCIENCE)
May 2010
Copyright 2010 Chanpreet Singh
ii
DEDICATION
To all those scientists, for whom finding THE truth was more important than any
personal comfort, gain or fame, for our lives are too meager as compared to the truth.
iii
ACKNOWLEDGEMENTS
Sir Issac Newton famously uttered the lines (originally attributed to Bernard of Chartes) –
“Dwarfs standing on the shoulders of giants” – so apropos to the scientific community. I
would like to thank those giants in the way they have contributed and influenced science
over the past several centuries. In my personal experiences, I would first like to thank my
advisor Dr. Richard F. Thompson, who has provided me with a lot of opportunities to
hone myself as a scientist. Working under his guidance was the best research experience
of my life so far. I would also like to acknowledge my committee members Dr. Roberta
Brinton, Dr. Michel Baudry, Dr. Larry Swanson, and Dr. Stephen Madigan for their
support, comments, and critiques of my research and this dissertation. In particular, I
would like to thank my collaborator Dr. Brinton who provided me with the opportunity to
work on allopregnanolone project. She was the brain behind the development of
allopregnanolone as a neurogenic therapeutic for Alzheimer‟s disease. I would like to
thank her for providing me with a lot of intellectual, scienitific and financial support
during my stay at USC. I would also like to thank people from Brinton lab, in particular
Dr. Jun Ming Wang and Dr. Ronald Irwin, for helping me a lot in allopregnanolone
project. In addition, I would like to thank my other collaborators – Jean Shih lab, Finch
lab and Longo lab in helping with so many diverse projects which helped me in learning
a lot during my graduate school. I would also like to thank Dr. Jean Shih for a wonderful
collaboration on monoamine oxidase knockout mice project. Further, I would like to
extend my thanks to Judith Thompson and Michael for all their help during my stay in
this lab. I would also like to thank my past and present lab mates – Dr. Michael Foy, Dr.
iv
Ka Hung lee, Dr. Narawut Pakaprot, Young Kyoung Kim and Soyun Kim for their help
in the lab. Finally, I would like to acknowledge the neuroscience graduate forum (NGF)
for making graduate school an enjoyable experience and teaching me a lot outside of my
research focus which will benefit me in my future endeavors.
v
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
CHAPTER ONE Introduction 1
1. Alzheimer‟s disease
What is Alzheimer‟s disease? 1
Symptoms of Alzheimer‟s disease 2
How is Alzheimer‟s disease diagnosed? 3
Risk factors and causes for Alzheimer‟s disease 4
Classic neuropathology of Alzheimer‟s disease 5
Current therapeutics for Alzheimer‟s disease 6
Neural stem cells as a possible therapeutics for AD 7
2. Adult neurogenesis
Neurogenic niches in an adult brain 8
Regulation of adult neural stem cells and neural progenitors 9
Implications of Adult neurgenesis in learning and memory 12
Status of adult neurogenesis in neurodegenerative diseases 16
3. Allopregnanolone 17
4. Eyeblink conditioning
Neural circuitry of eyeblink conditioning 21
Adult neurogenesis and eyeblink conditioning 27
Alzheimer‟s disease and eyeblink conditioning 27
5. Triple-transgenic Alzheimer‟s disease mice 30
6. Monoamine oxidase
What are monoamines? 32
MAO A and MAO B mutant mice 34
Absence of MAO and human disorders 37
7. Fear conditioning 38
Neural circuitry of fear conditioning 39
Fear conditioning and stress 42
Fear and anxiety 44
vi
CHAPTER TWO Allopregnanolone Reverses the Neurogenic and Cognitive
Deficits in Mouse Model of Alzheimer‟s disease
Chapter two abstract 47
Introduction 48
Methods 50
Results 59
Discussion 76
CHAPTER THREE Allopregnanolone Induced Neurogenesis Enhances the
Learning and Memory of Adult Triple Transgenic Alzheimer‟s disease Mice
Chapter three abstract 83
Introduction 84
Methods 86
Results 91
Discussion 104
CHAPTER FOUR Effect of Allopregnanolone on Delay Eyeblink
Conditioning in Mice
Chapter four abstract 108
Introduction 109
Methods 111
Results 114
Discussion 120
CHAPTER FIVE Absence of Monoamine Oxidase A and B leads to
Enhanced Emotional and Motor Learning
Chapter five abstract 123
Introduction 124
Methods 126
Results 131
Discussion 140
CHAPTER SIX Conclusions and Future Directions 143
REFERENCES 150
vii
LIST OF TABLES
Table 1: Ongoing Clinical Trials for Treating Alzheimer‟s disease. 7
Table 2: Correlation between cognitive function and hippocampal neurogenesis. 13
Table 3: Effects of combined mutations in APP, PS and tau on neurogenesis in 17
AD models.
viii
LIST OF FIGURES
Figure 1: A hypothetical model for neural circuitry of trace eyeblink 26
conditioning.
Figure 2: Neural progenitor proliferation is deficient in 3-month-old male
3xTgAD mice. 61
Figure 3: APα concentration in plasma and cortex. 63
Figure 4: APα reversed the neurogenic deficits in dentate gyrus of 3-month-old
3xTgAD mice. 65
Figure 5: APα increased the expression of cell proliferating markers. 67
Figure 6: Phenotypic characterization of the BrdU-positive cells in mouse
dentate gyrus. 69
Figure 7: APα reversed the learning and memory deficits of 3xTgAD mice. 72
Figure 8: APα-induced neurogenesis is specific to APα and not induced by
trace eyeblink conditioning training. 74
Figure 9: APα reversal of memory deficit is correlated with neurogenesis in
3xTgAD mice. 76
Figure 10: Experimental design for the aging study. 93
Figure 11: AP reversed the learning and neurogenic deficits in 6-month-old
3xTgAD mice. 96
Figure 12: AP reversed the learning and neurogenic deficits in 9-month-old
3xTgAD mice. 99
Figure 13: AP showed no significant effect in both 12-month-old non-Tg and
3xTgAD mice. 102
Figure 14: AP reversed the learning and neurogenic deficits in 15-month-old
non-Tg mice. 104
Figure 15: Experimental design for delay conditioning. 116
ix
Figure 16: APα enhanced the learning of both 3xTgAD and non-Tg mice in
delay eyeblink conditioning. 119
Figure 17: Crossing behavior of wild-type and MAO A/B KO mice. 133
Figure 18: MAO A/B KO mice showed enhanced freezing during training and
Context test. 135
Figure 19: MAO A/B KO mice showed less exploration in a novel chamber and
Higher freezing in response to the tone. 137
Figure 20: Delay eyeblink conditioning in MAO A/B KO and wild-type mice. 139
x
ABSTRACT
This dissertation seeks to investigate the memory abnormalities that result from
Alzheimer‟s disease and anxiety disorders. In particular, this dissertation will focus on
investigating if neurogenic deficits in the hippocampus contribute to the etiology of
Alzheimer‟s disease by studying a hippocampal-dependent associative behavior test.
Additionally, we will study the potential of allopregnanolone(APα), a metabolite of
progesterone, to increase neurogenesis in the hippocampus of mice and to determine the
functional significance of these new neurons. Our data has demonstrated that APα
reverses the neurogenic and cognitive deficits of Alzheimer‟s disease mice to levels of
age-matched normal mice. This study suggests that APα could serve as a regenerative
therapeutic to prevent or delay neurogenic and cognitive deficits associated with mild
cognitive impairment and Alzheimer‟s disease. As a second part of this thesis, we have
investigated memory abnormalities in a mouse model of anxiety. This mouse model does
not have any monoamine oxidase A and B and hence, has significantly elevated levels of
monoaminergic neurotransmitters. Previous study has shown that MAO A/B KO mice
have anxiety-like behavior. As a further characterization of these mice, we have found
that there is a significant enhancement of emotional memories in these mutants and point
to these mice as an interesting animal model to study the role of monoamines in fear-
related behaviors and post-traumatic stress disorder. Furthermore, MAO A/B KO mice
have shown enhanced level of learning in cerebellar-dependent delay eyeblink
conditioning, further shedding light on the possible role of monoaminergic afferents in
the modulation of motor learning in the cerebellum.
1
CHAPTER ONE
Introduction
Mammalian nervous system is the result of millions of years of evolution. The
complexity that we observe while studying the nervous system is immense. This
complexity is optimized by a tightly regulated cellular and molecular system. Any
deviation from this regulation resulting from random DNA mutations, genetic
predisposition, environmental factors, and cellular toxicity over time leads to abnormal
manifestation in the form of neurological, psychiatric and neurodegenerative disorders.
This thesis seeks to understand some aspects of two such instances – Alzheimer‟s disease
and anxiety.
1. Alzheimer’s Disease
What is Alzheimer’s disease?
Dementia is characterized by loss of or decline in memory and other cognitive abilities. It
is caused by various diseases and conditions that result in damaged brain cells. The most
common type of dementia that accounts for 60 to 80 percent of cases is Alzheimer‟s
disease (AD) (Alzheimer‟s Association 2009). Currently, the Alzheimer‟s disease
Association estimates that approximately 4.5 million Americans have AD, a number that
could quadruple in the next 4 decades (http://www.alz.org). In AD, as in other types of
dementia, increasing numbers of nerve cells deteriorate and die. A healthy brain has 100
billion nerve cells, or neurons, with long branching extensions connected at 100 trillion
2
points. At these connections, called synapses, information flows in tiny chemical pulses
released by one neuron and taken up by the receiving cell. Different strengths and
patterns of signals move constantly through the brain‟s circuits, creating the cellular basis
of memories, thoughts and skills.
In AD, information transfer at the synapses begins to fail, the number of synapses
declines and eventually cells die. Brains with advanced AD show dramatic shrinkage
from cell loss and widespread debris from dead and dying neurons.
Symptoms of Alzheimer’s disease
Most people‟s memory declines a little with age, so the line between normal age-related
forgetfulness and earliest signs of AD can be fine – so fine that a category of „mild
cognitive impairment‟, or MCI, has been created, in part to avoid diagnosing AD in
people with more benign memory impairments (Ganguly et al., 2001). However, many
people with MCI progress to AD (70–80%). Typically, AD shows itself as a gradual loss
of episodic memory (for instance, forgetting that a conversation took place the day
before). This is more apparent to others than to the patient. But AD can also present as
word-finding difficulties, getting lost in familiar neighborhoods, or more complex
behavioral changes, sometimes brought on suddenly by a change in environment (such as
hospitalization). The most common symptom pattern begins with gradually worsening
difficulty in remembering new information because disruption of the brain cells usually
begins in regions involved in forming new memories. As damage spreads, individuals
also experience confusion, disorganized thinking, impaired judgement, trouble expressing
3
themselves and disorientation to time, space and location, which may lead to unsafe
wandering and socially inappropriate behavior. In advanced AD, people need help with
bathing, dressing, using the bathroom, eating and other daily activities. Those in the final
stages of the disease lose their ability to communicate, fail to recognize loved ones and
become bed-bound and reliant on 24/7 care. Alzheimer‟s disease is ultimately fatal.
How is Alzheimer’s disease diagnosed?
Diagnosing AD with 100% certainty requires a detailed post-mortem microscopic
examination of the brain. But nowadays, AD can be diagnosed with more than 95%
accuracy in living patients by using a combination of tools. These including taking a
careful history from patients and their families, and assessing cognitive function by
neuropsychological tests (Albert et al., 2001). Other causes of dementia must be ruled
out, such as low thyroid function, vitamin deficiencies, infections, cancer and depression.
It‟s also crucial to differentiate AD from other neurodegenerative dementias, including
frontotemporal dementia, Lewy-body dementia and Creutzfeldt-Jakob disease. Brain
imaging (Kepe et al., 2006) and tests of cerebrospinal fluid (CSF) (for review see
Blennow et al., 2010) can help to distinguish AD from these conditions. Patients with AD
typically show shrinkage of brain regions involved in learning and memory on magnetic
resonance images (Devanand et al., 2007), as well as decreased glucose metabolism
(Nordberg et al., 2010); and increased uptake of radioligands that detect abnormal protein
deposits (amyloid) on positron emission tomography scans (for review see Rabinovici
4
and Jagust 2009). CSF abnormalities include low levels of amyloid-β (Aβ) peptides and
increased levels of the protein tau (for review see Mattsson et al., 2009).
Risk Factors and causes for Alzheimer’s disease
Although the causes of AD are not yet known, most experts agree that AD, like other
common chronic conditions, probably develops as a result of multiple factors rather than
a single cause. The greatest risk factor for AD is advancing age. Most Americans with
AD are aged 65 or older, although individual younger than age 65 can also develop the
disease. When AD or other dementia is recognized in a person under age 65, these
conditions are referred to as “younger-onset” or “early-onset” Alzheimer‟s or “younger-
onset” or “early-onset” dementia. A small percentage of AD cases, probably less than
5%, is caused by rare genetic variations found in a small number of families worldwide.
These patients have inherited autosomal dominant mutations in genes whose protein
products – Amyloid Precursor Protein (APP), Presenilin 1(PS1) or PS2 – are involved in
the production of Aβ peptides. In these inherited forms of Alzheimer‟s, the disease tends
to develop before age 65, sometimes in individuals as young as 30 (Campion et al.,
1996).
A genetic factor in late-onset AD (Alzheimer‟s disease developing at age 65 or older) is
apolipoprotein E-ε4 (APOE-ε4) (Mahley et al., 2006). APOE-ε4 is one of the three
common forms of the APOE gene, which provides the blueprint for a protein that carries
cholesterol in the bloodstream. Everyone inherits one form of the APOE gene from each
of his or her parents. Those who inherit one APOE-ε4 gene have increased risk of
5
developing AD. Those who inherit two APOE-ε4 genes have an even higher risk.
However, inheriting one or two copies of the gene does not guarantee that the individual
will develop AD. Certain variants of genes encoding another lipid carrier, clusterin
(apoJ), the intracellular trafficking protein PICALM, or complement component (3b/4b)
receptor 1 also modulate AD risk, possibly by affecting Aβ levels, synaptic functions or
inflammation (Van et al., 2009).
The risk of AD may be increased by other non-genetic factors such as a low level of
education, severe head injury, cerebrovascular disease, diabetes and obesity. It is likely
that AD-predisposing genes interact with other disease genes and environmental factors.
Classic neuropathology of Alzheimer’s disease
Neuropathologically, AD is characterized by brain region-specific deposition of β-
amyloid protein (Aβ) which creates senile plaques, hyperphosphorylation of the
cytoskeletal protein tau that forms lesions called neurofibrillary tangles (NFTs) and
neuropil threads, glial activation which is associated with inflammatory responses, and
both synaptic and neuronal loss (Braak and Braak, 1990; Hardy, 1997, Akiyama et al.,
2000; Hardy and Selkoe 2002; LaFerla and Oddo, 2005). Although the mechanisms of
AD pathogenesis remain to be fully understood, the leading hypothesis posits that the
disease is initiated and driven by prolonged elevation of Aβ levels (Hardy and Higgins,
1992). Aβ is a proteolytic byproduct of the metabolism of amyloid precursor protein, a
widely expressed protein with numerous functions ranging from axonal transport to gene
transcription (Turner et al., 2003). As a consequence of amyloid precursor protein
6
expression, Aβ is normally found as a soluble protein at low levels in fluids and tissues
throughout the body. Alterations in either the production or clearance of Aβ can lead to
an increase in the levels of Aβ that can lead to the development of AD (Hardy and Selkoe
2002). The accumulation of Aβ encourages its abnormal assembly into oligomeric
species that exhibit an altered structural conformation and can induce a range of
neurodegenerative effects (Haass and Selkoe, 2007). Consequently, enormous effort has
been expended on identifying factors that regulate Aβ accumulation and or affect its
neurodegenerative properties.
Current Therapeutics for Alzheimer’s disease
Currently prescribed medicine for AD fall into three groups: inhibitors of
acetylcholinesterase; an antagonist of a receptor for the neurotransmitter glutamate; and
drugs from the psychiatric toolbox to control depression and other behavioral
abnormalities. The neurotransmitter acetylcholine is depleted in AD brain (Smith and
Swash 1978), and inhibiting acetylcholinesterase, its degrading enzyme, leads to an
improvement in cholinergic neurotransmission. Excitotoxicity resulting from
overstimulation of the NMDA receptors may also contribute to AD and hence
memantine, an NMDA antagonist is used for blocking these receptors (Fleischhacker et
al., 1986). The beneficial effects of these drugs are not large and do not seem to arrest or
reverse AD. Research over the past two decades has reached to a stage where rather than
treating the symptoms we can hope to slow down the progression of AD. Several drugs
7
are under being tested in clinical trials (Table 1). More and more patients are being
enrolled in carefully controlled prospective clinical trials.
Neural stem cells as a possible therapeutics for AD
The discovery of neural stem cells (NSCs) has raised hope for a stem-cell based therapy
for AD. Contrary to other neurodegenerative disorders such as Parkinson disease,
ischemia, and spinal cord injury where a single neuronal subtypes and/or a restricted
anatomical region is affected, AD is characterized by multiple pathologies that afflict
several neuronal subtypes across multiple brain regions. This approach was successfully
tested in transgenic AD models recently (Blurton-Jones et al., 2009) and might pave way
for future studies and clinical trials.
Table 1: Ongoing Clinical Trials for Treating Alzheimer‟s disease (adapted from –
Mucke 2009)
Approach or drug Proposed mechanism of action Phase
β-Secretase inhibiton Decreases formation of Aβ from amyloid precursor
protein
II
γ-Secretase inhibition Decreases formation of Aβ from amyloid precursor
protein
II/III
Active immunization
with Aβ peptides
Generates anti-Aβ antibodies that interact with Aβ
and remove it from the brain by uncertain downstream
mechanisms
II
Passive
immunization with
anti-Aβ antibodies
The antibodies interact with Aβ and remove it from
the brain by uncertain downstream mechanisms
III
Intravenous pooled
immunoglobulins
May enhance clearance of Aβ and other harmful
proteins from the brain; may decrease harmful
inflammatory processes.
III
8
Table 1: Ongoing Clinical Trials for Treating Alzheimer‟s disease (adapted from –
Mucke 2009), Continued
Scyllo-inositol Decreases formation and stability of pathogenic
Aβ assemblies
II
Latrepirdine Prevents mitochondrial dysfunction III
Inhibiton of receptor for
advanced glycation end
products (RAGE)
Blocks stimulation of the cell-surface receptor
RAGE, which binds Aβ, decreasing Aβ levels in
the brain and preventing Aβ from activating
pathogenic pathways.
II
Stimulation of insulin
signaling
Prevents hyperglycaemia; may overcome insulin
resistance in the brain
II
Selective oestrogen-
receptor modulator
Promotes neuroprotective effects of oestrogen
without eliciting its harmful effects
II
2. Adult Neurogenesis
Neurogenic niches in an adult brain
In the developing brain, most stem cells and microenvironments are spatially shifting and
are temporally transient as the cellular and molecular programs of neurogenesis and
morphogenesis are “assembled and disassembled” (Alvarez-Buylla and Lim, 2004). In
contrast to the undulating milieu of stem cell zones in the embryonic nervous system, the
adult brain restricts neural stem cells and their proliferation to select microenvironments.
These specialized domains, the subventricular zone (SVZ) of the lateral ventricle and the
dentate gyrus subgranular zone (SGZ) of the hippocampus, retain developmental
potential throughout the life span (Alvarez-Buylla and Lim, 2004).
9
Regulation of adult neural stem cells and neural progenitors
Adult hippocampal neurogenesis is a complex process originating in the proliferation of
neural progenitor cells (NPCs) located in the subgranular zone (SGZ). A majority of
these neural progenitor cells undergo initial differentiation and migrate into the inner
granule cell layer within a week of their birth and become dentate granule cells (DGCs).
Between 1 and 4 weeks after birth, newborn DGCs undergo a long process of
morphological and physiological maturation and display characteristics distinct from
mature DGCs before they fully integrate into the neural circuits and become
indistinguishable from mature DGCs. During this time, newborn DGCs project their
axons to CA3 and grow dendrites into the molecular layer, starting to form both afferent
and efferent synapses (Hastings and Gould, 1999; Zhao et al., 2006; Toni et al., 2007,
2008; Faulkner et al., 2008). The immature DGCs start to receive GABAergic inputs
within 8 d of birth and glutamate inputs by 18 d and have a lower threshold for long-term
potentiation (LTP) induction and enhanced synaptic plasticity (Schmidt-Hieber et al.,
2004; Esposito et al., 2005; Ge et al., 2006).
Similar to SGZ neurogenesis, SVZ neurogenesis is a very complex process. SVZ
progenitors are adjacent to the ependymal cell layer of the lateral ventricles. New neurons
in the adult SGZ migrate over a short distance and become integrated into the granule cell
layer. In contrast to that, adult-born neurons in the SVZ migrate over a longer distance
through the rostral migratory stream. Similar to the new neurons in SGZ, newborn
neurons in the SVZ undergo morphological and physiological development before
10
integrating as granule neurons in the granule cell layer (GCL) and as periglomerular
neurons in the glomerular layer (GL) (Zhao et al., 2008).
Adult neurogenesis in SGZ and SVZ is sensitive to several factors that are present in the
neurogenic niche and affect the process of adult neurogenesis directly or interact
indirectly with the microenvironment of the neural stem/progenitor cells. Hippocampal
astrocytes may play an important role in SGZ neurogenesis, acting through Wnt signaling
pathway (Song et al., 2002; Lie et al., 2005). Antagonizing signaling by Noggin may
promote SVZ neurogenesis by blocking bone morphogenetic proteins (BMPs) (Lim et al.,
2000). Proliferating cells in both SGZ and SVZ are closely associated with the
vasculature indicating that factors released from the blood vessels may have a direct
impact on adult neural progenitors (Alvarez-Buylla and Lim, 2004; Palmer et al., 2000).
In fact, vascular endothelial growth factor (VEGF) promotes cell proliferation in SGZ
and SVZ and is required for increased neurogenesis in adult mice exposed to an enriched
environment (Cao et al., 2004). Neural progenitors in the SGZ and SVZ are influenced by
other neurotransmitters as well as by neural peptides (Jang et al., 2007). Growth factors
such as epidermal growth factors (EGFs) and fibroblast growth factor 2 (FGF2) are
potent factors for the maintenance of adult neural stem cells (NSCs) in vitro (Kuhn et al.,
1997). Adult neural progenitors are also regulated by a variety of other extrinsic factors
(Zhao et al., 2008). Signaling through the sonic hedgehog pathway may regulate adult
neurogenesis. The neurotrophin brain-derived neurotrophic factor (BDNF) is one of the
key positive regulators of adult neurogenesis. In addition to the canonical intracellular
11
signaling pathways downstream of the growth factors, neurotrophins, and morphogens, a
variety of other intracellular mechanisms have been implicated in the regulation of adult
neurogenesis (Zhao et al., 2008). Among these, several transcription factors have been
shown to play critical roles in postnatal neurogenesis. TLX, an orphan nuclear receptor,
and Bmi-1 are required for the maintenance of adult forebrain NSCs. Pax6 promotes
neuronal differentiation of SVZ progenitors, whereas Olig2 has an opposite effect. Adult
neurogenesis is also subject to epigenetic regulation (Hsieh and Eisch 2010). In addition,
genes involved in cell cycle regulation, DNA repair and chromosome stability are
required for the proper function of adult neural progenitors. The early processes of adult
neurogenesis might also be influenced by somatic gene insertions, as the retrotransposon
long interspersed nuclear element-1 (LINE-1) is expressed in adult hippocampal neural
progenitors in vitro (Muotri et al., 2009). In addition to all these factors discussed above,
aging and stress are two major negative regulators of SGZ neurogenesis. While the
regenerative potential of the mammalian brain is sustained throughout the life span, the
magnitude of the proliferative efficacy of neural progenitors declines with age (Kuhn et
al., 1996; Rao et al., 2005). The decline in neurogenic potential is evident as early as
middle age and is one of the early changes in the aging hippocampus (Kuhn et al., 1996).
Early neurogenic decline is most likely due to a decline in the concentration of
neurotrophic factors, such as the steroids and peptide growth factors or a concomitant
decline in receptor density or effector signaling
12
In essence, numerous extrinsic and intracellular pathways have been implicated in
regulating adult NSCs and neural progenitors.
Implications of Adult neurogenesis in learning and memory
Mammalian brain produces thousands of new cells in the hippocampus each day, most of
which will differentiate into neurons, provided that they survive (Cameron et al., 1993;
Eriksson et al., 1998; Kempermann, 2005). These new cells are more likely to survive if
they exist in the hippocampus of an animal that has learned compared to the controls with
no learning (Gould et al., 1999; Waddell and Shors, 2008). However, not all learning will
rescue these neurons from death. Nearly 10 years ago Gould and associates reported that
learning does impact neurogenesis and soon thereafter that neurogenesis might also be
involved in learning (Shors et al, 2001). Since then, many additional studies have
appeared, some of which support a relationship and others that do not. A correlation
between hippocampal neurogenesis and learning in a spatial memory task (the Morris
water maze) was observed in mice of different strains. In addition, some mutant mice
with decreased SGZ neurogenesis have impaired performance on hippocampus-
dependent learning tasks (Zhao et al., 2008). Both voluntary exercise and environmental
enrichment promotes the survival of 1- to 3-week-old immature neurons and also
improve the performance of young and aged mice in the Morris water maze, even though
a correlation between SGZ neurogenesis and performance in Morris water maze was
observed in individual aged animals in some but not in other studies. Shors and
colleagues have shown that SGZ neurogenesis plays a critical role in hippocampal
13
dependent trace eyeblink conditioning, while no correlation was detected in delay
eyeblink conditioning that depends only on the cerebellum (Shors et al., 2001). When
neurogenesis was blocked in mice, they showed a learning deficit when required to learn
to associate discontiguous events across time. In another study using hippocampal
irradiation, decrements in contextual fear conditioning (Saxe et al., 2006; Winocur et al.,
2006) were observed but no such decrements were observed in mice when a genetic
approach was used to reduce the number of neural stem cells. As a whole, multiple
studies indicate that neurogenesis is not necessary to learn simple associations between
aversive events and the context. New neurons may become more engaged as the
associations with the environment become more ambiguous. Another question that is still
unanswered is about the role of these new neurons once they become incorporated as
mature neurons into the hippocampus. The hippocampus is mostly involved in learning
and is only temporarily involved in the retention of most memories. If the hippocampus is
not required to express the memories (like contextual fear conditioning and trace eyeblink
conditioning) after a few days of learning, then the new neurons within the hippocampus
cannot be necessary for retention. This matter needs to be further studied. A summary of
the studies done to investigate the correlation between cognitive function and
hippocampal neurogenesis is given in table 2.
Table 2: Correlation between cognitive function and hippocampal neurogenesis
Neurogenes
is
Factors Tasks Correlation Reference
↓ Irradiation Water maze X Meshi et al., 2006
↓ Irradiation Water maze √ Rola et al., 2004
14
Table 2: Correlation between cognitive function and hippocampal neurogenesis,
Continued
↓ Irradiation Barnes maze √ Raber et al., 2004
↓ Irradiation Water maze X Raber et al., 2004
↓ MBD 1-/- Water maze √ Zhao et al., 2003
↓ NT-3 -/- Water maze √ Shimazu et al.,
2006
↓ Immunodeficiency Water maze √ Ziv et al., 2006
↓↑ Strain Water maze √ Kemperman and
Gage 2002
↓ Neurokinin-1-R -/- Trace/context
ual fear
conditioning
X Morcuende et al.,
2003
↓ Neurokinin-1-R -/- Water maze X Morcuende et al.,
2003
↓ Irradiation and
NSC ablation
Contextual
fear
conditioning
√ Saxe et al., 2006
↓ Irradiation and
NSC ablation
Water maze X Saxe et al., 2006
↓ Irradiation and
NSC ablation
Radial maze Inverse
correlation
Saxe et al., 2007
↓ Temozolomide Water maze
flexibility
√ Garthe et al., 2009
↓ Irradiation Radial arm
maze and
touch screen
√ Clelland et al., 2009
↑ Environment
enrichment
Water maze √ Kempermann et al.,
1997
↑ Environment
enrichment
Water maze √ Kempermann et al.,
1998
↑ Exercise Water maze √ Van Praag et al.,
1999
↑ Exercise Water maze √ Van Praag et al.,
2005
↑ Exercise Water maze √ Clark et al., 2008
↑ Exercise Contextual
fear
conditioning
X Clark et al., 2008
15
A lot of attention has been given to the role of SVZ neurogensis in odor learning. The
new cells added to the olfactory bulb (OB) undergo functional integration and this
process is thought to be important for learning and memory. Some studies have suggested
that new neurons are not required for olfactory learning, whereas others have implicated
adult-generated neurons in a number of olfaction functions. For example, olfaction has
been shown to be unaffected in Bax-knockout mice (Kim et al., 2007) and in mice
producing a neuron-specific enolase-diphtheria toxin (Imayoshi et al., 2008). On the other
hand, both mice lacking neural cell-adhesion molecule (NCAM) and mice with the brain-
derived neurotrophic factor (BDNF) Val66Met knock-in display impaired OB
neurogenesis and odor discrimination (Gheusi et al., 2000; Bath et al., 2008). Olfactory
discrimination is also impaired in aging rodents, mice heterozygous for leukemia
inhibitory factor receptor (Lifr +/-), and waved-1 mutant mice (a hypermorph of TGF-α),
in which OB neurogenesis level is reduced (Enwere et al., 2004). Finally, a correlation
has been found between the degree of OB neurogenesis and olfactory memory (Rochefort
et al., 2005; Scotto-Lomassese et al., 2003)), providing further support for the hypothesis
that adult neurogenesis plays an important role in olfaction. A very recent study, using
focal SVZ irradiation in adult mice, has shown that the continuous recruitment of adult-
generated OB neurons are not required for any of the olfactory functions except for long-
term olfactory memory, which was less robust after irradiation (Lazarini et al., 2009)
16
Status of adult neurogenesis in neurodegenerative diseases
Conflicting observations have been reported regarding the level of neurogenesis in
models of AD (Verret et al., 2007 and the references therein). Although most mouse
models of AD based on mutations in the amyloid precursor protein (APP) have shown
decreased neurogenesis, transgenic mice expressing presenilin mutations have either
increased or decreased neurogenesis. The wild-type presenilin and the soluble form of
APP have both been implicated in the function of adult neurogenesis, but the deregulated
signaling of APP and presenilin may only contribute partially to the changes in adult
neurogenesis in AD. Mouse models of Parkinson‟s disease have been generated by
overexpressing the wild-type or mutant forms of human α-synuclein, which accumulates
in neurodegenerative diseases including Parkinson‟s disease, dementia with Lewy bodies
and multiple system atrophy. Over expression of wild-type human α-synuclein
significantly decreases the survival of newborn neurons in both SGZ and SVZ, without
affecting cell proliferation, whereas expression of mutant α-synuclein primarily inhibited
cell proliferation in the SVZ (Winner et al., 2007).
Cell proliferation is increased in the SGZ of postmortem AD patients, decreased in the
SGZ and SVZ of Parkinson‟s patients, and increased in the SVZ of Huntington‟s patients.
A recent study has shown the Aβ leads to an imbalance between GABAergic and
glutamatergic transmission that leads to impairment in SGZ neurogenesis in an animal
model of AD (Sun et al., 2009). A deficit in both SGZ and SVZ neurogenesis has also
17
been observed in the most widely used animal model of AD, triple-transgenic mice
(Rodriguez et al., 2008; Rodriguez et al., 2009).
Table 3: Effects of combined mutations in APP, PS and tau on neurogenesis in AD
models
Promoters-mutations Changes in
neurogenesis
Altered neurogenesis
parameters
Reference
APP X PS Knockin-
APP
SWE
x PS1
P264L
↓ Reduced DG
stem/progenitors and
neuroblasts
Zhang et al., 2007
PrP-APP
SWE
x PS1
dE9
NC Proliferation in SGZ Verret et al., 2007
PrP-APP
SWE
x PS1
dE9
↓ Survival of new born
cells and neuronal
differentiation in
SGZ
Verret et al., 2007
PrP-APP
SWE
x PS1
dE9
↓ Proliferation and
neuroblasts in GCL at
9 months but not 5
months
Taniuchi et al.,
2007
PrP-APP
SWE
x PS1
dE9
↓ Proliferation in SGZ
but not in SVZ at 9
months
Niidome et al.,
2008
PrP-APP
SWE
x PS1
dE9
↓ Survival in SGZ at 9
months
Niidome et al.,
2008
Thy1-APP
SWE
x PS1
L166P
↓ Neuroblasts in DG Ermini et al., 2008
Thy1-APP
SWE
x PS1
L166P
↓ NPCs in DG Ermini et al., 2008
APP x PS x Tau
Thy 1.2- Tau
P301L
x
APP
SWE
x knockin
PS1
M146V
↓ Mitotic marker in
SGZ and SVZ
Rodriguez et al.,
2008; Rodriguez et
al., 2009
3. Allopregnanolone
Allopregnanolone (APα), with a steroidal chemical structure and low molecular weight of
318, is a reduced metabolite of progesterone. During fetal development, APα is
18
synthesized throughout the embryonic period, is present in multipotential progenitor cells
(Gago et al., 2004; Lauber and Lichtensteiger, 1996) as well as in neurons (Agis-Balboa
et al., 2006; Pinna et al., 2004) and reaches its highest concentration in late gestation
(Pomata et al., 2000). APα also can be generated de novo in the central nervous system
(CNS) (Nguyen et al., 2004a, b), independent of maternal supply and of the
hypothalamic-pituitary-adrenal axis.
APα produced within the multipotential cells (Gago et al., 2004; Lauber and
Lichtensteiger, 1996) also declines with age and disease. In the aged and AD brain, both
the pool of NSCs and their proliferative potential are markedly diminished (Bernardi et
al., 1998; Genazzani et al., 1998). In parallel, APα content is diminished in the brains of
AD patients as compared with age-matched controls (Marx et al., 2006; Weill-Engerer et
al., 2002).
Recent reports have shown that APα limits CNS damage, reduces loss of neural tissue
and improves functional recovery following injury (Cutler et al., 2006; Djebaili et al.,
2004, 2005; He et al., 2004a,b; Vanlandingham et al., 2006). APα with a steroidal
chemical structure, (3α-hydroxy-5α-pregnan-20-one, low molecular weight of 318.49 and
hydrophobic properties (LogP value = 4.2)) easily penetrates the blood-brain barrier. APα
induces several CNS effects including antianxiolytic, antiseizure and neurogenesis. A
region-specific expression pattern of progesterone converting enzymes P450scc, 5α
reductase and 3α hydroxysteroid dehydrogenase, in brain is evident in both hippocampus
and cortex (Baulieu and Robel, 1990; Mellon and Griffin, 2002a,b). Likewise, the
19
enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase required to convert
progesterone to its 3α metabolites, are present and functional in pluripotential progenitors
(Lauber and Lichtensteiger, 1996; Melcangi et al., 1996). Because APα is an endogenous
metabolite of progesterone, humans are exposed to APα throughout their lifetime.
Substantial toxicity and pharmacokinetic analyses were conducted in animals and Phase I
safety analyses were conducted in humans (Laxer et al., 2000) and have shown no
toxicology issues in healthy human volunteers.
Recently, it was shown that APα promoted in vitro proliferation of human and neural
progenitors and mouse hippocampal neurogenesis in vivo in a dose-dependent and
steroid-specific manner (Wang et al., 2005). The efficacy of APα as a neurogenic factor
was comparable to that induced by bFGF + heparin (Wang et al., 2005).
In the past decades, several groups have investigated the effects of APα on behavior,
particularly on learning and memory. Mayo et al. (1993, 2005) and Johansson et al.‟s
(2002) groups demonstrated amnesic effects by T-maze and Morris water maze,
respectively. However, several other groups demonstrated mnemonic effects, for
example, APα enhances the behavioral recovery as tested by Morris water maze in male
rats after traumatic brain injury (He et al., 2004a, b). Other groups have shown that APα
enhances object recognition in naturally cycling and ovariectomized, hormone-replaced
rats (Frye et al., 2007; Walf et al., 2006).
It is well documented now that APα interacts with GABA
A
R. The reaction pathways are
different in mature and immature neurons. In mature neurons, APα acts as an allosteric
20
modulator of the GABA
A
R to increase chloride influx thereby hyperpolarizing the
neuronal membrane potential and decreasing neuron excitability (Brinton, 1994; Gee,
1988). In immature neurons, the flux of chloride is opposite to that of mature neurons. In
immature neurons, the high intracellular chloride content leads to an efflux of chloride
through the GABA
A
R, depolarization of the membrane and opening of L-type voltage-
dependent Ca
++
channels (Van Den Pol, 2004)
4. Eyeblink Conditioning
Classical conditioning has been widely used as model system to study the neural
substrates of associative learning. One form of classical conditioning, eyeblink
conditioning (EBCC), has received considerable attention by the neuroscience
community. In EBCC, repeated pairings of a neutral conditioned stimulus (CS), such as a
tone, and an unconditioned stimulus (US), such as an airpuff or a mild shock on the
eyelid muscle, eventually result in the CS alone eliciting conditioned responses (CRs),
suggesting that an association between the CS and US has been learned.
Delay and trace conditioning are two different procedures that vary in timing. In delay
conditioning, the CS and US co-terminate but differ in their onset whereas in trace
conditioning, a silent period (called the trace interval) elapses between offset of the CS
and delivery of the US. Considerable evidence from animal and human research suggests
that the hippocampus and other forebrain structures play a critical role during trace
conditioning whereas the cerebellum is involved in both delay and trace conditioning
21
(Disterhoft et al., 2003; Christian and Thompson, 2003; Gerwig et al., 2007; Woodruff-
Pak and Disterhoft, 2008).
Neural circuitry of eyeblink conditioning
A substantial body of data has demonstrated that the cerebellar interpositus nucleus
ipsilateral to the conditioned eye is essential to the acquisition and maintenance of
eyeblink conditioning (Christian and Thompson, 2003). In delay eyeblink conditioning,
conditioned stimulus (CS) and unconditioned stimulus (US) information are transmitted
to the cerebellum via mossy fibers originating in the pontine nuclei and climbing fibers
originating in the inferior olive, respectively (Mauk et al., 1986; Steinmetz et al., 1986,
1987, 1989). This CS and US information converge upon (1) Purkinje cells in the
cerebellar cortex and (2) the cerebellar interpositus nucleus (Gould et al., 1993; Steinmetz
and Sengelaub, 1992; Thompson, 1986; Tracy et al., 1998). Repeated pairings of this
convergent information are hypothesized to yield robust synaptic plasticity (e.g., long-
term depression – LTD; long-term potentiation – LTP) within each cerebellar region,
resulting in learning of the contingent CS-US relationship (Hansel et al., 2001; Linden &
Connor, 1995; Nores et al., 2000; Pugh & Raman, 2006). Several mouse models
including the Purkinje cell degeneration (pcd) mouse, with functional lesion of the entire
cerebellar cortex (Chen et al., 1996) and “waggler” – a mutant mouse which lacks brain-
derived neurotrophic factor (BDNF) in cerebellar granule cells (Bao et al., 1998) have
shown that the cerebellar cortex is normally involved in delay eyeblink conditioning but
is not essential. Similar findings of impaired (but not abolished) eyeblink conditioning in
22
the delay paradigm have been shown in various mutant and transgenic mouse models
exhibiting impaired cerebellar cortical LTD including glial fibrillary acidic protein
(GFAP) (Shibuki et al., 1996) knockout mice, PTPMEG (a cytoplasmic protein-tyrosine
phosphatase expressed in Purkinje cells) (Kina et al., 2007), and δ2 glutamate receptor
(GluRδ2) (Kakegawa et al., 2008), phospholipase C β4 (PLCβ4) (Kishimoto, Hirono et
al., 2001) and metabotrophic glutamate receptor 1 (mGluR1) (Aiba et al., 1994) mutant
mice all show impairments in cerebellar cortical LTD and delay eyeblink conditioning.
Recently, Lee et al. (2009) demonstrated impaired retention but unimpaired acquisition in
calcium/calmodulin-dependent protein kinase type IV (CaMKIV) knockout mice,
behavioral effects that parallel findings of normal acquisition but unimpaired
maintenance of LTD in mice deficient in CamKIV. Furthermore, genetically modified
mice that exhibit alterations in cerebellar cortical functioning without impairing LTD are
unimpaired in delay eyeblink conditioning (Endo et al., 2009; Tanaka et al., 2008). These
findings provide substantial evidence that LTD at Purkinje cell synapses is the primary
cerebellar cortical mechanism by which normal acquisition and retention of delay
eyeblink conditioning is produced and maintained.
In trace eyeblink conditioning acquisition of the learning requires the integrity of
forebrain areas such as the hippocampus (Kim et al., 1995; Moyer et al., 1990; Solomon
et el., 1986) and prefrontal cortex (Oswald et al., 2006; Weible et al., 2000) in addition to
the cerebellum. Consistent with findings in delay eyeblink conditioning paradigms,
lesions of the cerebellar interpositus nucleus abolish trace eyeblink conditioning in
23
rabbits (Pakaprot et al., 2009; Woodruff-Pak et al., 1985) whereas lesions of the
cerebellar cortex produce only transient impairments in retention of trace eyeblink CRs
(Woodruff-Pak et al., 1985). Recent findings in humans further suggest that trace
eyeblink conditioning is not dependent on cerebellar cortex integrity, as patients with
cerebellar cortical lesions displayed comparable levels of conditioning to normal controls
in the acquisition of a forebrain-dependent trace eyeblink conditioning task (Gerwig et
al., 2008).
Mounting evidence indicates that the cerebellar cortex may be differentially engaged in
delay relative to trace eyeblink conditioning. In a recent study, rats trained in a delay
eyeblink conditioning task showed higher levels of metabolic activity in regions of the
cerebellar cortex compared to rats trained in a trace eyeblink conditioning task (Plakke et
al., 2007). Perhaps the most compelling evidence for differential engagement of
cerebellar cortical mechanisms between these tasks, however, comes from studies
comparing delay and trace eyeblink conditioning in various mutant and transgenic mouse
models. Specifically, the aforementioned GluRδ2 knockout and PLCβ4 mutant mice
(with selective deficiencies of cerebellar cortical components critical for the induction of
LTD at the parallel fiber-Purkinje cell synapse) showed robust impairments in delay
eyeblink conditioning while trace eyeblink conditioning was unimpaired (Kishimoto et
al., 2001; Kishimoto, Kawahara et al., 2001; Kishimoto, Kawahara, Fujimichi et al.,
2001). Similarly, a mouse strain with a selective knockout of Purkinje cells Scn8a sodium
channels – a condition that disrupts normal firing patterns of Purkinje cells (Raman et al.,
24
1997) – was impaired in delay but not in trace eyeblink conditioning (Woodruff-Pak et
al., 2006). All these findings suggest that cerebellar cortical integrity is important for
delay, but not for trace eyeblink conditioning. A very recent study has investigated trace
eyeblink conditioning in pcd mutant mice (Brown et al., 2009) and has shown that trace
eyeblink conditioning is unimpaired in these mice providing considerable support that the
cerebellar cortex is not important for the acquisition and maintenance of this paradigm.
While there is a possibility that these transgenic and knockout (KO) mice were unique
and have developed or utilized unique alternate brain pathways to compensate for
abnormal cerebellar cortical function, more recent studies using rats and human patients
with cerebellar cortical lesions continue to support the mouse genetic manipulation
studies and indicate that the cerebellar cortex is more critical for delay than for trace
eyeblink conditioning.
The studies with transgenic mice raise the obvious possibility that the essential
involvement of the hippocampus, medial prefrontal cortex and other forebrain regions in
trace eyeblink conditioning eliminates or supersedes the role that cerebellar cortex plays
in delay eyeblink conditioning. Weiss and Disterhoft (1996) have hypothesized that
forebrain regions act to bridge the trace interval between the CS and US in trace
conditioning (also see Weiss et al., 2006). The forebrain regions that are recruited to
„bridge the trace interval‟ project to the pontine nuclei and from there to the cerebellar
cortex and deep nuclei. These projections appear to be able to support trace conditioned
responses via input to the pontine nuclei.
25
In vivo single-neuron recording studies have demonstrated that CA1 hippocampal
pyramidal neurons in the dorsal hippocampus change before the initial appearance of
behavioral CRs during learning (McEchron and Disterhoft, 1997; Weible et al., 2006).
Studies in which animals were trained to behavioral criterion before hippocampal brain
slices were prepared have demonstrated that hippocampal pyramidal neurons from
trained animals show increases in excitability, as indicated by reduced slow postburst
afterhyperpolarization and reduced spike frequency accommodation (Disterhoft et al.,
1986; Moyer et al. 1996; Thompson et al., 1996). These excitability increases, mediated
by reductions in calcium-activated potassium currents, are localized to the output neurons
of the hippocampus and are present during the acquisition and initial consolidation of the
trace eyeblink CR (Disterhoft and Oh, 2006). In vivo single-neuron recording studies
indicate that neurons in the caudal anterior cingulate cortex change very early in trace
conditioning, perhaps mediating attention to the presentation of the paired CS and US
(Weible et al., 2003). Later in training, the sensory cortex processing the trace CS is
required for initial acquisition of the trace CR and apparently stores at least some
component of the CR for the longer term (Galvez et al., 2006, 2007). Activity in direct
projections from forebrain regions such as the caudal anterior cingulate to the lateral
pontine nuclei (Weible et al., 2007), from the sensory cortices or indirectly from the
hippocampus could prolong the effect of the CS by the continuing to stimulate the lateral
pontine nuelci after CS offset in the trace paradigm. Figure 1 shows how the forebrain
structures play a role in trace eyeblink conditioning.
26
Figure 1: The diagram illustrates that the pontine nuclei mediate the CS afferent information to
the cerebellar cortex and deep nuclei. The CS information in trace eyeblink is preprocessed in
forebrain regions including the sensory cortices, the hippocampus and the prefrontal cortex,
which act as the bridge between the CS and the US during the stimulus-free trace interval, before
projecting the CS to the pontine nuclei. During delay conditioning, the CS information is
projected directly into the pontine nuclei via sensory system collaterals. Both trace and delay
conditioning utilize similar cerebellar circuits to mediate the CR output. The above discussion
indicates that delay conditioning requires an LTD-like process involving the cerebellar cortex.
However, during trace conditioning, forebrain inputs via the pontine afferents can bypass the
cerebellar cortex and drive CRs by direct activation of the deep cerebellar nuclei (Adapted from
Woodruff-Pak and Disterhoft 2008)
27
Adult neurogenesis and eyeblink conditioning
In a remarkable piece of work, Shors, Gould, and associates (2001) explored the fate of
new adult-generated neurons in the hippocampal dentate gyrus in trace and delay
eyeblink conditioning in the rat. New neurons were labeled with BrdU injection, a
thymidine analog 1 week before training; delay and trace groups were given the same
number of trials and both learned to the same asymptote. The trace procedure resulted in
a significantly and substantially higher number of new neurons in the dentate gyrus
compared to the delay and control conditions. Further injection of a toxin for proliferating
cells (MAM) markedly impaired trace but not delay acquisition. These results and many
more recent studies have raised the possibility that new adult-generated neurons play a
role in trace memory formation in the hippocampus.
Eyeblink conditioning, AD and Aging
Extensive research has been done to study the status of eyeblink conditioning in several
AD models and human AD patients. The first mouse eyeblink conditioning study using a
mouse model of AD was performed on PDAPP V717F mice (Weiss et al., 2002). These
mice over express APP with a platelet-derived (PD) growth factor promoter. PDAPP
mice took significantly higher number of trials to reach the learning criterion. The dorsal
part of the caudal hippocampus is significantly smaller in these AD mice. Since the
hippocampal volume is decreased in these mice and trace eyeblink conditioning is
hippocampal dependent, it would have been interesting to study it in these mice. Another
study was done using the AD model, APPLd2, with over expression of APP695 with a
28
Thy-1 promoter that resembled the mutation found in familial forms of AD V6421,
“London” mutation. No difference was observed in the young mice but 10 months old
APPLd2 mice did not learnt trace eyeblink conditioning even after 10 days of training.
However, a deficit was observed in the UR amplitude raising questions about the normal
blinking than learning deficits. Studies done with the AD model, APP+PS1 exhibited
deficits in hippocampus-dependent trace eyeblink conditioning only in case of greatest
amyloid pathology.
The results of using AD models of mice in eyeblink conditioning are inconsistent with
each other at this point. Both types of APP transgenic mice are impaired in delay (Weiss
et al., 2002) and trace (Rodriguez-Moreno et al., 2004) eyeblink conditioning, but the
APP+PS1 double transgenic mice acquire both delay and trace eyeblink conditioning at
levels similar to their wild-types littermates (Ewers et al., 2006). It seems to suggest that
over expressing PS1 is having some compensatory effect on the learning deficits induced
from APP over expression.
Eyeblink conditioning and stress
Stress response is a state of alarm, initiated by an environmental threat that promotes an
array of autonomic and endocrine changes designed to aid self-preservation. The effect of
stress on motor learning using eyeblink conditioning has been thoroughly studied in rats
(Shors et al., 2000). However, in case of mouse, there is just one stress model studied for
eyeblink conditioning (Weiss et al., 2005). After restraint and tail shock, the
corticosterone levels were significantly increased immediately and one hour after the
29
stress. This increase was not significantly different from the naïve control group after 24
hours. Stressed mice learned trace eyeblink conditioning better than the no-stress control
group. Stress reduced the amplitude of the post-burst afterhyperpolarization of CA1
hippocampal pyramidal neurons after 1 and 24 hours. Stress also reduced the spike
frequency accommodation (more spikes could be generated) of the same neurons and
time intervals. Several studies have shown that stress impairs hippocampal LTP (Weiss et
al., 2005), but the trace eyeblink conditioning result in this study showed enhancement,
suggesting that along with hippocampal LTP other processes are also important and
might be playing a critical role in this enhancement.
Studies using rats have shown that exposure to an inescapable stressor facilitates
acquisition of delay eyeblink conditioning (Shors and Servatius 1995), with
glucocorticoids playing a critical role in the process. Moreover, stress facilitation of
classical eyeblink conditioning is gender dependent – it facilitates in males while it
impairs in females through activational effects of ovarian hormones (Wood and Shors
1998). Studies in humans have not been that consistent in relating the effect of acute
stress to eyeblink conditioning. One study showed an enhancement in trace eyeblink
conditioning after exposure to acute stress, similar to the observations from animals. A
recent study, however, showed impairment in males after exposure to stress, contrary to
what has been observed in previous studies (Wolf et al., 2009).
30
5. Triple – transgenic Alzheimer’s disease mice
Triple-transgenic AD (3xTgAD) mouse is the most comprehensive model of AD to date.
This model incorporates mutations in three genes most commonly used to express AD-
like pathology – APP, PS-1 and FTDP-17 (Oddo et al., 2003). Specifically, this mouse
over-expresses the APP
SWE
, PS1
M146V
, and tau
P301L
. These mice were backcrossed to the
same strain (129/C57BL6), thereby reducing strain differences that can affect APP
phenotype (Carlson et al., 1997) and behavior (Crawley et al., 1997). Based on the initial
characterizations from several labs, the 3xTgAD mice display an age-dependent, region-
specific progression of AD-like pathology and memory deficits that is very consistent
with the human AD condition (Oddo et al., 2003; Billings et al., 2005; Caccamo et al.,
2005). Intraneuronal Aβ deposits, dense extracellular Aβ deposits, tau
hyperphosphorylation, tau aggregates (although no true “tangles”), dystrophic neuritis,
and signs of astrogliosis accumulate with age in only the following regions; cortex, CA1-
3, subiculum, dentate gyrus, entorhinal cortex, and amygdala. The first sign of AD-like
pathology appears in the neocortex at 3 months of age as intraneuronal Aβ deposits that
spreads to the CA1 pyramidal neurons, subiculum, entorhinal cortex, and amygdala – the
areas that are observed to be affected in human AD patients. These mice start to exhibit
long-term potentiation (LTP) deficits at an age between 3-6 months old, indicative of
synaptic disruption. Moreover, 6 months old 3xTgAD mice begin to show deficits in the
Morris water maze (Billings 2005) and fear conditioning (Caccamo et al., 2006). By 6
months of age, the first extracellular Aβ deposits become apparent as diffuse plaques in
31
layers 4 and 5 of the frontal cortex. The observation that memory deficits are apparent
before dense Aβ plaque formation is also consistent with observations of human AD
patients. Early markers of p-tau were apparent in the CA1 pyramidal neurons of the
hippocampus and co-localized to Aβ immunopositive neurons. Intraneuronal Aβ levels
increases with age as measured by the levels of Aβ 40 and 42 using ELISA. Dense
extracellular deposits are observed by 12 months of age in the hippocampus, subiculum,
entorhinal cortex and amygdala. By 12-15 months age, tau hyperphosphorylation is
observed in the hippocampus and by 18 months, tau aggregates and dystrophic neuritis
spread to the cortex. The appearance of Aβ accumulation prior to tau aggregation is
consistent with the amyloid cascade hypothesis. Moreover, 18 months old 3xTgAD mice
display astrogliosis as assessed by GFAP levels, another aspect of human AD
pathogenesis (Oddo et al., 2003). Taken together, the 3xTgAD mouse could be
considered the most comprehensive model of AD that most closely mimics the human
AD condition in an age-dependent and region specific manner. However, there are a few
concerns regarding the 3xTgAD mouse as an appropriate model of AD. There is no cell
loss in this model, which is one crucial aspect of AD. But this has been a limitation of
most of the previously generated transgenic models since the mechanism of cell death in
AD has yet to be elucidated. But studies have shown that tau pathology correlates with
cell death more closely than Aβ accumulation. In spite of these concerns 3xTgAD mouse
model is an excellent model to study AD and is widely used in therapeutics‟
development.
32
6. Monoamine Oxidase
What are monoamine neurotransmitters?
Monoamine neurotransmitters are the neurotransmitters and neuromodulators that contain
one amino group that is connected to an aromatic ring by a two-carbon chain (-CH
2
-CH
2
-
). Monoamines are derived from aromatic amino acids like phenylalanine, tyrosine,
tryptophan, and the thyroid hormones by the action of aromatic amino acid decarboxylase
enzymes. The most important members in this group are the neurotransmitter group
catecholamines (including dopamine, epinephrine, and norepinephrine) and the
indoleamine serotonin. The other important aromatic amine is β-phenylethylamine.
Monoamines seem to contribute to stable moods, and an excess or deficiency of
monoamines seems to cause or result from several disorders. The rapid degradation of
these molecules ensures the proper functioning of synaptic transmission and is critically
important for the regulation of emotional behaviors and other brain functions.
Monoamines are moved into or out of cells via a class of proteins called monoamine
transporters. Antidepressant and psychoactive drugs often affect the monoamine
transporters rather than the monoamine itself.
Monoamine oxidase
Monoamine oxidase (MAO) is a mitochondrial bound enzyme, which catalyzes the
oxidative deamination of dietary amines, monoamine neurotransmitters and hormones.
MAO works on many different substrates including several notable biogenic molecules:
33
indoleamines such as serotonin (5-hydroxytryptamine, 5-HT) and tryptamine;
catecholamines, such as dopamine (DA), norepinephrine (NE) and epinephrine; trace
amines, such as β-phenylethylamine (PEA), tyramine and octopamine.
As with other neurotransmitters, rapid removal and degradation of brain monoamines,
such as 5-HT, NE and DA is essential for the correct functioning of synaptic
neurotransmission. As discussed before, monoaminergic signaling plays a very critical
role in the modulation of mood and emotion, as well as the control of motor, perceptual
and cognitive functions. MAO degrades the monoamines into the corresponding
aldehydes, which are then oxidized into acids by aldehyde dehydrogenase (ALDH) or
converted into alcohols or glycols by aldehyde reductase (ALR). The byproducts of these
reactions include a number of potentially neurotoxic species, such as hydrogen peroxide
and ammonia. In particular, hydrogen peroxide can trigger the production of reactive
oxygen species (ROS) and cause mitochondrial damage and neuronal apoptosis.
MAO has two different isoforms – MAO A and MAO B. The two isoforms have different
substrate sensitivities. MAO A displays a higher affinity for 5-HT and NE, while MAO B
prfers PEA. The metabolism of DA and other monoamines (such as tryptamine and
tyramine) is generally contributed by both isoforms. DA degradation is mainly mediated
by MAO A in the rodent brain, while MAO B plays a substantive role in this process in
humans and other primates. Pharmacological distinction is done by using different
inhibitor sensitivities – MAO A is selectively inhibited by low doses of clorgyline
(Johnston, 1968), whereas MAO B is blocked by low doses of deprenyl (selegiline)
34
(Knoll and Maryar, 1972). Genes for MAO A and MAO B are located on the same
chromosome X (Xp 11.23) (Lan et al., 1989), in opposite direction with tail-to-tail
orientation, and display identical number of exons (Ou et al., 2006) and intron-exon
organization, suggesting that the two genes are likely derived from the duplication of a
common ancestral gene. The deduced primary sequences of the twp isoenzymes have
70% identity (Bach et al., 1988). Both proteins are predominantly located in the outer
membrane of mitochondria, to which they are anchored by the C-terminal domain
(Rebrin et al., 2001). Recently, it has been shown that MAO A and MAO B are activated
and repressed by different transcription factors (for review see Shih et al., 1999a), which
may account for the differences in the localization of these two isoenzymes (Grimsby et
al., 1990). In the brain, MAO A is predominantly localized in catecholaminergic neurons,
whereas MAO B is mainly expressed in serotonergic and histaminergic neurons, as well
as in astrocytes (Westlund et al., 1988; Saura et al., 1994; Jahng et al., 1998).
MAO Knockout (KO) mice
Total inactivation of each isoenzyme is not possible using the inhibitors because of
selectivity issues. To address this issue, several lines of transgenic mice have been made
using nonsense mutations for each MAO isoform. These mice have played a very
significant role in our understanding of the role of MAO in the regulation of behavior and
brain functions.
MAO A KO mice were engineered to study the functions of MAO A in vivo (Cases et al.,
1995). These mice showed a significant increase in brain levels of 5-HT and NE and a
35
modest increase in DA. Behaviorally, these mice show a dramatic enhancement of
aggressive traits, as measured both in the resident-intruder paradigm (Cases et al., 1995;
Shih et al., 1999b) and in encounters with cage mates. Additionally, MAO A KO mice
also exhibit higher retention of aversive or fear-related memories than WT animals,
including freezing response after footshock (Kim et al., 1997) and retention of
conditioned passive avoidance (Dubrovina et al., 2006). In general, the impact of MAO A
deficiency in the brain seems to induce a general increase in the resistance to the effects
of environmental stressors. For example MAO A KO mice display a typical reduction in
immobility in the forced swim test (Cases et al., 1995), which is a validated animal model
of depression (Cryan et al., 2001). MAO A KO mice also typically exhibit a reduction in
acoustic startle reaction (Cases et al., 1995). Overall, these results suggest that MAO A
KO mice may display impairments in the perception of external stress and in the
mediation of adaptive responses to stress, which could also underpin the dramatic
increase in aggressive behaviors against both intruders and cage mates. These alterations
may be underpinned by sensory dysregulation, as suggested by the disruption of the
barrel fields in the somatosensory cortex. Both aggressive phenotype and the alterations
of the barrel fields were rescued by a forebrain-specific knock-in approach of MAO A
into MAO A KO mice (Chen at al., 2007).
MAO B KO mice show increased levels of PEA, but not 5-HT, NE and DA. This
alteration in the levels of the neurotransmitters was not associated with significant
changes in locomotor patterns in the open field and in the elevated-plus maze, but it did
36
reduce immobility time in the forced swim test, upon single or repeated presentation
(Grimsby et al., 1997). MAO B KO mice do not display apparent alterations in DA
release or uptake. The phenotypical alterations in MAO B KO mice observed so far are
more subtle than those in MAO A KO mice, since PEA is present in very small
concentrations in the brain and is thought to serve amphetamine-like functions in the
modulation of the neurotransmission and signaling of DA and NE (Sabelli et al., 1975;
Risner and Jones 1977). PEA is co-synthesized with dopamine by L-DOPA
decarboxylase (Juorio 1988). Recent studies have shown that PEA is the main activator
of the trace-amine-associated-receptor 1 (TAAR1) (Xie and Miller 2008; Wolinsky et al.,
2007), which, in turn, modulates catecholaminergic signaling (Wolinsky et al., 2007).
MAO B KO mice display several abnormalities that may be linked to the alterations
induced by high levels of PEA – for example, they display attenuation of the
hyperlocomotive effects of amphetamine (Yin et al., 2006), as well as alterations in the
distribution of cerebral blood flow (Scremin et al., 1999). PEA is also likely responsible
for a reduction in D2-like receptors and a super sensitivity of D1 receptors in the striatum
of these mutant mice (Chen et al., 1999). In essence, MAO B KO mice represent a unique
model to study the long-term consequences of high levels of PEA in behavior and brain
function.
A recently characterized line of MAO AB KO mice has is the basis of the current study.
These mice arose out of a spontaneous mutation in MAO A in a mice line carrying
ablation of MAO B. Levels of PEA, 5-HT, NE and DA are highly increased in
37
comparison to MAO A and MAO B (Chen et al., 2004). Behaviorally, these mice are
characterized by low novelty-induced locomotion, high levels of anxiety-like behaviors
in the elevated-plus maze, and low latency to attack in the resident-intruder paradigm
(Chen et al., 2004).
MAO and human disorders?
MAO inhibitors are the first category of antidepressant and show high mood-enhancing
efficacy. They generally work by inhibiting MAO A and countering the reduction in 5-
HT, NE and DA levels that characterizes depression. MAO inhibitors are also indicated
for some anxiety-spectrum disorders, namely social phobia, panic disorder, post-
traumatic stress disorder (PTSD) and obsessive-compulsive disorder (OCD). MAO B
inhibitors have a stimulant effect and ameliorate hyperactive behavior, showing a general
improvement of ADHD symptoms. This might be mediated by an increased level of
PEA, which is assumed to act as an endogenous amphetamine. MAO B inhibitors are
used in Parkinson‟s disease (PD) because DA is preferentially deaminated by this
isoenzyme in the human nigrostriatal dopaminergic system. Using MAO B inhibitors
increase the DA levels to compensate for the nigrostriatal deficits in this
neurotransmitter. Additionally, hydrogen peroxide, which is produced as a byproduct of
MAO-mediated reaction and contributes to the formation of other ROS triggering
mitochondrial damages and neuronal death, is reduced due to MAO inhibition. Age-
related increases in MAO B activity, as well as the neuroprotective effects of its
inhibitors, have been considered as rational bases to use MAO B inhibitors in
38
Alzheimer‟s disease (AD). MAO B blockade induced cognitive improvement in AD. In
essence, MAO A and MAO B have a very important role in maintaining a balance in the
CNS.
7. Fear Conditioning
Evolution has endowed each species with an array of instinctual defense mechanisms to
help organisms cope with environmental threats and other challenges and safety and well-
being. The term fear refers to both a psychological state and a set of bodily responses that
occur in response to threat (LeDoux JE 1995) A lot of research has been done to
understand how fear is organized in the brain through studies of Pavlovian fear
conditioning, a laboratory model for studying fear. Pavlovian fear conditioning is a
behavioral procedure in which an emotionally neutral conditioned stimulus (CS), such as
an auditory tone, is paired with an aversive conditioned stimulus (US), typically a foot
shock. After one or several pairings, the CS comes to elicit defensive behaviors,
including freezing behavior, as well as increased arousal in the brain and secretion of
norepinephrine (NE) and glucocorticoids (GCs) peripherally. These conditioned
responses (CRs) are hard-wired and occur in response to both innate and learned threats.
Thus, fear conditioning does not create conditioned fear responses but allows
environmental stimuli to come to elicit such responses. In fear conditioning; responses
elicited by the CS are often measured during acquisition and retrieval tests. Acquisition
refers to the initial learning of the associated between the CS and the US during the
training phase. Retrieval involves recalling the association between the CS and the US
39
and is often tested by the presentation of the CS alone to measure how well the animal
remembers the aversive stimuli (US) that was paired with the neutral stimulus (CS)
during the training. Retrievals test that occur within a few hours after acquisition measure
short-term memory (STM), whereas those that occur later (typically 24 hours after
learning) measure long-term memory (LTM). STM and LTM are used to study memory
reconsolidation, the process though which an unstable STM is converted into a more
stable and enduring LTM (Dudai 2004; McGaugh 2000; Rodrigues et al., 2004).
Retrieval tests are also used to study reconsolidation. Reconsolidation occurs when fear
memories are retrieved and enter a labile state and require conversion from an unstable
STM trace into an enduring LTM trace (Dudai 2006; Nader 2003; Nader et al., 2000). In
addition, retrieval tests are used to measure extinction, which is the gradual reduction in
the ability of the CS to elicit fear CRs that occur when the CS is presented repeatedly in
the absence of the US (Quirk and Mueller 2008; Sotres-Bayon et al., 2004). Unlike the
disruption of reconsolidation, which is robust and long lasting, extinction involves a new
learning process. As a result, after extinction training, the CS can spontaneously produce
CRs again after the animal is re-exposed to the US or is placed in a novel context
(Bouton et al., 2006; Ji and Maren 2007).
Neural circuitry of fear conditioning
The pathways have been mapped from the CS sensory processing to the CR motor
control systems. A key link in this circuitry is the amygdala, which sits between the
sensory input systems on one hand and the motor output systems on the other (Fanselow
40
and Poulos 2005; Lang and Davis 2006; LeDoux 1995, 2007; Maren 2001, 2005; Pare et
al., 2004; Rodrigues et al., 2004). However, other brain structures also contribute to fear
conditioning.
The amygdala contains heterogeneity of distinct nuclei, differing by cell type, density,
neurochemical composition, and connectivity (LeDoux 2007; Pitkanen et al., 2000;
Swanson 2003). The lateral nucleus (LA), receiving auditory, visual, gustatory, olfactory,
and somatosensory (including pain) information from the thalamus and the cortex
(olfactory and taste information is transmitted to other nuclei, as well), is viewed as the
sensory gateway to the amygdala. The thalamo-amygdala pathway is an emergency
shortcut of sorts, transmitting rapid and crude information about fear-eliciting stimulus
without the opportunity to filter by conscious control (LeDoux 1995). The cortico-
amygdala pathway, in contrast, provides slower, but more detailed and sophisticated
sensory information. The convergence of the CS and US seems to occur in the dorsal half
of the LA (Romanski et al., 1993). and studies have shown that cellular plasticity occurs
in the dorsal LA during fear conditioning. Moreover, molecular changes in the LA
consolidate the fear memory, converting STM into LTM traces (Dudai 2004; Rodrigues
et al., 2004). The central nucleus (CE) of the amygdala, in contrast, is viewed as the
major output region of the amygdala. The CE controls the expression of the fear reaction,
including behavioral, autonomic, and endocrine responses via projections to downstream
areas, including the hypothalamus, central gray, and the dorsal motor nucleus of the
vagus (Fanselow and Poulos 2005; Lang and Davis 2006; LeDoux 1995, 2007; Maren
41
2001, 2005; Pare et al., 2004; Rodrigues et al., 2004). The LA communicates with the CE
directly but other nuclei also help mediate between these two. For instance, the LA
projects to the basal nucleus (B), which in turn projects to CE (LeDoux 2000; Pitkanen et
al., 1997). Information about the context of a fearful situation is conveyed to the LA and
B by the hippocampus (Pitkanen et al., 2000). Thus, although fear conditioning to
discrete sensory cues, such as a tone or a light, are hippocampal independent,
conditioning to contextual information, including details about the environment, are
hippocampal dependent. The LA, B and accessory basal nuclei are sometimes grouped
together as the BLA (basolateral amygdala). Although the LA is required for fear
conditioning under standard training conditions, it appears that, with overtraining, fear
conditioning can be mediated by circuits that do not depend on the LA (Lee et al., 2005;
Maren 1999). With overtraining, weak connections to the CE appear to induce fear
conditioning (Zimmerman et al., 2007), but such pathways are not normally used.
Moreover, although the B is not required for conditioning, it nevertheless appears to
contribute to the appearance of the fear response (Anglada-Figueroa and Quirk 2005)
The expression of fear responses can be regulated by the medial prefrontal cortex
(mPFC) via projections to the LA, B and ITC (intercalated region), an inhibitory network
that connects with the CE and get projections from LA and B. The infralimbic subregion
(IL) of the mPFC is especially important for fear extinction (Quirk et al., 2006;
Rosenkranz et al., 2003; Sotres-Bayon et al., 2004) because it inhibits the output of the
amygdale (Vidal-Gonzalez et al., 2006). However, the nature of the role of prefrontal
42
cortex and amygdala interactions in fear extinction is still debated. The hippocampus also
plays a key role in the contextual modulation of fear extinction (Ji and Maren 2007; Kim
and Fanselow 1992; Phillips and LeDoux 1992) and thus the learning to suppress a
previously learned fear.
Fear conditioning and stress
Once the brain‟s fear system detects a threat, it gets activated and initiates the stress
response in both the brain and the body. Amygdala, as discussed above, is responsible for
the detection of threat and orchestration of stress responses in the brain and body.
Outputs from the amygdala lead to the activation of a variety of target areas that control
both behavioral and physiological responses designed to address the threat (Lang and
Davis 2006; LeDoux 1998, 2002). Along with the expression of defensive behaviors,
such as freezing (Blanchard and Blanchard 1969; Fendt and Fanselow 1999), amygdala
activation leads to responses in the brain and the body that support the fear reaction. In
the brain, monoaminergic systems are activated, resulting in the release of
neurotransmitters such as NE, acetylcholine, serotonin, and dopamine throughout the
brain. These neurotransmitters lead to an increase in arousal and vigilance and, in
general, an enhancement in the processing of external cues (Aston-Jones and Cohen
2005; LeDoux 2007; Ramos and Arnsten 2007; Talarovicova et al., 2007). Detection of
threat by the amygdala also has endocrine consequences. Amygdaloid signaling causes
secretion of corticotrophin-releasing hormones (CRH) from the periventricular nucleus of
the hypothalamus. CRH, along with other hypothalamic secretagogs, causes the release of
43
adrenocorticotropic hormone from the pituitary, which in turn stimulates the secretion of
GCs from the adrenal cortex. Circulating GCs bind to the high-affinity mineralocorticoid
receptor (MR) and the low-affinity glucocorticoid receptor (GR) in tissues throughout the
body and brain (de Kloet 2004; Korte 2001).
Finally, detection of threat by the amygdala has autonomic consequences. Connections
from the amygdala to the brainstem leads to the activation of the sympathetic nervous
system, involving the release of epinephrine and NE from the adrenal medulla and NE
from the terminals of sympathetic nerves throughout the body. Adrenal medullary
hormones and sympathetic nerves produce an array of effects, including increasing blood
pressure and heart rate, diverting stored energy to exercising muscle, and inhibiting
digestion (McCarty and Gold 1996; Sapolsky 2003). Coming full circle, fear circuitry is
profoundly influenced by these endocrine and autonomic stress responses. Thus, GCs
travel in the circulation to the brain and affect amygdala and a variety of other structures.
Although NE does not cross the blood-brain barrier, it may affect the brain indirectly by
binding to visceral afferent nerves, which transmit to the brain (McGaugh 2002). Thus,
the detection of threat and the consequent activation of the fear system elicit a variety of
effects that feed back onto the system that initiated and modulates emotional processing
(Sapolsky 2002).
An extensive literature has demonstrated the ways in which stress, and the endocrine
mediators of the stress response, can impact explicit, declarative memory processes
(McEwen 1999). Stressful experiences, NE, and GCs alter the morphological and
44
electrophysiological characteristics of neurons in areas that play a vital role in fear
processing, including the amygdale (LA and B), hippocampus, and PFC. As a result, they
can have a powerful influence on fear conditioning. Existing studies show that stress
before or after training boosts LTM of fear conditioning. In addition, stress before, but
not after reactivation enhances reconsolidation. Finally, stress before fear conditioning,
but not before extinction training, interferes with extinction LTM. Altogether, these
findings suggest that during initial learning, stress activates events that can influence later
the consolidation and extinction processes.
Fear conditioning and anxiety disorders
Anxiety is a psychological and physiological state characterized by cognitive, somatic,
emotional and behavioral component. These components combine to create an unpleasant
feeling that is typically associated with uneasiness, fear, or worry. Anxiety is a
generalized mood condition that occurs without an identifiable triggering stimulus. As
such, it is distinguished from fear, which occurs in the presence of an observed threat.
Fear is related to specific behaviors of escape and avoidance, whereas anxiety is the
result of threats that are perceived to be uncontrollable and unavoidable. Another view is
that anxiety is a “future-oriented mood state in which one is ready or prepared to attempt
to cope with upcoming negative events” suggesting that it is a distinction between future
vs. present dangers that divides anxiety and fear. Anxiety is considered to be a normal
reaction to stress. When anxiety becomes excessive, it may fall under the classification of
Generalized Anxiety Disorder (GAD). It is characterized by excessive, exaggerated
45
anxiety and worry about everyday life events with no obvious reasons for worry. The
exact causes of GAD are not known, but a number of factors – including genetics, brain
chemistry and environmental stresses – appear to contribute to its development. Several
studies have shown that human subjects with anxiety disorders and animal models of
anxiety display enhanced response in fear conditioning (Barkus et al., 2010). As
previously suggested, anxiety arises when there is a conflict between potential response
options available to the animal. In laboratory tests of anxiety, such as the elevated plus
maze or the successive alleys test, this manifests as an approach/avoidance conflict with
respect to exploration of the open arms. It has been suggested that the distinction between
fear and anxiety is best encapsulated by the concept of defensive direction (Gray and
McNaughton, 2000). Fear is a behavioral state that functions to remove the animal from a
dangerous situation (active avoidance), or to deal with that dangerous situation by
adopting the appropriate stimulus-specific response in the presence of a stimulus that has
become associated with danger (such as freezing in response to a cue associated with
footshock). Anxiety, on the other hand, functions to limit whether or not the animal
should enter into a potentially dangerous situation (passive avoidance). In other words,
fear is the response to a threat that is present, whereas anxiety is the response to a
potential threat. These different behavioral responses to present or potential danger
differentially depend on the amygdala and ventral hippocampus respectively, and exist
within a hierarchical defense system that is arranged to protect the animal from harm.
Gray has suggested that the septo-hippocampal formation is the seat of anxiety in the
brain, and that it acts, first, to detect situations of conflict or uncertainty, and then second,
46
to resolve those conflicts and thus protect the animal from danger (and/or maximize its
chances of reward; see Gray and McNaughton, 2000). It has long been suggested that the
hippocampal formation could potentially act as a comparator, allowing novel or
unexpected events to be detected by comparing the current state of the world with what
would be expected on the basis of information retrieved from memory, and thus allow
situations of conflict to be identified (Gray, 1981; Gray and McNaughton, 2000). Having
detected a conflict (a novel or unexpected event), the hippocampal formation acts to
resolve the conflict by increasing levels of attention and arousal, and through behavioural
inhibition of prior, on-going motor programs. These behavioural responses constitute
anxiety and they allow the animal to gather more information in order to resolve the
conflict before responding appropriately. Gray and MacNaughton (2000) suggested that
the hippocampal system resolves the conflict by increasing the weighting given to
affectively negative information. In other words, in a normal animal the hippocampal
system will act to favor avoidance behavior over approach behavior.
47
CHAPTER TWO
Allopregnanolone Reverses the Neurogenic and Cognitive Deficits in Mouse Model
of Alzheimer’s Disease
Chapter Two Abstract
Our previous analyses demonstrated that allopregnanolone (APα) significantly
increased proliferation of rodent and human neural progenitor cells in vitro (Wang et al.,
2005). In this study, we investigated the efficacy of APα in promoting neurogenesis in
the hippocampal subgranular zone (SGZ), to reverse learning and memory deficits in the
3 month-old male triple transgenic mouse model of Alzheimer‟s (3xTgAD) and the
correlation between APα-induced neural progenitor cell survival and memory function in
3xTgAD mice. Neural progenitor cell proliferation was determined by unbiased
stereological analysis of BrdU incorporation and survival determined by FACS for
BrdU+ cells. Learning and memory function was assessed using the hippocampal-
dependent trace eye-blink conditioning paradigm. At 3 months, basal level of BrdU+
cells in the SGZ of 3xTgAD mice was significantly lower relative to non-Tg mice,
despite the lack of evident AD pathology. APα significantly increased, in a dose
dependent manner, BrdU
+
cells in SGZ in 3xTgAD mice and restored SGZ proliferation
to normal magnitude. As with the deficit in proliferation, 3xTgAD mice exhibited deficits
in learning and memory. APα reversed the cognitive deficits to restore learning and
memory performance to the level of normal non-Tg mice. In 3xTgAD mice, APα-
induced survival of neural progenitors was significantly correlated with APα-induced
memory performance. These results suggest that early neurogenic deficits, which were
48
evident prior to immuno-detectable Aβ, may contribute to the cognitive phenotype of AD
and that APα could serve as a regenerative therapeutic to prevent or delay neurogenic and
cognitive deficits associated with mild cognitive impairment and Alzheimer‟s disease.
This work was published in the Proceedings of National Academy of Science, USA in the
early edition March 2010-03-15. Work presented here was done in collaboration with
Brinton lab at USC and parts of the project except behavioral studies were done in her
lab.
Introduction
Allopregnanolone (APα, 3α-hydroxy-5α-pregnan-20-one), a metabolite of progesterone,
is synthesized de novo in the embryonic and adult CNS (Brinton 1994; Baulieu et al.,
1990; Mellon and Griffin, 2002) and in pluripotential progenitor cells (Gago et al., 2004).
Previously we demonstrated that APα significantly increased proliferation of rodent and
human neural progenitor cells in vitro in culture via a GABAA receptor and L-type Ca
++
channel dependent mechanism (Wang et al., 2005). In this study, we investigated the
efficacy of APα as a neurogenic agent in vivo and its effects on learning and memory
using the triple transgenic Alzheimer‟s disease mouse (3xTgAD).
The adult brain has two stable regions of mitotic activity, the subventricular zone (SVZ)
of the lateral ventricle in the frontal cortex and the subgranular zone (SGZ) of the dentate
gyrus in the hippocampus (Toni et al., 2008; Jessberger and Gage, 2009). While the
regenerative potential of the mammalian brain is sustained throughout the life span,
49
proliferative capacity of neural progenitors declines with age and diseases, such
Alzheimer‟s disease (AD) (Kuhn et al., 1996). Parallel to the diminution in neurogenesis,
is the decline of neurosteroids in the aging and AD brain (Genazzani et al., 1998; Weill-
Engerer et al., 2002; Marx et al., 2006). Consistent with a decline in growth factors
(Weill-Engerer et al., 2002; Hattiangady and Shetty, 2008; Shetty et al., 2005), APα
content is significantly lower in aged subjects (Genazzani et al., 1998) and in brains of
AD patients compared with age-matched controls (Weill-Engerer et al., 2002; Marx et al.,
2006). In contrast, during fetal development when neural progenitor proliferation is
maximal, APα is synthesized throughout the embryonic period and in multipotential
progenitor cells (Gago et al., 2004; Lauber and Lichtensteiger, 1996).
To test the hypothesis that APα can function as a regenerative factor with an impact on
cognitive function in AD, we investigated the efficacy of APα as a neurogenic agent in
vivo using the triple transgenic Alzheimer‟s disease mouse (3xTgAD) and the non-
transgenic control mouse (non-Tg). The 3xTgAD mouse carries mutations in two human
familial AD genes (APP
Swe
, PS1
M146V
) and one frontal temporal dementia linked tau
mutation (tau
P301L
) and manifests age-dependent neuropathology of both β-amyloid
plaques and neurofibrillary tangles (Oddo et al., 2003). In addition to expressing
neuropathological markers of AD, the 3xTgAD mouse exhibits early learning and
memory deficits, (LaFerla et al., 2007). Using this AD model, we characterized APα
concentration within brain and serum, basal level of neurogenesis and cognitive function
at 3 months of age. These analyses were conducted in parallel to an investigation of the
50
impact of APα on both neurogenic and cognitive function using unbiased stereology,
immuncytochemistry (IHC), FACS, real-time RT-PCR, Western blot, and a hippocampal
dependent associative learning task, trace eyeblink conditioning.
Materials and methods
Animals and treatment
Breeding pairs of the triple transgenic Alzheimer‟s disease mouse (3xTgAD,
homozygous mutant of human APPswe and tauP301L and PS1M146V) and its
background strain (129/Sv x C57BL/6) were obtained from Dr. Frank LaFerla (UC
Irvine) and the colonies were established at USC. The characterization of amyloid and tau
pathologies, as well as synaptic dysfunction in this line of mice has been described
previously (Oddo et al., 2003) and confirmed in our laboratory. The mice were genotyped
regularly to confirm the purity of the colony. Experiments were performed using 3-
month-old male 3xTgAD and non-Tg. The number of mice per condition is indicated
within the results section. Mice were maintained under a 12 h light/12 h dark cycle with
continuous access to food and water.
Allopregnanolone (APα, 3α-hydroxy-5α-pregnan-20-one) (aka APα, Allo or THP) stock
solution was prepared in pure ethanol and diluted in PBS before injection (with a final
ethanol concentration of 0.002 % of the body weight). Mice received a subcutaneous
(s.c.) injection of APα at different concentrations or vehicle as indicated. One hour after
APα injection, mice were intraperitoneally (i.p.) injected with BrdU at a concentration of
100 mg/kg. In all acute experiments, mice were sacrificed the next day after injection and
51
sampled as described in the supporting information. All experiments used minimal
number of animals conformed to the Animal Welfare Act, Guide to Use and Care of
Laboratory Animals, and the U.S. Government Principles of the Utilization and Care of
Vertebrate Animals Used in Testing, Research, and Training guidelines on the ethical use
of animals.
Animal dissection and tissue collection
Prior to sacrifice, mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg
xylazine. Approximately 0.2-0.4 ml of blood was collected from each mouse by cardiac
puncture into EDTA coated 1.5 ml centrifuge tube and centrifuged at 10,000 rpm for 5
min immediately which yielded 100-200 µl of plasma. Mice were perfused with PBS and
brains were dissected into two hemispheres. One hemisphere was fixed immediately in
cold 4% paraformaldehyde for immunohistology and unbiased stereological analyses.
From the remaining brain hemisphere, the cortical and hippocampal regions were
collected and quickly frozen on dry ice and then stored at -80
o
C until use. Tissues were
processed for mRNA, protein, and steroid level analyses. The hemispheres for histology
were embedded into blocks (40 brain hemispheres per block) for cryostat sectioning by
NeuroScience Associates (Knoxville, TN). The 40-brain hemisphere blocks were
sectioned into a series of 40 µm coronal sections and sections processed under the same
conditions during the histological and phenotypic immuno-labeling and stereological
analyses.
52
Immunohistochemistry
Prior to labeling, all slides were coded and the codes were not broken until analyses were
completed. BrdU histolabeling was performed by NeuroScience Associates (Knoxville,
TN) and labeling conducted in every sixth section in the series. Briefly, brain sections
were denatured with 0.08 N HCI for 1 h at 37
o
C, neutralized with 0.1 M boric acid, pH
8.5, for 2 x 5 min, and incubated with NOVUS monoclonal anti-BrdU antibody (1: 1500)
for 24 h at 4
o
C. After HRP-conjugated secondary antibody incubation, the section was
color reacted with DAB/Ni. Nonspecific antibody binding was blocked by incubation in
blocking buffer (10% normal serum derived from the same species as the secondary
antibody raised) for 30 min at room temperature. For identifying the phenotype of the
newly formed BrdU positive cells, adjacent sections were incubated overnight with the
following primary antibodies in a proper triple staining combination: rat monoclonal
antibody for BrdU (AdB Serotec, Raleigh, NC), Rabbit polyclonal antibody for glial
fibrillary acidic protein (GFAP, 1:1000, DAKO, Carpinteria, CA); Monoclonal antibody
for NeuN (1: 800, Chemicon, Temecula, CA); goat anti-Doublecortin polyclonal
antibody (DCX, 1:400, Santa Cruz Biotechnology). After being washed with PBS, slides
were incubated for 30 min in a mixture of secondary antibodies containing anti-rat IgG
conjugated with FITC (1: 500; Vector Laboratories), anti-mouse IgG conjugated with
Cy5 (1:500; Vector Laboratories), anti-goat IgG conjugated with Cy3 or anti-rat IgG
conjugated with FITC, anti-rabbit IgG conjugated with Cy5 (1:400, Vector Laboratories),
and anti-mouse IgG conjugated with Cy3 (1:1000; Vector Laboratories) according to the
53
requirements of the first antibody used and co-staining combination. For evaluating AD
pathology development, sections were first incubated with monoclonal antibody for
Amyloid-beta 6E10 (1: 1000, Signet, Berkeley, CA) overnight and then with anti-mouse
IgG conjugated with FITC for 30 min (1:500, Vector Laboratories). The slides were
washed 3 x 10 min with PBS, rinsed with water, then mounted under cover slips with
anti-fading mounting medium (Vector Laboratories). Fluorescent signals were detected
using a Zeiss LSM 510 Meta NLO imaging system equipped with Argon, red HeNe and
green HeNe lasers. Panels were compiled in Adobe Photoshop 7.0 (Adobe Systems Inc,
Mountain View, CA). All analyses were performed in sequential scanning mode and
double or triple labeling was confirmed by comparing the fluorescent IR signal intensity
profile and three-dimensional reconstructions of z-series.
Real-time reverse transcription-PCR
Real-time reverse transcription (RT)-PCR was performed to determine APα regulation of
cell cycle gene expression. Total RNA from mouse hippocampus was isolated using
TRIzol reagent (Invitrogen, Carlsbad, CA) as described previously (Wang et al., 2005;
Wang et al., 2006). cDNA was synthesized using Super-Script III reverse transcriptase
(Invitrogen, Grand Island, NY) and oligo-dT primer in accordance with the protocols of
the manufacturer. The expression of related genes was quantified using the SYBR green
reagent (2x SYBR Green Supermix; Bio-Rad, Hercules, CA) following the instructions
of the manufacturer on a Bio-Rad iCycler. PCR was performed in multi-replicates under
optimized conditions: 95°C denatured for 3 min, followed by 40 cycles of 45 s at 94°C,
54
45 s at 55°C, and 45 s at 72°C. The primers for proliferating cell nuclear antigen (PCNA)
(GenBank accession number NM_011045), forward, 5‟-
TTGGAATCCCAGAACAGGAG - 3‟, reverse, 5‟- CAGTGGAGTGGCTTTTGTGA -3‟
generated a 287 bp DNA fragment. No other products were amplified because melting
curves showed only one peak. Fluorescence signals were measured over 40 PCR cycles.
The cycle number (Ct) at which the signals crossed a threshold set within the logarithmic
phase was recorded.
For quantitation, we evaluated the difference in cycle threshold (ΔCt) between the APα-
treated group and vehicle control of each gene. Efficiency of amplification of each pair of
primers was determined by serial dilutions of templates and all were >0.93. Each sample
was normalized with the loading references β-actin and GAPDH. Ct values used were the
means of triplicate replicates. Experiments were repeated at least three times. The results
were presented as % changes vs. vehicle.
Western blot
Protein extracts from mouse hippocampus and cortex were separated by SDS gel as
described before (Wang et al., 2005). After transfer, PVDF membrane was plotted with
monoclonal antibody for proliferating cell nuclear antigen (PCNA, 1:500, Zymed
Laboratories Inc, San Francisco, CA), polyclonal antibody for CDC2/p34 (1:500, ab7953;
Novus Biologicals, Littleton, CO), monoclonal antibody for amyloid-β 6E10 (1:800,
Signet, Berkeley, CA); polyclonal antibody for amyloid-β 1-42 (1:200, Chemicon,
Temecula, CA); monoclonal antibodies for human tau PHF AT8 and HT7 (1:400, and
55
1:200, respectively, Pierce, Rockford, IL). Membrane was then incubated with a
horseradish peroxidase conjugated secondary antibody which is complementary to the
primary antibody. Results were visualized by the ECL Plus Western Blotting Detection
System (GE Healthcare, Amersham, Buckinghamshire, UK). Optical density was
measured using BioRad Chemidoc system (Segrate (Milan), Italy) and analyzed by
BioRad Quantity One software. The percent protein expression vs. control normalized by
loading control β-actin was represented with mean ± SEM values. The statistical
significance was analyzed with a one way or two-way (according to the number of
variables in different experiments) ANOVA followed by a Neuman-Keuls.
Unbiased Stereology
Number of BrdU labeled cells were determined in every sixth section in a series of 40 µm
coronal sections using unbiased stereology (optical dissector). The first section of each
hemisphere was randomly started at the beginning of olfactory and serial sections were
collected till the end of the cerebellum. Systematic samplings of unbiased counting
frames of 50 µm on a side with a 200 µm matrix spacing were produced using a
semiautomatic stereology system (Zeiss Axiovert 200M fluorescent microscope as part of
the 3iMarianas digital microscopy and a 60× SPlan apochromat oil objective (1.4
numerical aperture). Positive cells that intersected the uppermost focal (exclusion) plane
and those that intersected the exclusion boundaries of the unbiased sampling frame were
excluded from analysis. Cells that met analysis criteria through a 20 µm axial distance
were counted according to the optical dissector principle. The granule cell layer reference
56
volume was determined by summing the traced SGZ, granule cell areas for each section
multiplied by the distance between sections sampled. The mean granule cell number per
disector volume was multiplied by the reference volume to estimate the total granule cell
number. The stereologically determined number of BrdU-positive cells was related to the
granule cell layer sectional volume and multiplied by the reference volume to estimate
the total number of BrdU-positive cells. Statistically significant differences were
determined by a one-way ANOVA followed by a post-hoc Neuman-Keuls analysis.
Extraction, Derivatization and Quantification of APα by GC/MS
Plasma and brain tissues were collected as described previously and stored at -80
o
C until
use. Brain tissue was homogenized in sterile distilled water (ca. 250µl/10mg tissue)
using a 25 gauge needle. Steroids in the plasma and brain homogenates were extracted
using solid phase extraction (100 mg C18 columns). The internal standard solution (20
µl) and acetonitrile (20 µl) were added to plasma/brain homogenates and the resulting
mixtures were diluted with 300 µl distilled water, and then extracted using 100 mg C18
cartridges equilibrated with methanol, then water. Samples were washed with water and
methanol/water (20/80, v/v), and the steroid fraction was eluted with methanol, followed
by evaporation to dryness at 70°C under nitrogen. Steroid trimethylsilyl derivatives were
synthesized by adding 25 µl of MSTFA/NH4I/dithioerythritol (1000/2/3) to the
evaporated samples and heating for 15 min at 70°C, and analyzed using gas
chromatography/mass spectrometry (GC/MS). The mass spectrometer (Agilent 5975
inert MSD) was operated in EI mode, 70 eV, splitless injection. The gas chromatograph
57
(Agilent 6890N) interfaced with the mass spectrometer is equipped with 18.1m x 0.32
mm (i.d.) cross-linked 100% dimethyl polysiloxane column with a 0.25 um thickness
(HP-1ms, Agilent, Santa Clara, CA). A linear temperature gradient from 140°C -300°C
was used, with helium carrier gas. Linear scan data were obtained from m/z 75–600. Four
ions were scanned using the SIM mode, the m/z 448.30, 463.30 and 447.40, 462.40 for
d4-pregnenolone and allopregnanolone respectively. The calibration curve using one ml
stock plasma or brain homogenate was prepared by spiking with 25 ng/ml d4-
pregnenolone (C/D/N ISOTOPES INC, Pointe-Claire, Québec), and allopregnanolone
(Steraloids, Inc., Newport, Rhode Island) at the following concentrations: 0, 0.5, 1, 5, 10,
25, 50 and 75 ng/ml. The concentration of each steroid was calculated by linear
regression of the peak area corresponding to the diagnostic ions (m/z) with the highest
intensity. For d4-pregnenolone and allopregnanolone the following ions were monitored:
m/z 448.30, 463.30, and 447.40, 462.40, respectively.
Trace eyeblink conditioning
Under deep anesthesia by intraperitoneal (i.p.) injection with ketamine (100mg/kg i.p.)
and xylazine (25 mg/kg i.p.), a 4 pin head stage (DIGI-KEY) was cemented to the skull
of 3 month old male mouse with dental acrylic. The connector has four Teflon-coated
stainless steel wires and one bare stainless steel wire (0.003” bare and 0.0055” coated, A-
M Systems, Inc.). The bare wire was attached via a gold pin (Time Electronics) to the
head stage. Coated wires were implanted s.c. in the orbicularis oculi dorsal to the left
upper eyelid to record the EMG and s.c. periorbitally to deliver the shock US. All animals
58
were then placed on a warm isothermal pad after surgery to recovery for 30 min. After
surgery, mice were individually housed, provided with ad libitum access to food and
water, and maintained on a 12 hr light/dark cycle.
After one week of acclimation to the colony room, mice with no obvious adverse
responses to surgery were randomly assigned to an experimental condition. Mice were
injected subcutaneously (s.c.) with 10 mg/kg AP or vehicle followed one hour later with
an IP injection of BrdU (100 mg/kg). Following injection of test compound, mice were
returned to their home cage for 7 days prior to onset of behavioral testing.
During the first day of training, mice were placed within Plexiglas cylinders in a sound-
attenuated chamber and were habituated to the test environment for one session
consisting of 30 stimulus-free trials at 30-60 sec inter-trial intervals while spontaneous
eye-blink activity was recorded using electromyographic (EMG) activity recorded from
the obicularis oculi dorsal to the orbit during each trial. EMG activity was rectified, and
integrated using custom designed computational Labview routines {Ohno, 2005 #30;
Tseng, 2004 #46}.
Following habituation to the test environment mice underwent a learning phase and were
trained for five days. Mice were trained by pairing delivery of a tone (CS, 250 msec, 2
kHz, 85 dB) as the conditioned stimulus followed by a 250 msec period of no stimuli,
followed by the periobital shock as the unconditioned stimulus (US, 100 msec). Mice
received two blocks of 30 trials per day (30–60-sec inter-trial intervals, 3–4-h inter-block
intervals). This trace eye-blink conditioning paradigm is subthreshold for inducing
neurogenesis {Gould, 1999 #81} {Leuner, 2004 #83}. The unpaired group received
59
random tone at the same magnitude as the paired conditioning and shock with 15-30 sec
as inter-trial intervals and a total of 60 trials for one session per day. Shock intensity was
adjusted daily for each mouse to elicit a head-movement response. Following the learning
phase, mice were returned to their home cage for eight days followed on the ninth day by
a single session to assess memory of the conditioned response. The percentage of CR was
computed as the ratio of the number of CRs to the total number of valid trials. Animals
were perfused at the end of the memory trial day.
Results
1. Neural progenitor proliferation in subgranular zone of the dentate gyrus (SGZ) is
deficient in 3 month old male 3xTgAD mice prior to onset of visible AD pathology.
To determine development of amyloid β deposition in 3xTgAD male mice, brain sections
were immunolabeled with anti-amyloid β antibody (6E10) (fig. 2A). At 3 months, intra or
extracellular Aβ immunoreactivity (IR) was not detectable in either hippocampus or
cerebral cortex whereas intraneuronal Aβ IR intensity was apparent at 6, 9 and 12 months
and increased with age consistent with that reported before (Oddo et al., 2003).
Extraneuronal Aβ IR was rarely observed in 9-month-old 3xTgAD hippocampi but was
consistently present in the hippocampus of 12-month-old 3xTgAD mice (Oddo et al.,
2003)
To determine basal level of proliferation, a comparative analysis of BrdU incorporation
was conducted in the SGZ of 3xTgAD and non-Tg mice. BrdU IHC was performed in
adjacent sections immunolabeled for Aβ. The majority of the BrdU-positive cells were
60
observed in the SGZ (fig. 2B). The distribution of the newly formed cells within the
3xTgAD and non-Tg mice was consistent with that observed in both rat and mouse
dentate gyrus (Kuhn et al., 1996). Results of unbiased quantitative stereological analyses
indicated that 3-month-old non-transgenic mice generated 4568 ± 1089 BrdU positive
cells which is consistent with that reported in C57BL/6 and SJL x C57BL/6 (19) (fig.
2C). Basal proliferation in the 3xTgAD dentate gyrus was significantly lower (2625 ±
426) compared to the non-Tg mouse (4568 ± 1089) dentate gyrus (p < 0.01; fig. 2C) and
is consistent with that reported for 3xTgAD mouse dentate (20). The 42 % decrease in
basal proliferation in the 3xTgAD dentate (figs. 2B and 2C) was evident prior to the
appearance of markers of AD pathology (fig. 2A). These data demonstrate a preexisting
deficit in basal neurogenesis in 3xTgAD mice SGZ that is evident prior to development
of overt AD pathology.
61
Figure 2. Neural progenitor proliferation is deficient in 3-month-old male 3xTgAD mice. A.
Representative immunocytochemical images of amyloid-beta (Aβ) expression in hippocampal
CA1 of 3xTgAD mouse at different ages. Aβ immunoreactivity (IR; 6E10 labeling appears as
green) was not observed in 3-month-old 3xTgAD mouse brain whereas intra-neuronal Aβ IR
within hippocampal CA1 region was evident at 6, 9 and 12-months of age. Extracellular Aβ IR
occurred rarely in 9-month-old mice. B. Representative image of BrdU positive cells in a
3xTgAD mouse hippocampal subgranular zone (SGZ) of mice treated with a single injection of
BrdU (100mg/kg) and sacrificed 24 hrs later. GCl = granular cell layer. C. Bar graph summarizes
results of unbiased stereology indicating a significant decrease in BrdU-positive cells in the
3xTgAD mouse SGZ relative to non-Tg mouse prior to detectable Aβ accumulation. Data are
presented as average ± SEM (n = 4/group), *p < 0.05 as compared to non-Tg. D. Total number of
hipppocampal cells determined by fluorescence activated cell sorting of propidium iodide
positive nuclei of both nonTg (n = 8) and 3xTgAD mouse hippocampi (n = 11) from mice that
had undergone vehicle treatment behavioral assessment. Data indicate that 3xTgAD mice have
significantly lower total hippocampal cell number relative to non-Tg mice. Data are presented as
average ± SEM, *p < 0.05 as compared to non-Tg.
62
2. APα concentration in plasma and cerebral cortex.
In both non-Tg and 3x TgAD mice, plasma and cortical levels of subcutaneously injected
(1, 10, or 20 mg/kg BW) APα were readily detectable by GC/MS 24 hours after
administration and exhibited a linear dose dependent increase with an increasing
magnitude commensurate with dose (fig. 3). APα is a lipophilic molecule (logP = 3.97
with a low molecular weight (318.5 Da). These two figures facilitate APα penetration of
the blood brain barrier.
Basal concentration of APα in plasma (0.47 ng/ml ± 0.88) was significantly lower (p <
0.05) than in cortex (10.36 ng/g ± 1.43) in non-Tg mice, indicating a higher brain
accumulation which is consistent with locally synthesized APα in hippocampus and
cortex (Baulie and Robel, 1990; Mellon and Griffin, 2002; Schumacher et al., 2003). In
contrast, 3xTgAD mice exhibited a lower basal level of APα in the cerebral cortex (6.49
ng/g ± 2.02 vs. non-Tg mice (10.36 ng/g ± 1.43) (fig. 3), suggesting either impairment of
APα synthesis or accelerated APα metabolism in 3xTgAD mice brain. Interestingly,
3xTgAD mice exhibited a consistently lower level of APα in cortex for both the vehicle
and APα treated mice relative to non-Tg. Given that both non-Tg and 3xTgAD mice
received the same doses of APα or vehicle, the lower levels of APα in the 3xTgAD
support accelerated APα metabolism in 3xTgAD mice which could be mediated by
increased expression of 17β-hydroxysteroid dehydrogenase (17β-HSD, aka ERAB,
ScHAD or ABAD) (Mellon and Griffin, 2002; Yang et al., 2005). In the 3xTgAD mouse
cortex, a 10mg/kg dose of APα resulted in a cortical level of 21.92 ± 8.57 ng/g APα / wet
63
tissue 24 hrs post injection which is comparable to the concentration reported for
pregnant day 19 rats (15.9 ng/g ± 2.5) (Concas et al., 1998) and mice (20.9 ng/g ± 2.6)
(Oberto et al., 2002).
Figure 3. APα concentration in plasma and cortex. Samples were collected 24 hrs following a
single subcutaneous injection of APα or vehicle. Subcutaneous injection of APα (1-20 mg/kg)
induced a dose dependent linear increase of APα in both plasma (upper panel) and cerebral cortex
(lower panel) as determined by GC/MS. In non-Tg mice, the basal level of APα concentration in
cerebral cortex was significantly higher than that in plasma (● p < 0.05) which was not evident in
3xTgAD mouse cortex vs. plasma. APα levels in the 3xTgAD mouse cerebral cortex were
significantly lower in both the APα treated and vehicle conditions relative to non-Tg (* p < 0.05)
with a similar pattern evident in plasma. Data are presented as means ± SEM (n ≥ 4 per group)
and were analyzed by two-way ANOVA followed by a post-hoc Neuman-Keuls. Inset depicts the
chemical structure of allopregnanolone (APα aka, AP, Allo, THP; α distinguishes the chemical
structure from other stereoisomers).
64
3. APα reverses the neurogenic deficit of 3xTgAD mouse.
To determine the impact of APα on neural progenitor cell proliferation, BrdU IHC and
unbiased quantitative stereology were conducted. BrdU positive cells were observed in
clusters located in the SGZ of both 3xTgAD and non-Tg mouse hippocampus (fig. 4A
and B). APα treatment was associated with a dose dependent rise in BrdU incorporation
in both non-Tg and 3xTgAD mice (fig. 4C). In the non-Tg SGZ, in which the basal level
of BrdU positive cells was high (4568 ± 1089), APα (10 mg/kg) exerted a modest but
non-significant increase in progenitor cell proliferation (5616 ± 614, p < 0.36). In contrast
in 3xTgAD SGZ and dentate gyrus, APα induced a significant increase in progenitor cell
proliferation with the greatest increase occurring at 10 mg/kg APα (55 ± 18 %, F
(3, 9)
=
3.86 p < 0.05 Neuman-Keuls, p < 0.05, n = 4/group) (fig. 4C). These findings were
supported by APα induced expression of two proliferation markers, proliferating cell
nuclear antigen (PCNA) and cyclin dependent kinase 1 (CDK1/cdc2), in the
hippocampus of 3xTgAD and non-Tg mice.
To ascertain whether APα increased proliferation beyond normal or restored
neurogenesis to normal, total number of BrdU positive cells was determined for non-Tg
and 3xTgAD at the three doses tested (fig. 4D). APα reversed the neurogenic deficit of
3xTgAD (from 2625 ± 426 to 5520 ± 633, p < 0.01) such that proliferation within the
SGZ of 3xTgAD was comparable to BrdU incorporation of non-Tg SGZ (4568 ± 1089, p
< 0.25).
65
These data indicate that APα treatment reversed the proliferative deficit of 3xTgAD mice
and restored NPC proliferation to that of the normal non-Tg SGZ (fig. 4D).
Figure 4. APα reversed the neurogenic deficits in dentate gyrus of 3-month-old 3xTgAD
mice. Stereological estimates of the total number of BrdU-labeled cells in the dentate gyrus of
non-Tg and 3xTgAD male mice treated with 1, 10, or 20 mg/kg APα or vehicle alone. A.
Representative immunolabeled BrdU positive cell images of hippocampal dentate gyrus in APα
or vehicle treated 3xTgAD mice. B. A representative image showing magnification at which
BrdU positive cells were analyzed. C. Summary of dose dependent effects of APα on BrdU
incorporation in non-Tg (upper panel) and 3xTgAD (lower panel) mice SGZ. Note that 10mg/kg
of APα exerted greatest and statistically significant efficacy. D. Comparison of total BrdU
positive cells in SGZ of non-Tg and 3xTgAD mice. Data indicated that treatment with APα
reversed the neurogenic deficits within SGZ of the 3xTgAD mouse to restore 3xTgAD mouse
proliferation to comparable levels of normal non-Tg mice. Bars represent mean ± SEM, n ≥ 4 in
each group, *p < 0.05 vs. vehicle control of the same genotype group.
66
4. APα increased expression of markers of cell cycle progression in the
hippocampus of 3xTgAD and non-Tg mice.
To verify that BrdU incorporation was associated with cell cycle activation and not an
artifact of DNA repair, biochemical and molecular biological analyses were performed to
determine APα regulation of two markers of proliferation, PCNA and CDK1/cdc2,
which were induced in vitro by APα in cultured rat hippocampal progenitor cells (Wang
et al., 2005). Brain samples derived from the same brains, that underwent unbiased
stereological analysis and GC/MC for APα detection, were analyzed using real-time RT-
PCR and Western blot for expression of PCNA and CDK1/cdc2 mRNA and protein
respectively. Results of these analyses indicate that APα induced a dose-dependent
increase in PCNA mRNA in 3xTgAD mice hippocampus (1 mg/kg = 38.7 ± 8.7, p =
0.21; 10 mg/kg = 179 ± 21, p < 0.05; and 20 mg/kg = 170 ± 16 %, p < 0.05, respectively,
vs. the vehicle control; fig. 5A). To confirm that mRNA was associated with increased
protein expression, Western blot analysis was conducted. Results of these analyses
indicated that 10 mg/kg APα induced a significant increase in PCNA protein expression
(128 ± 16 % increase vs. vehicle in non-Tg, p = 0.17 and 162 ± 24 vs. control in
3xTgAD, p < 0.03) and CDK1/cdc2 protein expression (119 ± 9 vs. non-Tg, p = 0.28 and
165 ± 25 vs. vehicle control, p <0.015) in the hippocampi of 3xTgAD relative (fig. 5B).
These data are consistent with the BrdU data and are indicative of entry into the cell
cycle (fig. 4C).
67
Figure 5. APα increased the expression of cell proliferating markers. APα significantly
increased PCNA and CDK1/cdc2 expression in hippocampus of 3-month-old male 3xTgAD mice
whereas APα had no effect on PCNA and CDK1/cdc2 expression in hippocampus of non-Tg
mice. A. Hippocampal mRNA expression of proliferating cell nuclear antigen (PCNA) detected
by real-time RT-PCR in APα or vehicle treated mice. B. Protein expression of PCNA and cyclin
dependent kinase 1 (CDK1/cdc2) detected by Western blot in hippocampi of non-Tg and
3xTgAD mice in the presence or absence of APα. Data are means ± SEM, n = 4/condition, * p <
0.05.
5. Newly formed NPCs in APα treated 3xTgAD mice express neuronal cell markers
in hippocampal dentate gyrus.
To verify the phenotype of the newly formed BrdU-positive cells in vivo, triple-
68
immunolabeling was conducted in sections of mouse hippocampi adjacent to those that
were stereologically analyzed and derived from 3-month-old 3xTgAD mice treated with
a single injection of 10 mg/kg APα. The following phenotypic markers were assessed:
doublecortin (DCX), to label young immature neurons; NeuN, to label mature neurons
and glial fibrillary acidic protein (GFAP), to label astrocytes. Confocal microscopy
identified BrdU positive cells colocalized with DCX alone (arrow head) or together with
NeuN (arrow) (figs. 6A and C). To confirm IR co-localization and sub-cellular
distribution, IR fluorescent intensity was digitally recorded across two BrdU and DCX
positive cells to generate a histogram of fluorescent intensity (fig. 6B). The fluorescent
intensity distribution profile demonstrated an overlap in cell of DCX (red) and BrdU
(green) cell positive cells. These results are consistent with the report by Kempermann et
al. that 24 hour after BrdU injection, a subset of the BrdU positive cells are NeuN
positive (Kempermann et al., 2003) indicating a neuronal lineage of these newly formed
cells. This observation was further confirmed by immunolabeling coronal sections
derived from APα (10mg/kg) treated 3xTgAD mouse dentate 21 days post APα treatment
and behavioral analyses (fig. 6D, see fig. 7A for behavioral paradigm). BrdU positive
cells were located in the middle of granular cell layer indicating the migration of newly
formed cells from the SGZ to the GCL. Collectively, these data indicate that newly
formed cells, generated following APα treatment, express a neuronal phenotype.
69
Figure 6. Phenotypic characterization of the BrdU-positive cells in mouse dentate gyrus. A.
Triple-immunolabeling to co-localize BrdU (a marker for newly formed cells), doublecortin
(DCX, an early progenitor cell marker), and NeuN (a marker for mature neurons) was performed
in sections adjacent to those used for stereological analysis with specific antibodies as described
in the Material and Methods. An illustrative BrdU
+
cell within the dentate gyrus (DG) is labeled
with DCX and NeuN (arrow heads) indicating transition from a young to mature neuron and
migration towards the granular cell layer (GCL) 24 hrs after APα and BrdU injections. B.
Confocal laser scanning histogram of IR fluorescent intensity profile verification of co-
localization. DCX red fluorescent signal in the cytoplasm and BrdU green fluorescent signal in
the nucleus overlapped with NeuN blue fluorescent signal (a nuclear neuronal marker). C.
Further, triple-immunostaining was conducted in mouse brain sections 21 days following APα
and BrdU injection. A fully mature BrdU positive cell located in the middle of the GCL was
positive for NeuN whereas colocalization with the glial cell marker GFAP was not observed. D.
A three-dimensional reconstruction of Z-series images of neuron shown in C.
70
5. APα reverses learning and memory deficits of 3xTgAD mice to performance level
of normal non-Tg mouse.
To determine whether APα-induced neurogenesis was associated with a functional
behavioral consequence, we assessed the impact of APα on a hippocampal-dependent
associative learning and memory task, trace eyeblink conditioning (Thompson and Kim,
1996) which has been shown to be dependent upon the generation of new neurons in the
dentate gyrus (Shors et al., 2001; Shors 2004). Three-month old male 3xTgAD and non-
Tg mice were prepared for behavioral testing and received a single subcutaneous
injection of APα (10mg/kg once) or vehicle 7 days prior to start of the learning trial. The
rationale for the 7 day interval between exposure to APα and the start of the behavioral
experiment was to allow time for the proliferation, migration, and integration of newly
generated neurons into the dentate gyrus (Aimone et al., 2009).
Results of behavioral analyses indicate that at 3 months, 3xTgAD mice exhibited a
learning deficit relative to the performance of normal non-Tg mice at the end of training
(fig. 7B, F
(1, 19)
= 8.177, p < 0.010). APα significantly increased the learning performance
of 3xTgAD mice (fig. 7B, F
(1, 21)
= 4.477, p < 0.04) to a level comparable to non-Tg mice
such that the performance of APα treated 3xTgAD mice was not statistically different
from normal non-Tg mouse (fig. 7B, F
(1, 20)
= 0.563, p < 0.5). Consistent with its modest
effect on neurogenesis in normal non-Tg mice, APα did not augment their learning
performance (fig. 7B, F
(1, 21)
= 0.676, p < 0.5).
71
Following a 9 day period of no training, mice were tested for memory of the learned
association. Vehicle treated 3xTgAD mice exhibited significantly impaired memory
performance relative to vehicle treated non-Tg performance (fig. 7C, F
(1, 14)
= 12.206, p <
0.01). APα treated 3xTgAD mice exhibited a significant increase in memory
performance (F
(1, 19)
= 11.204, p < 0.01) compared to vehicle-treated 3xTgAD mice.
Memory performance of APα treated 3xTgAD mice was restored to a level comparable to
the normal non-Tg mice (Figure 5C, F
(1, 17)
= 0.279, p< 0.5). As in the learning trial, APα
did not significantly augment memory performance of non-Tg mice (fig. 7C, F
(1, 14)
=
0.083, p < 0.8). A two-way ANOVA analysis indicated significant differences in learning
by genotype (F
(1, 126)
= 19.787, p < 0.0001) and days of training (F
(5, 126)
= 8.729, p <
0.00001). No interaction was observed between days of training and genotype (F
(5, 126)
=
1.671, p < 0.2). Relative to vehicle treated 3xTgAD mice, APα treated 3xTgAD mice
exhibited a significantly higher learning and memory performance (F
(1, 138)
= 11.086, p <
0.01) (figs. 7B and C).
To determine the specificity of APα on associative learning, 3xTgAD mice underwent
either paired or unpaired trace eye-blink conditioning. Results of these behavioral
analyses indicated that APα significantly increased learning and memory performance of
3xTgAD mice that received paired trace eye-blink conditioning while exerting no effect
upon performance in the unpaired condition (p = 0.48) (figs. 7B and C).
72
Figure 7. APα reversed the learning and memory deficits of 3xTgAD mice. A. Experimental
behavioral paradigm. 3xTgAD and non-Tg background mice received a single s.c. injection of
APα (10mg/kg) or vehicle followed one hour later by a single injection of BrdU (100mg/kg).
Animals were returned to home cages for 7-days to allow for regenerative neurogenesis and
migration of newly generated cells to dentate gyrus. Following the 7-day interim regeneration
period, behavioral testing commenced with a 5-day (2 x 30 trials/day) training/learning phase,
mice were returned to home cage for 9 days followed by a memory test of the learned association.
B. At the end of the learning phase, 3xTgAD male mice displayed significantly fewer conditioned
responses than non-Tg mice indicating impaired learning performance (grey square vs. grey
triangle, *p < 0.01). APα significantly increased the conditioned response / associative learning of
3xTgAD mice (grey square vs. black square, *p < 0.04) to a level comparable to non-Tg mice
(black square vs. grey triangle, p < 0.46). In the non-Tg mice, APα did not augment conditioned
response performance (grey triangle vs. black triangle, p < 0.5). C. Nine days following learning
phase, 3xTgAD mice exhibited significantly decreased memory performance (28%; *p < 0.005)
relative to non-Tg mice performance (50% conditioned response). APα significantly increased
memory performance of 3xTgAD (54% p < 0.004 vs. vehicle control) to a level of conditioned
response comparable to that of non-Tg mice. Memory performance of non-Tg mice was
unaffected by APα treatment. APα-induced increase in learning and memory performance was
specific to associative paired trace conditioning as APα had no effect on performance of 3xTgAD
in the unpaired trace conditioning on either learning (B) or memory (C). Two way ANOVA
analysis indicated significant differences in learning for genotype (p < 0.001) and days of training
(p < 0.001).
73
Classical trace conditioning can, independent of exogenous growth factors, increase the
number of newly formed BrdU positive cells in rat hippocampus (Shors et al., 2001). A
higher number of trials (> 800 /day) can increase BrdU positive cells in SGZ whereas a
lower number of trials (200 trials /day) does not (Leuner at al., 2004). To evaluate the
contribution of trace conditioning training trials versus APα on the number of newly
generated BrdU positive cells in 3xTgAD mouse and behavioral response, a comparative
analysis was conducted in which 3xTgAD mice received either no-training ± APα or
training ± APα. Mice were sacrificed following completion of the memory test. Results
of this analysis indicated no statistical difference (p < 0.9) in the total number of BrdU
positive cells under the no-training (624.8 ± 219) and training vehicle treatment
conditions (633.6 ± 115) (fig. 8). These data indicate that the training paradigm did not
induce an increase in proliferation. In contrast, APα induced a significant increase in the
number of BrdU positive cells in the SGZ of mice under both the no-training (1228 ±
280) and training (1132 ± 210) conditions (p < 0.05 vs. vehicle control, fig. 8). These
results indicate that APα-induced proliferation is independent of training and is directly
related to APα exposure. Further, these data indicate that APα treatment increased the
survival of the newly formed BrdU cells, as only the APα treated mice exhibited
significantly more BrdU positive cells in both non-trained and trained mice (p < 0.05, fig.
8). These data indicate that the trace conditioning training paradigm (2 x 30 trials per day
for 5 days) used in this study, did not induce a rise in BrdU incorporation and thus did not
contribute to the number of surviving BrdU-positive cells (21 days after APα and BrdU
exposure) (fig. 8).
74
Figure 8. APα-induced neurogenesis is specific to APα and not induced by trace eye-blink
conditioning training. To distinguish between the contribution of APα and training on BrdU
incorporation, 3xTgAD mice were either trained for 2 x 30 trials/day for 5 days or underwent
preparation for training but did not receive training sessions. APα and BrdU injections occurred
as per experimental paradigm described in Figure 5 with animals sacrificed at the end of the
memory test session. BrdU positive cells were analyzed by unbiased quantitative stereology. APα
induced a significant increase of BrdU
+
cells compared to vehicle controls in both the no-training
and trained conditions (1228 ± 280 vs. 625 ± 219, and 1132 ± 210 vs. 633.6 ± 115, * p < 0.05)
whereas no effect of vehicle occurred in either the no-training or trained conditions. These data
indicate a direct effect of APα on neural progenitor cell proliferation whereas the training
paradigm had no effect (p = 0.87) on proliferation. Bars represent mean ± SEM.
The number of surviving BrdU positive cells 21-days after BrdU injection (fig. 8) is
approximately 5 times less than that detected 1-day after BrdU injection (figs. 2 and 4).
This ratio is in agreement with that reported by Gage‟s group (Kempermann et al., 2003)
75
that the number of BrdU positive cells is reduced by about a factor of 5 within one month
after BrdU injection in C57BL/6 mice. Furthermore, the majority of the newly formed
cells will undergo apoptosis within one week without stimulation (Aimone et al., 2009).
The above results demonstrate that APα reversed the deficits in neuroprogenitor cell
proliferation and associative learning and memory of 3xTgAD mice. To determine the
relationship between increased neurogenesis and memory enhancement, a correlational
analysis was performed between memory performance and magnitude of BrdU positive
cells within each 3xTgAD mouse. The number of BrdU positive cells per dentate in the
3xTgAD vehicle treated group was 672.8 ± 88, whereas in 3xTgAD APα treated group
the number of BrdU+ cells was 1136 ± 138 and the average percent increase in
conditioned response (CRs) was 28.33 ± 4.7 and 53.96 ± 9.8, respectively. Pearson
correlation analysis indicated a significant correlation between BrdU positive cells and
increased memory performance in APα treated group (r = 0.80, p < 0.01) which was not
apparent in the vehicle treated group where number of BrdU+ cells were not correlated
with memory performance (r = 0.13, p < 0.3) (fig. 9). These data indicate that memory
performance in the vehicle treated group did not require survival of newly generated cells
whereas memory performance in the APα treated 3xTgAD was correlated with and likely
required the survival of APα-induced generated neurons.
76
Figure 9. APα reversal of memory deficit is correlated with neurogenesis in 3xTgAD mice.
A. Scatterplots of total number of BrdU positive cells (x axis) vs. percentage of conditional
responses (y axis) of each 3xTgAD mouse tested. Correlation (r value) between number of
surviving BrdU positive cells and memory performance in vehicle (r = 0.13) and APα treated
3xTgAD mice (r = 0.80). B. Pearson correlation analysis indicated a highly significant correlation
(p < 0.01) between the number of surviving BrdU positive cells and memory performance in APα
treated 3xTgAD mice. Performance in vehicle treated 3xTgAD mice was not significantly
correlated (p < 0.2) with BrdU labeled cells.
Discussion
APα reversed the neurogenic and cognitive deficits that typify the male 3xTgAD mouse
prior to the onset of overt Alzheimer‟s pathology. APα-induced neural progenitor
proliferation in vivo is consistent with earlier in vitro analyses in which APα significantly
77
increased proliferation of human and rat neural progenitors in a dose dependent and
steroid specific manner via a GABAA receptor and L-type Ca
++
channel mechanism
unique to neural progenitor cells (Wang et al., 2005). In vivo a single EC
100
neurogenic
dose of APα was functionally relevant and reversed hippocampal-dependent associative
learning and memory deficits. The significant increase in associative memory induced by
APα was highly correlated with APα-induced neurogenesis.
Neurogenic and cognitive deficit of 3xTgAD mouse
The 3xTgAD mouse, carrying homozygous mutant genes (APP
Swe
, PS1
M146V
and
tau
P301L
), mimics multiple aspects of AD neuropathology in AD-relevant brain regions
(Oddo et al., 2003). Results of our analyses demonstrated a significant neurogenic deficit
as early as 3 months in the 3xTgAD mouse SGZ. Consistent with our finding, several
studies indicate a SGZ neurogenic deficit in transgenic and knock-in mice that express
mutated human APP (Donovan et al., 2006; Haughey et al., 2002) or PSEN1 and APP
(Feng et al., 2001; Wen et al., 2004). In the 3xTgAD SGZ, a neurogenic deficit was
detected at 9 months (Rodriguez et al., 2008) whereas our data indicate a significant
deficit in the SGZ by 3 months. In our study, neurogenic deficit was detected using
unbiased quantitative stereology which was confirmed by analyses of cell cycle gene
expression whereas Rodriguez and colleagues quantified phosphorylated Histone 3
(Rodriguez et al., 2008). However, a substantial decrement in phosphorylated Histone 3
positive cells was evident at 3 months but failed to reach statistical significance due to
high variability (Rodriguez et al., 2008). Our results confirm these findings but more
78
importantly indicate that the deficit in neurogenesis can occur prior to the development of
overt AD pathology.
Emerging evidence indicates that neurogenic deficits are paralleled by cognitive deficits
and that associative learning and memory require temporal encoding that is dependent
upon the generation of new neurons in the dentate gyrus (Shors et al., 2001; Shors et al.,
2004; Aimone et al., 2009; Yamasaki et al., 2007). The deficiency in hippocampus-
dependent associative learning in the 3-month-old 3xTgAD mice is consistent with other
cognitive deficits in these animals, including dysfunction in synaptic plasticity, deficits in
LTP and paired-pulse facilitation and early Alzheimer's disease-related cognitive deficits
(Oddo et al., 2003; LaFerla et al., 2007). The coincidence of neurogenic and cognitive
deficiency prior to overt evidence of AD pathology suggests a close link between a
deficit in neurogenesis and deficits in cognitive function. The neurogenic deficit of the
3xTgAD mouse is not unique to the subgranular zone of the dentate gyrus. We have
detected a comparable deficit in proliferative capacity of the 3xTgAD subventricular
zone (Liu et al., 2008). To date, we have no evidence of a neurogenic deficit in the
cerebellum which influences eyeblink conditioning. However, analyses are currently
underway to investigate this issue.
APα in aging and Alzheimer’s disease
Proliferation of neural progenitor cells (NSCs) is markedly diminished in the aged and
AD brain (Weill-Engerer et al., 2002; Hattiangady and Shetty, 2008). While underlying
mechanisms which mediate the age and AD associated decline in regenerative potential
79
of neural progenitor cells remains to be fully determined, peptide growth factors and
neurosteroids have been shown to play a key role. In middle-aged rat hippocampus, the
average concentration of several peptide growth factors, FGF-2, IGF-1 and VEGF are
substantially lower relative to that in young rat hippocampus (Hattiangady and Shetty,
2008). Consistent with a decline in growth factors, a significant decrease in APα was
evident in aged and AD brains (Genazzani et al., 1998; Weill-Engerer et al., 2002; Marx
et al., 2006). Interestingly, APα in the 3xTgAD mouse plasma and cortex was
consistently lower in the 3xTgAD mouse compared to the non-Tg. As both non-Tg and
3xTgAD mice were treated with the same dose of APα under exactly the same
conditions, the decrease in APα level must be attributable to either decreased absorption
or increased metabolism. Our working hypothesis is that the decrease in APα is due to
metabolism via increased expression of 17β-hydroxysteroid dehydrogenase (17β-HSD,
aka ERAB and ABAD) (Mellon and Griffin, 2002; Yang et al., 2005; Lustbader et al.,
2004). This enzyme is significantly elevated in APP transgenic AD mice but not in
normal controls (Lustbader et al., 2004; Takuma et al., 2005). Elevation of 17β-HSD /
ABAD in both liver and brain would be consistent with increased metabolic conversion
of APα to 5alpha-dihydrotestosterone leading to lower levels of APα in both plasma and
brain. Together, these data suggest that the changes in the local biochemical milieu of
neurosteroids and peptide growth factors could contribute to neurogenic deficits in early
stages of AD (Brinton et al., 2008; Wang et al., 2007).
Cell cycle gene expression in neural progenitor cells is an obligatory requirement for
80
neurogenesis and ultimately regeneration. However, aberrant entry into the cell cycle has
been reported to precede neuronal death in the cortex and CA3 regions at all stages of
AD, from MCI to late stage AD and within AD mouse models (Herrup and Yang, 2007).
Further, expression of the ectopic cell cycle proteins ultimately predicts the demise of
these neurons (Yang et al., 2006). These findings are especially challenging for
therapeutics targeting the regenerative potential of endogenous neural stem/progenitor
populations as an unintended side effect may be to promote ectopic entry of neurons into
the cell cycle and thereby exacerbate neuron demise. Herrup‟s group recently
demonstrated that aberrant entry into the cell cycle is triggered by Aβ oligomers which
first appear in the frontal cortex layers II/III (Varvel et al., 2008). In contrast, LaFerla‟s
group recently reported that neuronal cell cycle reactivation is not induced by Aβ or tau
pathology, but rather appears to be triggered by acute neuronal loss (Lopes et al., 2009).
As the 3xTgAD mouse does not exhibit Aβ accumulation or neuronal loss at 3-months of
age, APα-induced cell cycle gene and protein expression, concomitant with markers of
proliferation and survival, is unlikely to be due to aberrant entry into the cell cycle. An
extensive series of analyses are currently underway to determine the impact of APα on
neural progenitor proliferation and cell cycle gene expression during the development of
AD pathology in the 3xTgAD mouse brain.
Mechanism of APα induced neurogenesis
Earlier in vitro analyses demonstrated that APα significantly increased proliferation of
both rodent and human neural progenitor cells (Wang et al., 2005). APα-induced
81
proliferation was mediated via GABAA receptor-activated voltage-gated L-type Ca
++
channels (Wang et al., 2005) leading to a rapid rise in intracellular calcium in neural
progenitors (Wang and Brinton, 2008). In neural progenitor cells, the high intracellular
chloride content leads to an efflux of chloride through the GABA
A
channels upon
opening, which leads to depolarization of the membrane and influx of Ca
++
through
voltage dependent L-type Ca
++
channels and activation of the CREB transcription factor
(Wang et al., 2005; Wang and Brinton, 2008; Jagasia et al., 2009). Through this pathway,
APα stimulation of GABA-mediated excitation and CREB signaling can activate a key
pathway in adult hippocampal neurogenesis, to promote the proliferation, survival and
differentiation of neural progenitor cells. While this APα-activated pathway is relevant to
induction of NPC proliferation, the mechanisms by which APα promotes survival of
rNPCs which could involve either delayed or prolonged actions of APα on gene
expression and neuron survival mechanisms remain to be determined.
An important mechanistic consideration is the impact of behavioral training which can
induce neural progenitor cell proliferation independent of exogenously administered
factors (Shors et al., 2001). We elected to use the trace eye-blink conditioning paradigm
based on the studies of Shors and colleagues (Shors et al., 2001) who demonstrated that
newly generated neurons within the dentate gyrus contribute to the association of stimuli
that are separated in time, which is a hippocampal-dependent associative learning and
memory function (Aimone et al., 2009). Unlike the high number of training trials used in
the Shors analyses (800 trials over three days in Leuner et al., 2004), which can promote
82
the survival of newly generated neural progenitors, we intentionally used a lower number
of training trials (2x30 trials/day for 5 days) which our data show does not change the
number of surviving BrdU
+
cells. Verification that the behavioral paradigm used in this
study did not induce proliferation was evident in the lack of difference between
proliferation in the trained and untrained vehicle treated 3xTgAD mice. As most of the
vehicle 3xTgAD mice failed to learn (average CRs of 28%), the subthreshold training
regime would not be expected to increase the number of surviving cells which is
consistent with Waddell and Shors (2008). Analyses to determine the impact of APα on a
broader array of behavioral measures are underway.
In conclusion, results of the current analysis demonstrate that APα reversed deficits in
SGZ neurogenesis, learning and memory in the 3xTgAD mouse model of Alzheimer‟s
disease to restore both regenerative and cognitive function to that of the normal non-
transgenic mouse. From a translational perspective, blood brain penetrance of APα,
efficacy of a single exposure and mechanism of APα action make it an ideal molecule
and a promising regenerative therapeutic candidate for promoting neural regeneration and
reversing cognitive deficits associated with the prodromal stage of Alzheimer‟s disease.
83
CHAPTER THREE
Allopregnanolone Induced Neurogenesis Enhances the Learning and Memory of
Adult Triple Transgenic Alzheimer’s disease Mice
Chapter Three Abstract
Previously, we demonstrated that allopregnanolone (AP ) increased proliferation of
neural progenitor cells in vitro (Wang et al., 2005) and in vivo to reverse both neurogenic
and cognitive deficits in the male triple transgenic mouse model of Alzheimer‟s disease
(3xTgAD) prior to appearance of AD pathology (Wang et al, 2010) In the current
analysis, we determined the efficacy of AP to promote neurogenesis and cognitive
function in 6, 9, 12 and 15-months-old 3xTgAD male mice and their non-Tg counterparts
following the onset of AD pathology in the transgenic mice. Neural progenitor cell
proliferation was determined by flow activated cell sorting (FACS) analysis of
hippocampal BrdU+ cells at the end of the behavioral tests. Cognitive performance was
assessed by an associative learning task - trace eyeblink conditioning. AP significantly
increased both learning and memory function and survival of BrdU positive cells in 6 and
9 months old 3xTgAD male mice when intraneuronal β-amyloid occurs with no effect in
the 12-months and older 3xTgAD mice when β-amyloid plaques are abundant and
neurofibrillary tangle pathology starts becoming apparent. Whereas the positive effect of
APα disappeared at an age of 12 months in 3xTgAD mice, the non-Tg mice started
showing a positive trend at this age and converted into a significant increase at an age of
15 months. These results provide preclinical evidence that AP can restore cognitive
84
performance and promote adult neurogenesis in the pre-plaque phase of AD pathology
and in normal aging.
Introduction
In the past decades studies from multiple laboratories have revealed the existence of
neurogenesis in restricted brain regions throughout adulthood across various species,
including rodents (Altman and Das, 1965; Gage et al., 1998; Kempermann et al., 1998),
songbirds (Alvarez-Buylla et al., 1988a; Alvarez-Buylla and Nottebohm 1988b), and
humans (Eriksson et al., 1998). In most mammals, adult neurogenesis is restricted to the
subventricular zone (SVZ) of the lateral ventricle (Alvarez-Buylla et al., 2008) and the
subgranular zone (SGZ) of the dentate gyrus in the hippocampus (Gage 2003; Gage et al.,
1998). The dentate gyrus is the projecting target of the perforant pathway, which is the
major cortical input from the entorhinal cortex to the hippocampus, and the newly
generated neurons in the dentate gyrus contribute to multiple forms of hippocampal-
dependent learning and memory (Aimone et al., 2006; Shors, 2008).
While the regenerative potential of the mammalian brain is sustained throughout the life
span, the magnitude of the proliferative efficacy of neural progenitors declines with age
and diseases, such as Alzheimer‟s disease (Hattiangady and Shetty 2008; Hattiangady et
al., 2007; Kuhn et al., 1996; Rao et al., 2005). Aging-associated decline in hippocampal
neurogenesis has been reported in multiple species by multiple laboratories ever since the
discovery of adult neurogenesis (Altman and Das 1965; Seki and Arai 1991; Kuhn et al.,
1996; Kempermannet al., 1998; Eriksson et al., 1998) and the majority of the studies
focused on cell proliferation and much less about the other stages of neurogenesis. The
85
effect of Alzheimer‟s disease (AD) burden on adult neurogenesis has been extensively
studied in various animal models on both cell proliferation and survival (Wen et al.,
2002; Wang et al., 2004; Chevallier et al., 2005; Haughey et al., 2002; Donovan et al.,
2006; Verret et al., 2007), as well as in human brains (Jin et al., 2004). Due to the animal
models and species differences, effect of AD burden on cell proliferation is complex and
sometimes contradictory. While age- and AD-related neurogenesis decline has been
reported in various studies and may contribute to age- and AD-associated cognitive
decline (Chevallier et al., 2005; Donovan et al., 2006; Jin et al., 2004; Kuhn et al., 1996;
Morgenstern et al., 2008; Rodriguez et al., 2008), the underlying mechanism for this
neurogenic deficit remains unclear. It is suggested one of the factors might be the
changes in local biochemical milieu, including growth factors, cytokines,
neurotransmitters, and neurosteroids (Brinton 2009).
Allopregnanolone (AP ), a metabolite of progesterone, is a neurosteroid synthrsized de
novo in both embryonic and adult CNS (Mellon and Griffin 2002) as well as
pluripotential progenitor cells (Gago et al., 2004), and exhibits an age- and AD-
associated decline (Bernardi et al., 1998; Brinton et al., 2008; Marx et al., 2006; Weill-
Engerer et al., 2002). Using a triple-transgenic mouse model of Alzheimer‟s disease
carrying APP
Swe
, PS1
M146V
, and tau
P301L
(3xTgAD)(Oddo et al., 2003), we have
previously shown that AP reversed both neurogenic and cognitive deficits in 3-month-
old 3xTgAD mice (Wang et al, 2010) in addition to its proliferative effect in vitro (Wang
et al., 2005). Since this mouse model exhibits age-dependent neuropathology of both β-
amyloid plaque formation and neurofibrillary tangles starting at 6-month-old of age
86
(Oddo et al., 2003), it would be important to determine the efficacy of AP when
pathological burden is apparent. We hence extended our study to 6, 9, 12 and 15-months-
old non-Tg and 3xTgAD mice to further elucidate the effect of AP on both
neurogenesis and cognitive performance.
Materials and methods
Steroid
Allopregnanolone used for this study was purchased from Steraloids Inc. (Newport,
Rhode Island, USA). The stock solution for APα was made in pure ethanol and was
diluted in PBS before injection.
Animals and treatment
Breeding pairs of the triple transgenic Alzheimer‟s disease mouse (3xTgAD,
homozygous mutant of human APP
Swe
, PS1M
146v
and tau
P301L
) and its background strain
(129/Sv x C57BL/6) were obtained from Dr. Frank LaFerla (University of California,
Irvine) and the colonies were established at University of Southern California. The
characterization of amyloid and tau pathologies, as well as synaptic dysfunction in this
line of mice has been described previously (Oddo et al., 2003; Billings et al., 2005) and
confirmed in our laboratory. Mice were genotyped regularly to confirm the purity of the
colony. Experiments were performed using 6, 9, 12 and 15-months-old male 3xTgAD
and non-Tg mice. The number of mice per condition is indicated in the results section.
Mice were maintained under a 12hr light/dark cycle with continuous access to food and
water. APα stock solution was prepared in pure ethanol and diluted in PBS before
injection (with a final ethanol concentration of 0.002% of the body weight). Mice of each
87
genotype and age received a subcutaneous (s.c.) injection of APα at a concentration of 10
mg/kg body weight (BW), an optimal dose of AP based on our previous studies. One
hour after APα injection, mice were intraperitoneally (i.p.) injected with 100 mg/kg BW
bromodeoxyuridine (BrdU). The experimental paradigms used was similar to the one
used in our previous study - APα treatment for behavior and cell survival assessment
(Fig.1B), in which animals received a single shot of APα/vehicle, followed by a 7-day
neurogenesis and migration phase, followed by a 5-day trace-conditioning test followed 9
days later by a 1-day memory test, and scarified 21days after a single APα injection at
which time FACS analysis of BrdU positive cells was performed to assess cell survival;
Timing of AP and BrdU injections, training diagrams, and perfusion were based on
previous studies showing that learning enhances the survival of newly born cells
generated 1 week before training (Gould et al., 1999; Aimone et al., 2006) and from our
previous analysis indicating that APα-induced neurogenesis significantly increased
learning of trace eyeblink conditioning in 3 month old 3xTgAD male mice following an
injection of AP one week prior to behavioral testing.
All experiments strictly conformed to the Animal Welfare Act, Guide to Use and Care of
Laboratory Animals, and the U.S. Government Principles of the Utilization and Care of
Vertebrate Animals Used in Testing, Research, and Training guidelines on the ethical use
of animals. In addition, the minimal number of required animals was used for these
experiments and pain was minimized.
88
Trace eyeblink conditioning
Under deep anesthesia by intraperitoneal (i.p.) injection with ketamine (100 mg/kg i.p.)
and xylazine (25 mg/kg i.p.), a 4 pin head stage (DIGI-KEY) was cemented to the skull
of 3 month old male mouse with dental acrylic. The connector has four Teflon-coated
stainless steel wires and one bare stainless steel wire (0.003” bare and 0.0055” coated, A-
M Systems, Inc.). The bare wire was attached via a gold pin (Time Electronics) to the
head stage. Coated wires were implanted s.c. in the orbicularis oculi dorsal to the left
upper eyelid to record the EMG and s.c. periorbitally to deliver the shock US (3). All
animals were then placed on a warm isothermal pad after surgery to recovery for 30 min.
After surgery, mice were individually housed, provided with ad libitum access to food
and water, and maintained on a 12 hr light/dark cycle.
After one week of acclimation to the colony room, mice with no obvious adverse
responses to surgery were randomly assigned to an experimental condition. Mice were
injected subcutaneously (s.c.) with 10 mg/kg AP or vehicle followed one hour later with
an IP injection of BrdU (100 mg/kg). Following injection of test compound, mice were
returned to their home cage for 7 days prior to onset of behavioral testing.
During the first day of training, mice were placed within Plexiglas cylinders in a sound-
attenuated chamber and were habituated to the test environment for one session
consisting of 30 stimulus-free trials at 30-60 sec inter-trial intervals while spontaneous
eye-blink activity was recorded using electromyographic (EMG) activity recorded from
the obicularis oculi dorsal to the orbit during each trial. EMG activity was rectified, and
integrated using custom designed computational Labview routines (Tseng et al., 2004).
89
Following habituation to the test environment mice underwent a learning phase and were
trained for five days. Mice were trained by pairing delivery of a tone (CS, 250 msec, 2
kHz, 85 dB) as the conditioned stimulus followed by a 250 msec period of no stimuli,
followed by the periobital shock as the unconditioned stimulus (US, 100 msec). Mice
received two blocks of 30 trials per day (30–60-sec inter-trial intervals, 3–4-h inter-block
intervals). This trace eye-blink conditioning paradigm is subthreshold for inducing
neurogenesis (Gould et al., 1999; Leuner et al., 2004). The unpaired group received
random tone at the same magnitude as the paired conditioning and shock with 15-30 sec
as inter-trial intervals and a total of 60 trials for one session per day. Shock intensity was
adjusted daily for each mouse to elicit a head-movement response. Following the learning
phase, mice were returned to their home cage for eight days followed on the ninth day by
a single session to assess memory of the conditioned response. The percentage of CR was
computed as the ratio of the number of CRs to the total number of valid trials. Animals
were perfused at the end of the memory trial day.
Animal dissection and tissue collection
Mice were sacrificed at the end of memory test for cell survival assessment (Fig.1A). On
the day of sacrifice mice were deeply anesthetized with a combination of ketamine (100
mg/kg) and xylazine (10 mg/kg), and perfused with PBS. Brains were dissected into two
hemispheres, and one hemisphere was fixed immediately in cold 4% paraformaldehyde
and was used for FACS analysis (cell survival).
90
Nuclei extraction and flow cytometry counting
Hippocampus was dissected from the fixed hemispheres from cell survival assessment
experiment using consistent anatomical landmarks as criteria for dissection as described
before (Bilsland, 2006). The rostral 1/3 of the hippocampus lobe was removed to avoid
the subventricular zone and rostral migratory stream proliferative pools. The extracted
hippocampi were homogenized using Next advance 24 sample homogenizer (Next
Advance Inc., NY) for 3 minutes on speed 7. This procedure lyses the plasma lemma
while preserves the nuclear envelope intact. The nuclei sample was collected into a
regular 1.5mL microcentrifuge tube by washing the beads and tube 4 times using 200μL
of PBS, and then centrifuged for 10 minutes at 10,000 rpm. Once all of the nuclei were
collected in a pellet, the supernatant was discarded. The pellet was then re-suspended in
600μL of PBS plus 0.5% Triton x-100. The number of nuclear density was estimated by
counting the propidium iodide (PI), a fluorescent molecule stoichiometrically binds to
DNA by intercalating between the bases with no sequence preference, positive particles.
Aliquots of 25μl were re-suspended in 200μL of a 0.2 M solution of boric acid, pH 9.0,
and heated for 1h at 75°C for epitope retrieval. After washed in PBS, the nuclei were
incubated for 24hours at 4ºC with primary mouse monoclonal anti-BrdU antibody (1:100,
Abcam, Ab12219) and subsequently with FITC-conjugated goat anti-mouse IgG
secondary antibody (1:100 in PBS; Vector Labs, FI-2001). The remainder of cell
suspension is diluted to 500μL and sent for flow cytometry assay using Beckman FC 500
System with CXP Software. Propidium iodide (PI) cells were first gated on a histogram;
the expressing cells were visualized on a forward/side scatter plot. PI cells were „back-
91
gated‟ on the forward/side scatter plot to eliminate debris prior to analysis; this also
eliminated auto fluorescence of the sample. Gates were always set using dissociates with
cell aliquots which lack of the first antibody, but which were incubated with second
antibody and processed alongside the experimental procedure. PI-labeled cells in a fixed
volume were gated, and the number of cells showing BrdU signal was analyzed. Data
were expressed as total positive cells per hippocampus.
Statistical analysis
Data were analyzed using a one-way ANONA followed by Neuman–Keuls post hoc
analysis. Data displayed in graphs were reported as mean ± SEM or fold change ± SEM.
P-values of < 0.05 were considered to be significant regardless of the statistical test used.
Results
1. Rationale for and design of AP treatment experiments.
Our previous analyses demonstrated that AP reversed cognitive and neurogenic deficits
of male 3xTgAD mice (3 months of age) prior to the onset of AD pathology (Wang et al,
2010). The current study was designed to determine the efficacy of AP to promote
cognitive and neurogenic function in 3xTgAD male mice with mild (6-month-old),
moderate (9-month-old) and severe (12-month-old) β-amyloid accumulation. 15 months
old 3xTgAD mice exhibiting extensive extracellular plaque pathology and NFTs were
used as a further control. Cognitive performance was assessed using the trace eyeblink
conditioning behavioral paradigm (fig. 10A) which provides an indication of associative
learning and memory that is dependent upon the generation of new neurons in the dentate
92
gyrus (Shors et al., 2001) and is sensitive to aging and Alzheimer‟s disease burden
(Kishimoto et al., 2001; Woodruff-Pak et al., 1990). As indicated in the experimental
design (fig.10B), animals received a single s.c. injection of APα (10mg/kg) or vehicle 7
days prior to the start of the learning trials, a time period sufficient for the proliferation,
migration, and integration of newly generated neurons into the dentate gyrus (Shors et al.,
2001). Cell survival assessment was conducted 21 days after APα or vehicle injection.
The 21-day time frame was specifically selected as newly generated neurons would
survive only when they integrate into existing neuronal network, which occurs
approximately 2-3 weeks after birth in SGZ of the hippocampus.
93
Figure 10. Experimental design - A. Experimental plan for behavior test and cell survival
determination. Mice were surgically implanted with electrodes to measure the electromyogram
(EMG) of the eyelid muscle. After 3~4 day recovery, mice were injected s.c. with either APα (10
mg/kg) or vehicle and 1 hr later with i.p. injection of BrdU (100 mg/kg). One week later, Mice
were subjected to 1-day habituation and subsequently 5-day behavior test using trace eyeblink
conditioning. At the end of the acquisition phase, mice were housed in their home cages and
tested for the retention of the memory after 9 days. Following the memory test, animals were
sacrificed and the brains were collected for assessment of cell survival. B. During trace eyeblink
conditioning, CS, a tone, was paired with US, periorbital shock), which elicits an eyeblink
response. The trace period between the CS offset and US onset was 250 ms. Eyeblinks were
detected by an increase in the magnitude of the EMG. Eyeblinks occurring during the trace
interval were a measure of learning and were considered CRs.
2. AP reversed cognitive and neurogenic deficits in 6-months-old 3xTgAD mice.
At 6-months of age, intraneuronal beta amyloid (A ) accumulation is apparent in the
94
hippocampus whereas A plaques are absent. In 6-month-old 3xTgAD male mice, AP
reversed the learning and neurogenic deficit of these animals with no effect in normal
non-Tg mice (figs. 11A-C). Vehicle-treated 6-month-old non-Tg mice achieved a
maximal conditioned response rate of 31.3 ± 6.1% (fig. 11A). AP -treated non-Tg mice
achieved a maximal conditioned response rate of 30.4 ± 7.7% which was not statistically
different than vehicle- treated (F
(1, 20)
= 0.010; p > 0.05). There was no significant
difference observed during the course of training within the vehicle-treated and AP -
treated groups (F
(1, 108)
= 0.292; p > 0.05). Learning performance was mirrored in their
memory performance 9 days post learning phase which was not statistically different
than vehicle-treated non-Tg mice (F
(1, 16)
= 0.038; p > 0.05) (figs. 11B & C). In contrast,
AP significantly increased learning performance of 6-months-old 3xTgAD mice from a
basal learning level of 17.4 ± 3.3 % to 39.2 ± 9.2% (F
(1, 12)
= 4.943; p < 0.05) at the end
of training (fig. 11B). During the entire course of training, AP -treated 3xTgAD mice
performed significantly better than vehicle-treated mice (F
(1, 68)
= 31.072; p < 0.001)
AP - treated 3xTgAD mice performed comparable to the vehicle-treated non-Tg mice at
the end of the training (F
(1, 15)
= 0.538; p > 0.05). AP also significantly increased the
retention of the conditioned response rate in the memory test conducted 9 days after the
acquisition phase in 3xTgAD mice (F
(1, 10)
= 5.560; p < 0.05) (fig. 11C). Vehicle-treated
3xTgAD mice showed a reduced basal level of learning as compared to the vehicle-
treated non-Tg mice but it was not statistically significant due to a large variation.
To determine whether cognitive performance was correlated with survival of
95
neuroprogenitor cells, hippocampi from behavioral test animals were analyzed for total
number of BrdU positive cells using fluorescence activated cell sorting (FACS). As BrdU
was administered one hour post AP injection at the start of the behavioral experiment
(fig. 10B), FACS analysis detected the total number of surviving BrdU+ cells and thus
can be used as a marker of neural progenitor cells that survived and integrated into the
hippocampal neuronal network. In 6-months-old vehicle treated non-Tg mice, BrdU+ cell
survival was 2003.6 ± 229.5 (fig. 11D). In AP -treated non-Tg mice, 2772.2 ± 534.9
BrdU+ cells survived which was not significantly different from vehicle-treated (F
(1, 13)
=
1.142; p > 0.05). 6-months-old 3xTgAD mice exhibited a significantly lower number of
surviving BrdU+ (574.1 ± 136.9) compared to non-Tg mice (F
(1, 15)
= 27.937; p < 0.001).
AP significantly increased BrdU+ in cell survival (1372.5 ± 326.2) (F
(1, 19)
= 4.069; p <
0.05) to promote cell survival by greater than 2 fold in 3xTgAD mice (fig. 11D).
96
Figure 11. AP reversed the learning and neurogenic deficits in 6-month-old 3xTgAD mice.
A. Learning curve of 6-month-old non-Tg mice - The non-Tg group showed a modest level of
learning (highest level: 31.3 ± 6.1%) and APα did not augment their learning. B. Learning curve
of 6-month-old 3xTgAD mice - The 3xTgAD group showed a reduced basal level of learning
when compared to non-Tg group (but not significant). APα treatment significantly increased both
the rate of learning and the highest CR% compared to vehicle-treated 3xTgAD mice, and the final
level was comparable to the non-Tg group (p > 0.05). C. Summary of the results from memory
test in 6-month-old animals, conducted 9 days after the acquisition phase - APα significantly
enhanced the memory retention in 6-month-old 3xTgAD mice while showed no effect on the age-
matched non-Tg mice. D. Summary of results from cell survival analysis in post-training brains
using fluorescent activated cell sorting (FACS) - 6-month-old 3xTgAD mice had a significantly
lower basal level of cell survival in SGZ as compared to the non-tg mice. Treatment with APα
significantly reversed the neurogenic deficiency in 6-month-old 3xTgAD mice while showed no
significant impact in the age-matched non-Tg group. * p < 0.05 as compared to 3xTgAD +
vehicle group; † p < 0.05 as compared to non-Tg + vehicle group. Data are presented as average
± SEM (n = 7 – 12).
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3. APα reversed the cognitive and neurogenic deficits in 9-months-old 3xTgAD mice.
At 9-months of age, greater intraneuronal A accumulation is apparent in the
hippocampus and in very rare instances A plaques have developed. Similar to the
effects in 6-months-old animals, AP reversed the learning and neurogenic deficits of
3xTgAD mice with no effect in non-Tg mice. 9-months-old non-Tg mice were impaired
in their learning in the initial phase (first 2 days) but improved to 27.8 ± 6.1% with
continuous practice over 5-days of training. Treatment with AP had no significant effect
on learning (F
(1, 17)
= 0.915; p > 0.05) (final level of learning was 20.6 ± 4.1%) although
an enhancing trend was observed in the initial phase (fig. 12A). Similarly, no significant
effect in the performance was observed in the memory test between vehicle- and AP -
treated 9-months-old non-Tg mice (F
(1, 16)
= 0.149; P > 0.05) (fig. 12C). Vehicle-treated
3xTgAD mice showed a deficit in the final level of learning as compared to the vehicle-
treated non-Tg mice (F
(1, 20)
= 6.034; p < 0.05). Vehicle-treated 9-months-old 3xTgAD
mice exhibited almost no learning with a response rate of 12.5 ± 2.4% (fig. 12B).
Compared to the vehicle-treated group, the final learning of AP -treated group was
significantly increased (F
(1, 25)
= 4.820; p < 0.05) to a level of 23.6 ± 4.0%. Similar to the
effects observed in 6-months-old 3xTgAD mice, AP significantly increased the learning
of 9-months-old 3xTgAD mice during the entire course of training (F
(1, 129)
= 24.837; p <
0.001). (fig. 12B). The enhancement in learning by AP was not a transient effect as it
persisted during the whole acquisition course, and was confirmed by the significantly
better memory performance after 9 days relative to vehicle-treated 3xTgAD mice F
(1, 24)
=
98
5.141; p < 0.05) (fig. 12C). Vehicle-treated 3xTgAD mice showed a significant deficit in
the memory test as compared to the vehicle-treated non-Tg mice (F
(1, 20)
= 15.237; p =
0.001), which was reversed by treatment with AP . AP -treated 3xTgAD mice
performed comparable to vehicle-treated non-Tg mice (F
(1, 21)
= 0.282; p > 0.05).
AP induced a significant increase in BrdU+ cell survival in 9-months-old 3xTgAD mice
similar to what occurred in the 6-months-old 3xtgAD mice. Vehicle-treated 9-months-old
non-Tg mice exhibited survival of BrdU+ cells (2288.2 ± 557.9) which was not
significantly different from that of 6-months-old non-Tg mice (F
(1, 13)
= 0.214; p > 0.05.
In the 9-months-old non-Tg mice, AP had no effect on BrdU+ cell survival (2255.3 ±
662.4) as compared to the vehicle-treated group (F
(1, 14)
= 0.001; p > 0.05) (fig. 12D). In
9-months-old vehicle-treated 3xTgAD mice, BrdU+ cell survival (243.7 ± 30.3) was
significantly reduced as compared to vehicle-treated non-Tg mice (F
(1, 15)
= 17.294; p =
0.001) (fig. 12D). AP significantly increased the number of surviving BrdU+ cells to
899.3 ± 313.1 (F
(1, 19)
= 7.234; p < 0.05) (fig. 12D) as compared to the vehicle-treated
group. However the treatment with AP in 3xTgAD mice was not able to reverse the
levels of BrdU+ cells back to that of vehicle-treated non-Tg mice (F
(1, 17)
= 7.288; p <
0.05).
99
Figure 12. AP reversed the learning and neurogenic deficits in 9-month-old 3xTgAD mice.
A. Learning curve of 9-month-old non-Tg mice - 9-months-old non-Tg mice showed lower
learning rate and basal learning level (highest level: 27.8 ± 6.1%) similar to 6-month-old non-Tg
mice. Treatment with APα did not significantly affect the overall acquisition phase of non-Tg
mice. B. Learning curve of 9-month-old 3xTgAD mice - Compared to the non-Tg group, 9-
month-old 3xTgAD mice showed a significant deficit in the highest level of learning (p < 0.05).
APα significantly increased the learning rate and the final levels of learning in 3xTgAD mice
after 5 days of training, to levels comparable to the non-Tg mice C. Summary of the results from
memory test in 9-month-old animals, conducted 9 days after the acquisition phase - 3xTgAD
mice group exhibited memory retention deficits as compared to non-Tg group (p < 0.001). APα
significantly enhanced the retention and restored the memory performance of 3xTgAD mice to a
level comparable to that of age-matched non-Tg group. No effect of AP was observed on the
retention of non-Tg mice. D. Summary of results from cell survival analysis in post-training
brains using fluorescent activated cell sorting (FACS) - 9-month-old 3xTgAD mice had a
significantly reduced cell survival rate in the SGZ as compared to non-Tg mice (p < 0.001). APα
treatment restored the neurogenic deficiency in 3xTgAD mice to that of age-matched non-Tg
group, and showed no significant impact on cell survival in the age-matched non-Tg group. * p <
0.05, as compared to 3xTgAD + vehicle group; † p < 0.001 as compared to non-Tg + vehicle
group. Data are presented as average ± SEM (n = 9 -14).
100
4. Loss of APα cognitive and neurogenic efficacy in 12-months-old 3xTgAD mice
parallels development of A plaques.
At 12-months of age, maximal A intraneuronal accumulation is apparent in the
hippocampus and A plaques are widespread. In contrast to the cognitive and
neurogenic efficacy of AP in the 6 and 9 months-old 3xTgAD mice, AP treatment in
12-months-old 3xTgAD mice was without effect. In parallel to the loss of efficacy in
3xTgAD mice, emergence of trend towards an AP effect in 12 months-old non-Tg mice
appears. In 12-months-old non-Tg mice a profound decline in associative learning ability
was apparent during the initial phase (first 2 days) which subsequently improved to a
level of 23.8 ± 5% with 5 days of training. The final level of learning in AP -treated non-
Tg mice was 25.4 ± 6%, which was not significantly different from the vehicle-treated
group. AP -treated non-Tg mice did not show a significant increase in their learning
during the course of training as compared to the vehicle-treated group (F
(1, 83)
= 2.449; p
> 0.05) (fig. 13A). However, AP -treated non-Tg mice showed a positive trend towards
increased learning on day 2 as compared to the vehicle-treated group but it was not
significant (F
(1, 15)
= 3.750; p > 0.05). AP -treated mice showed a modest improved
memory performance which was not significantly better than vehicle-treated group (F
(1,
15)
= 1.115; p > 0.05) (fig. 13C). Vehicle-treated 3xTgAD mice showed a deficit in the
final level of learning as compared to the vehicle-treated non-Tg mice. Vehicle-treated
12-months-old 3xTgAD mice exhibited almost no learning with a final response rate of
17.5 ± 2.6% (fig. 13B). Compared to the vehicle-treated group the final learning of AP -
101
treated group was not significantly increased and the final level was 14.9 ± 3.6%. The 12-
months-old 3xTgAD mice exhibited no improvement in learning over the 5 days of
training (F
(1, 113)
= 0.632; p > 0.05) (fig. 13B) which was also evident in the memory
performance (F
(1, 21)
= 0.019; p > 0.05) (fig. 13C). Vehicle-treated 3xTgAD mice showed
a significant deficit in the memory test as compared to the vehicle-treated non-Tg mice
(F
(1, 17)
= 4.963; p < 0.05), which was not reversed by treatment with AP . AP -treated
3xTgAD mice showed an equally significant deficit to vehicle-treated non-Tg mice (F
(1,
19)
= 4.287; p = 0.05).
As in the learning and memory analyses, AP induced a modest but not statistically
significant trend towards an increase in BrdU+ cell survival (fig. 13D). Basal level of
BrdU+ cell survival was significantly decreased in 12-months-old 3xTgAD mice (201.1 ±
51.5) as compared to age-matched non-Tg mice (F
(1, 16)
= 135.58; p < 0.001) (1460.8 ±
99.3). In cell survival experiment, basal level of BrdU+ cells in 12-months old non-Tg
mice was not significantly different from that of 6-months old (F
(1, 14)
= 4.209; p > 0.05)
and 9-months old (F
(1, 14)
= 2.438; p > 0.05) non-Tg mice. AP exerted no significant
effect on BrdU+ cell survival in 12-months-old non-tg mice (F
(1, 15)
= 0.329; p > 0.05) nor
12-months-old 3xTgAD mice (F
(1, 19)
= 2.977; p > 0.05).
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Figure 13. AP showed no significant effect in both 12-month-old non-Tg and 3xTgAD
mice. A. Learning curve of 12-month-old non-Tg mice - 12-month-old non-Tg mice showed low
learning rate and maximum level (highest level: 23.8 ± 5.0%) during the training. Treatment with
APα did not significantly alter the learning of non-Tg mice although a positive trend was
observed in the initial phase of training. B. Learning curve of 12-month-old 3xTgAD mice - 12-
month-old 3xTgAD mice showed almost no learning with the highest levels of learning
remaining around 15% at the end of the acquisition phase. APα affected neither the learning rate
nor the final levels of learning after 5 days of training. C. Summary of the results from memory
test in 12-month-old animals, conducted 9 days after the acquisition phase - 12-month-old
3xTgAD mice exhibited significant lower memory retention than age-matched non-Tg mice (p <
0.05). APα did not affect the retention of the learned responses in both 3xTgAD and non-Tg
mice. D. Summary of results from cell survival analysis in post-training brains using fluorescent
activated cell sorting (FACS) - 12-month-old 3xTgAD mice had a significantly reduced cell
survival rate in the SGZ as compared to non-Tg mice. APα treatment showed no significant
impact on cell survival in both the 3xTgAD and non-Tg group. * p < 0.05,** p < 0.00000001 as
compared to non-Tg + vehicle group. Data are presented as average ± SEM (n = 8 - 12).
103
5. APα reversed the cognitive and neurogenic deficits in 15-months-old non-Tg mice.
At 15-months of age, greater intraneuronal A accumulation and extracellular Aβ plaques
are present in the hippocampus and tau hyperphosphorylation is observed in the
hippocampus of 3xTgAD mice. Since no significant effect of APα was seen in 12-
months-old 3xTgAD mice, analyses of 15-months-old 3xTgAD mice is not reported here.
However, our results have shown that APα was not effective in increasing the learning
and neurogenesis levels in these mice. AP reversed the learning and neurogenic
deficits of 15-months-old non-Tg mice. Vehicle-treated mice showed a very less basal
level of learning (final level of learning was 20.3 ± 3.4%). Treatment with APα led to a
significant increase in the learning of 15-months-old non-Tg mice (final level of learning
was 36.5 ± 5.5%) as compared to the vehicle-treated group (F
(1, 19)
= 4.941; p < 0.05) (fig.
14A). A similar effect of APα treatment was seen in the memory test, where APα-treated
mice performed significantly better (F
(1, 19)
= 6.152; p < 0.05), with the levels of CR%
increasing from 18.8 ± 2.9% to 36.1 ± 6.1% (fig. 14B). Also a similar significant
increase was seen in the BrdU positive cells (data is not shown here)
104
Figure 14. AP reversed the learning and neurogenic deficits in 15-month-old non-Tg mice.
A. Learning curve for 15-month-old non-Tg mice – 15-month-old non-Tg mice showed low
learning rate and maximum level (highest level: 20.3 ± 3.4%) during the training. Treatment with
APα significantly enhanced the learning of non-Tg mice to a maximum level of 36.5 ± 5.5%. B.
Memory test in 15-month-old animals showed that APα enhanced the performance of these mice
significantly as compared to the vehicle – treated group (p < 0.05). Data are presented as average
± SEM (n = 8 - 12).
Discussion
Using a triple transgenic mouse model of Alzheimer‟s disease (3xTgAD) and its
background strain (non-Tg), we showed that aging and Alzheimer‟s disease could
differentially affect adult neurogenesis in the SGZ of the hippocampus and such
105
neurogenic deficits are closely related to the cognitive deficits. Further, we have shown
that APα could reverse the neurogenic and cognitive deficits in the aged mice, an effect
consistent with previous findings in vitroin human and rat neural progenitor cells (Wang
et al., 2005) and in vivo in 3-month-old 3xTgAD mice (Wang et al., 2010). Importantly,
the effect of APα appears to be specifically targeting the deficiences induced by
Alzheimer‟s disease rather than by aging until a very old age. APα seems to work when
there is an intrinsic deficiency and functions to restore the levels to that of age-matched
non-Tg mice. Results from our study shed light on the important role that neurosteroids
play in the regenerative capability of the central nervous system, how they regulate such
activities in response to neurodegenerative disease.
We previously showed that APα reverses both neurogenic and cognitive deficits in 3-
month-old 3xTgAD mice (Wang et al., 2010). In the present study, we have extended our
investigation to the effect of APα on 6, 9, 12 and 15-month-old mice. Our observations
have shown that APα is equally effective in 6 and 9-month-old 3xTgA mice, inspite of
the presence of β-amyloid. Both the neurogenic and cognitive deficits were reversed,
after treatment with APα, to the levels of age-matched non-Tg mice. APα-induced
augmentation in neurogenesis and learning and memory failed to persist beyond 12-
months of age in 3xTgAD mice, when the plaque distribution became widespread in the
hippocampus and the cortex (Oddo et al., 2003; Chen et al., 2009) and tau
hyperphosphorylation starts to become apparent. Appearance of plaque is indicative of a
high concentration of soluble Aβ levels and the reason of inefficacy of APα – induced
neurons to survive and affect learning in 12 months and older 3xTgAD mice could be due
106
to an altered balance between the GABAergic and glutamatergic transmission in the
dentate gyrus (Sun et al., 2009).
Since the effect of APα appeared to be targeting the decline associated with AD, it is
possible that APα specifically antagonizes the effector or pathway through which AD
pathology induces neurogenic and cognitive deficits, such as Aβ. Indeed APα has been
shown to regulate AD progression in 3xTgAD mice and decrease pathological burden,
including intraceullular Aβ and extracellular plaques (Chen et al., 2009). Alternatively,
the fact that APα treatment only affects 3xTgAD mice might represent a pathological
change in APα level in AD, thus external supply of APα in 3xTgAD mice simply
diminished APα shortage and accompanying neurological deficits. This is further
supported by previous studies showing that APα is significantly lower in this AD model
and also in AD patients (Weill-Engerer et al., 2002) and has been suggested as a potential
biomarker for AD.
Reported herein, one of the neurosteroids, APα, promotes neurogenesis and cognitive
performance in an AD mouse model. This discovery sheds light on the possibility that
neurosteroids could serve as regenerative agents for neurodegenerative diseases, such as
AD, to restore CNS activity and cognitive function. Additionally our results have shown
that APα promotes cell survival in 6 and 9-month-old mice. Majority of the newly-
generated and survived cells exhibited a neuronal phenotype (Wang et al., 2010). Further
APα improved the performance of 3xTgAD mice in trace eyeblink conditioning, an
associative learning task shown to be dependent upon the generation of new neurons in
the dentate gyrus (Shors et al., 2001; Shors et al., 2002; Tseng et al., 2004). Overall our
107
results provide preclinical evidence that APα can restore cognitive performance and
promote neural progenitor cell survival in the pre-plaque phase of AD pathology.
108
CHAPTER FOUR
Effect of Allopregnanolone on Delay Eyeblink Conditioning in Mice
Chapter Four Abstract
Our previous work has shown that allopregnanlone (APα), a metabolite of progesterone,
is a potent enhancer of trace eyeblink conditioning in young and adult 3xTgAD and old
non-Tg male mice. Our current hypothesis for these observations, as shown by our
experimental results is that APα is mediating these effects by increasing the neurogenesis
levels in the hippocampus. Apart from the hippocampus, the cerebellum plays a very
important role in trace eyeblink conditioning. A recent study has shown the persistence of
adult neurogenesis in the cerebellum of rabbits. These factors indicate that APα could be
affecting the neurogenesis in the cerebellum of 3xTgAD mice, if present, and enhancing
their learning in trace eyeblink conditioning. To investigate this possibility, 3 months old
3xTgAD and non-Tg mice underwent delay eyeblink conditioning, a much simpler
version of eyeblink conditioning that depends only on the cerebellum and its associated
circuitry. The experimental conditions used were exactly same as in our previous studies,
except that the tone (CS) and shock (US) were delivered without any trace period but
were co-terminating. Results of our analyses showed that both 3xTgAD and non-Tg mice
did not show any learning, when very few conditioning trials were used. However, APα
enhanced the learning of both 3xTgAD and non-Tg mice. Analyses are underway to find
the number of BrdU positive cells in the cerebellum, following the behavioral study.
These observations further support our previous results that enhancement in trace
109
eyeblink conditioning was not due to differences in the cerebellum but in the
hippocampal neurogenesis. A single dose treatment enhancing the delay eyeblink
conditioning, a motor task, further supports APα as a very interesting molecule, whose
therapeutic potential should be studied in even greater details.
Introduction
Adult neurogenesis has been shown to persist in the subgranular zone (SGZ) of the
hippocampus and subventricular zone (SVZ) of the lateral ventricle in the mammals. In
marked contrast, adult cerebellum is known as one of the most static structures in the
central nervous system (CNS) under the profile of cell renewal. Cerebellar neurons in
mammals are generated both centrifugally and centripetally from different sources, at
different developmental stages. Purkinje neurons and interneurons originate from the
neuroepithelium of the fourth ventricle whereas granule ells come from actively
proliferating cell precursors which ccumulate in the external granule layer (EGL) after
tangential migration from the rhombic lip (Altman and Bayer 1997). The external granule
layer (EGL) persists after birth on the cerebellar surface until it provides the granule cell
population by radial migration during early postnatal periods whose duration is strictly
dependent on the species (Fujita et al., 1966; Rakic 1971; Abraham et al., 2001). On the
other hand, the genesis of cerebellar cortex interneurons occurs by migration through the
subjacent white matter and is completed in rodents before the end of granule cell genesis.
All neuroepithelium-derived, GABAergic interneurons, including basket, stellate, and
Golgi II cell precursors are produced by a common pool of progenitors (Leto et al., 2006)
and express the paired box transcription factor Pax2 (Maricich and Herrup 1999).
110
Recently it was shown that in the rabbit cerebellum between the fourth and the fifth
postnatal week the external granule layer (EGL) is replaced by a proliferative layer which
then persists beyond puberty (called as subpial layer, SPL) (Ponti et al., 2006). SPL is
characterized by the occurrence of tangential chains of neuroblasts, similar to those
described in the forebrain subventricular zone (SVZ). The rabbit SPL is completely
exhausted around the sixth month of postnatal life (Ponti et al., 2006). A remarkable
genesis of cells, mostly neuronal precursors, is detectable within the cerebellar cortical
layers at peri-puberal stages. In fact analysis of older rabbits also showed the presence of
a remarkable number of cells belonging to both the interneuron and synantocyte type
(Butt et al., 2005), even in the absence of a proliferative SPL (Ponti et al., 2008).
The presence of adult neurogenesis in the rabbit cerebellum has sparked an interest in
identifying similar processes in the cerebellum of other mammalian species including
mice. Whether adult neurogenesis plays a role in cerebellum dependent learning is still
unknown. Delay eyeblink conditioning, an associative task that depends critically on the
cerebellum, could be a very useful task to study such a role.
Allopregnanolone (AP ), a metabolite of progesterone, is a neurosteroid de novo
synthesized in both embryonic and adult CNS as well as pluripotential progenitor cells,
and exhibits an age- and AD-associated decline. APα plays a very critical role in
immature cerebellar granule cells proliferation (Keller et al., 2004) and also acts to
potentiate the GABA
A
receptors and can be suitable for physiological modulation of tonic
inhibitory neurotransmission via extrasynaptic GABA
A
receptors (Maksay et al., 2007).
The significant decline in APα in Alzheimer‟s disease could be one of the reasons for
111
motor impairments in Alzheimer‟s disease (Weill-Engerer et al., 2002). Having multiple
possibilities, the interaction of APα with the cerebellum needs to be further elucidated.
Using a triple-transgenic mouse model of Alzheimer‟s disease carrying APP
Swe
,
PS1
M146V
, and tau
P301L
(3xTgAD)(Oddo et al., 2003), we have previously shown that AP
reversed both neurogenic and cognitive deficits in 3-month-old 3xTgAD mice (Wang et
al, 2010) in addition to its proliferative effect in vitro (Wang et al., 2005). Since trace
eyeblink conditioning depends both on the hippocampus and the cerebellum (Christian
and Thompson 2003), the enhancement in learning, seen as a result of APα could be due
to changes in either of these structures. To validate our previous observations, we have
used delay eyeblink conditioning as the behavioral task and have investigated the effect,
if any, of APα on cerebellar-dependent learning.
Materials and methods
Animals and treatment
Breeding pairs of the triple transgenic Alzheimer‟s disease mouse (3xTgAD,
homozygous mutant of human APPswe and tauP301L and PS1M146V) and its
background strain (129/Sv x C57BL/6) were obtained from Dr. Frank LaFerla (UC
Irvine) and the colonies were established at USC. The characterization of amyloid and tau
pathologies, as well as synaptic dysfunction in this line of mice has been described
previously (Oddo et al., 2003) and confirmed in our laboratory. The mice were genotyped
regularly to confirm the purity of the colony. Experiments were performed using 3-
month-old male 3xTgAD and non-Tg. The number of mice per condition is indicated
within the results section. Mice were maintained under a 12 h light/12 h dark cycle with
112
continuous access to food and water.
Allopregnanolone (APα, 3α-hydroxy-5α-pregnan-20-one) (aka APα, Allo or THP) stock
solution was prepared in pure ethanol and diluted in PBS before injection (with a final
ethanol concentration of 0.002 % of the body weight). Mice received a subcutaneous
(s.c.) injection of either APα or vehicle at a concentration of 10 mg/kg. One hour after
APα injection, mice were intraperitoneally (i.p.) injected with BrdU at a concentration of
100 mg/kg. All experiments used minimal number of animals conformed to the Animal
Welfare Act, Guide to Use and Care of Laboratory Animals, and the U.S. Government
Principles of the Utilization and Care of Vertebrate Animals Used in Testing, Research,
and Training guidelines on the ethical use of animals.
Delay eyeblink conditioning
Surgeries: Under deep anesthesia by intraperitoneal (i.p.) injection with ketamine
(100mg/kg i.p.) and xylazine (25 mg/kg i.p.), a 4 pin head stage (DIGI-KEY) was
cemented to the skull the mouse with dental acrylic. The connector has four Teflon-
coated stainless steel wires and one bare stainless steel wire (0.003” bare and 0.0055”
coated, A-M Systems, Inc.). The bare wire is attached via a gold pin (Time Electronics)
to the head stage. The coated wires were implanted s.c. in the orbicularis oculi of the left
upper eyelid (Tseng et al., 2004). All animals were placed on a warm isothermal pad after
surgery to recover for 30 mins. Following the surgery, mice were individually housed,
provided with ad libitum access to food and water, and were maintained on a 12 hour
light/dark cycle.
113
Training paradigm: During the training, mice were placed within Plexiglas cylinders in a
sound-attenuated chamber and received a single habituation session for 1 day without
tone and shock and the spontaneous eyeblink activity was recorded. Following
habituation mice were trained with delay eyeblink conditioning for a period of 5 days
with a 352 msec tone (2kHz, 80-85 dB SPL) as the conditioned stimulus (CS), co-
terminating with a 100 msec shock (100 Hz, biphasic) as the unconditioned stimulus
(US). The US level was adjusted daily for each mouse to give a small head turn before
each day of training. The inter-stimulus interval (ISI) was kept at 252 ms and the inter-
trial interval was randomized between 20 and 40 sec (average 30 sec). Every training
session consisted of 30 trials in 3 blocks. Each block consisted of 1 tone-alone (1
st
) trial
and 9 paired trials with shock always following the tone. Following 5 days of acquisition
mice were left in their home cages and tested for their memory after 9 days.
Data collection and analysis: EMG activity during each trial was collected, rectified, and
integrated using specially designed Labview routines on a computer (Tseng et al., 2004;
Ohno et al., 2005). The eyeblink electromyographic (EMG) activity was amplified 104
times, filtered at 100-5000k Hz. The EMG data was then analyzed by using a custom-
made program described by Lee and Kim (2004). Briefly, the conditioned responses
(CRs) were determined in all the trials when the baseline was lower than 0.1 units,
without unstable activity or an unconditioned startle response (see Lee and Kim 2004 for
further details). The % of CR is defined as the ratio of the number of CRs in valid trials in
both CS/US paired trials and CS-only trials.
114
Animal dissection and tissue collection
Mice were sacrificed at the end of the memory test for cell survival assessment (Fig.1A).
On the day of sacrifice mice were deeply anesthetized with a combination of ketamine
(100 mg/kg) and xylazine (10 mg/kg), and perfused with PBS. Brains were dissected into
two hemispheres, and one hemisphere was fixed immediately in cold 4%
paraformaldehyde and was used for FACS analysis (cell survival). The analyses for BrdU
positive cell count is still in process and is not reported here.
Statistical analysis
Data were analyzed using a one-way ANOVA followed by Neuman–Keuls post hoc
analysis. Data displayed in graphs were reported as mean ± SEM or fold change ± SEM.
p-values of < 0.05 were considered to be significant regardless of the statistical test used.
Results
Rationale for and design of AP treatment experiments
Our previous analyses demonstrated that AP reversed cognitive and neurogenic deficits
of male 3xTgAD mice (3 months of age) (Wang et al, 2010). The current study was
designed to determine the efficacy of AP to affect a much simpler version of eyeblink
conditioning that does not depend on the hippocampus and to explore the possibility for
the presence of adult neurogenesis in the mice cerebellum.
Cognitive performance was assessed using the delay eyeblink conditioning behavioral
paradigm (fig. 15B) which provides an indication of associative learning and memory
that is dependent upon the cerebellum.
As indicated in the experimental design (fig. 15A), animals received a single s.c.
115
injection of APα (10mg/kg) or vehicle 7 days prior to the start of the learning trials, a
time period that was used in our previous studies. Cell survival assessment was
conducted 21 days after APα or vehicle injection. The 21-day time frame was specifically
selected to be consistent with our previous studies. In contrast to the trace paradimg that
was used in our prvious studies, tone and shock are delivered in a fashion so that they
both co-terminate as is shown in fig. 15B. The time gap between the tone onset and shock
onset used was 252 msec.
116
Figure 15. Experimental design - A. Experimental plan for behavior test and cell survival
determination. Mice were surgically implanted with electrodes to measure the electromyogram
(EMG) of the eyelid muscle. After 3~4 day recovery, mice were injected s.c. with either APα (10
mg/kg) or vehicle and 1 hr later with i.p. injection of BrdU (100 mg/kg). One week later, Mice
were subjected to 1-day habituation and subsequently 5-day behavior test using delay eyeblink
conditioning. At the end of the acquisition phase, mice were housed in their home cages and
tested for the retention of the memory after 9 days. Following the memory test, animals were
sacrificed and the brains were collected for assessment of cell survival. B. During delay eyeblink
conditioning, CS (a tone), was paired with US (periorbital shock), which elicits an eyeblink
response. The time gap between the CS onset and US onset was 252 ms, but both co-terminated.
Eyeblinks were detected by an increase in the magnitude of the EMG.
117
APα enhanced the learning of both 3xTgAD and non-Tg mice in delay eyeblink
conditioning
This study was done as a control for our previous studies to explore the effect of APα on
cerebellar-dependent learning. In the previous studies we showed that APα enhanced
learning in trace eyeblink conditioning for young and adult 3xTgAD mice and for aged
non-Tg mice. Since trace eyeblink conditioning requires both the hippocampus and the
cerebellum, the observed enhancement could have been due to changes in either of the
cerebellum or the hippocampus. Hence in this study, we investigated the performance of
3-month-old 3xTgAD and non-Tg mice on a simpler version of eyeblink conditioning
that depends only on the cerebellum to determine if APα affects the cerebellar-dependent
learning. As can be seen in fig. 16A the training paradigm was not sufficient to cause an
increase in the % CR in non-Tg mice. Usually the mice require a high number of trials
(100 in each session) to show an increase in their % CR. To be consistent with out
previous studies, we kept the number of trials to 30 in each session and used 2 sessions
per day, separated by 3 – 4 hrs. Non-Tg mice reached a maximum % CR of 12.2 ± 4.2,
which was not significant from their spontaneous blinking rate (p > 0.05), indicating that
there was no learning in these mice. However, the mice that were treated with APα
showed a significantly higher % CR compared to the vehicle – treated mice (F
(1, 8)
=
5.398; p < 0.05), reaching a maximum of 38.4 ± 10.5.
3xTgAD mice also performed similarly to the non-Tg mice in the paradigm used. Vehicle
– treated 3xTgAD mice did not show any learning and reached a maximum CR% of 8.7 ±
2.2 (fig. 16B), which was not different from their spontaneous blinking rate (p > 0.05).
118
APα – treated 3xTgAD mice showed significantly higher learning than vehicle – treated
mice (F
(1, 8)
= 10.844; p < 0.01), reaching a maximum % CR of 35.9 ± 8.0. Vehicle –
treated non-Tg and 3xTgAD mice did not differ in their final learning (F
(1, 8)
= 0.540; p >
0.05). Similarly, APα – treated non-Tg and 3xTgAD mice did not differ in their final
levels of learning (F
(1, 8)
= 0.036; p > 0.05).
119
Figure 16. APα enhanced the learning of both 3xTgAD and non-Tg mice in delay eyeblink
conditioning. A. Learning curve for 3-month-old non-Tg mice – vehicle-treated mice did not
show any significant learning at the end of the training. Treatment with APα significantly
enhanced the levels of learning in non-Tg mice as compared to the vehicle-treated group. B.
Learning curve for 3-month-old 3xTgAD mice – vehicle-treated mice did not show any
significant learning, and were similar to non-Tg in their performance. APα-treated 3xTgAD mice
performed in a similar manner to APα-treated non-Tg mice and their learning was significantly
enhanced as compared to the vehicle-treated 3xTgAD mice. * p < 0.05 as compared to vehicle-
treated non-Tg mice, ** p < 0.01 as compared to vehicle-treated 3xTgAD mice. Data are
presented as average ± SEM (n = 5)
120
Discussion
This study was done to validate our previous observations and show that APα-mediated
enhancement in trace eyeblink conditioning in 3xTgAD mice and aged non-Tg mice is
due to the involvement of the hippocampus and not because of its effects on the
cerebellum. We have recently shown that APα, a reduced metabolite of progesterone,
reverses the neurogenic and cognitive deficits of young (Wang et al., 2010) and adult
3xTgAD mice and old non-Tg mice (Singh et al., 2010, in preparation). The cognitive
task used in our previous studies was trace eyeblink conditioning that requires an intact
hippocampus, cerebellum and other forebrain structures (Woodruff-Pak and Disterhoft
2008). Moreover, trace eyeblink conditioning requires the newly born neurons in the
dentate gyrus of the hippocampus. The recent discovery of adult neurogenesis in rabbits
cerebellum (Ponti et al., 2006) has raised the question about the presence of a similar
environment in the cerebellum of other mammalian species. Since APα increases the
proliferation of neural progenitor cells from hippocampus (Wang et al., 2005), the effects
seen in our previous studies could be because of a similar increase in neural progenitor
cells in the cerebellum of the mice. The samples are being analysed at present in our lab.
APα, DHEAS and other neurosteroids have been shown to be significantly lower in AD
(Weill-Engerer et al., 2002). Treatment with APα in our previous studies might have
affected the cerebellum of the 3xTgAD mice and could have led to an enhancement in
their learning. Additionally, APα has been shown to be a major positive modulator at the
neurosteroid sites in the cerebellum. APα acts as an antagonist of cerebellar GABA
A
receptors with nanomolar affinity (Maksay et al., 2007). Hence treatment with APα could
121
have led to changes at the molecular level, affecting/modifying the GABA receptors.
Considering all these possibilities, this study was really crucial to be done.
To be consistent with our previous studies, we used smaller number of trials, divided in
two sessions everyday. A typical delay conditioning paradigm, however, comprises 100
trials everyday for 7 – 8 days. Considering that we used a much less number of training
trials, we were not surprised to see almost no learning in either of the genotype used. But
that does not rule out the possibility of 3xTgAD mice being impaired in delay eyeblink
conditioning. Even though, in previous studies employing several AD models showed no
deficit in delay eyeblink conditioning, no such report exist for 3xTgAD mice. A
significant decline in APα concentration was observed in our previous analysis of
3xTgAD mice and that might cause a deficit in cerebellar-dependent learning (Wang et
al., 2010). Treatment with APα was effective in enhancing the learning of both 3xTgAD
and non-Tg mice. Moreover, 3xTgAD and non-Tg mice did not learn any association
between the tone and shock and performed equally poorly. It will be very interesting to
study how these mice behave in a paradigm using a higher number of trials. The
enhanced learning in APα - treated 3xTgAD and non-Tg mice was not different between
the two groups. The present observation was a result of a single treatment with APα,
which indicates that the changes that happened because of this treatment persisted for a
long time and affected the learning later on, which is quite similar to the hippocampal
neurogenesis. At the time of submission of this report, we were still in the process of
analyzing the samples for markers of new neurons and BrdU – labeled cells. As discussed
before, the possibility of alterations in the GABA
A
receptors also needs to be studied
122
before we can conclude the exact reason for our current observations. Moreover, since
GABA
A
receptors are present widely in the brain, the action of APα on other structures
could have a modulatory effect on delay eyeblink conditioning and it needs to be further
studied. Since APα is a naturally occurring neurosteroid, its role in cerebellar – dependent
learning will be quite interesting to study for future research. Most importantly our results
suggest that APα exerted its effect on trace eyeblink conditioning by affecting the
neurogenesis in the hippocampus and not by affecting the cerebellum.
123
CHAPTER FIVE
Absence of Monoamine Oxidase A and B leads to Enhanced Emotional and Motor
Learning.
Chapter Five Abstract
Monoamine oxidases (MAOs) A and B are key enzymes in the metabolic pathways of
monoamine neurotransmitters. In spite of their similar structure and gene organization,
these two isoforms differ greatly for substrate specificity: MAO A has a higher affinity
for serotonin (5-HT), norepinephrine (NE) and dopamine (DA), while MAO B prefers
phenylethylamine (PEA) as substrate. Previous works have shown that mice carrying a
genetic ablation of both MAO A and MAO B display an array of behaviors reminiscent
of anxiety-related traits. In this study, we have extended the behavioral characterization
of these knockout mice further and have studied emotional learning and motor learning.
Results of our analyses have shown that MAO A/B KO mice display enhanced emotional
and higher levels of motor learning. Additionally, these KO mice exhibited no significant
alterations in the acute responses to different forms of mild environmental stress:
including tail suspension in a brightly lit environment, exposure to novel marbles in the
cage, and startle reflex elicited by 120-dB acoustic bursts. These results point to MAO
A/B KO as an interesting animal model for the study of the role of monoamines in fear-
related behaviors and post-traumatic stress disorder. Furthermore, MAO A/B KO mice
shows enhanced level of learning and impairment extinction in cerebellar dependent
eyeblink conditioning, further shedding light on the role of monoaminergic afferents in
the modulation of motor learning in the cerebellum.
124
Introduction
Monoamines, which include catecholamines such as dopamine, norepinephrine (NE), and
epinephrine, and indolamines such as serotonin (5-HT), play a very important role in a
variety of behaviors ranging from sleep to ingestion (Feldman and Quenzer 1984). Any
aberration from the normal levels of these neurotransmitters is correlated with mental
dysfunctions (e.g. schizophrenia and depression) and neurological disorders (e.g.
Alzheimer and Parkinson diseases) (Snyder et al., 1974; Palmer and DeKosky 1993;
Schildkraut 1965; Hornykiewicz 1974). The major source of these neurotransmitters lies
in the brainstem regions (e.g., NE in the locus ceoruleus, dopamine in the substantia
nigra, and 5-HT in the raphe nucleus) and these brainstem nuclei exert their influences by
sending both ascending and descending projections to various regions of the brain and the
spinal cord (Elliott et al., 1977).
Two monoamine oxidase isoenzymes (MAO A and MAO B) exist closely linked in
opposite orientation on the X chromosome (Shih 1990; Cases et al., 1995; Cases et al.,
1996) and are expressed on the outer mitochondrial membrane. MAO A and MAO B
oxidize these monoamines and indolamines. MAO A and B have different substrate
specificities. MAO A prefers serotonin (5-HT), norepinephrine (NE), and dopamine (DA)
as substrates while MAO B prefers phenylethylamine (PEA) as substrate. Previous
studies with MAO A knockout mice have shown that MAO-A deficiency leads to
manifestation of cognitive deficits – there was a selective enhancement of emotional
learning while no effects were observed in motor learning (Kim et al., 1997). Low levels
125
or absence of MAO A expression are associated with violent, criminal, or impulsive
behavior in humans and aggression in mice.
MAO-B KO mice exhibit deficient habituation of locomotor activity while no increase in
aggression was observed. Both MAO A and B knockouts have a specific biochemical and
behavioral phenotype.
MAO A/B KO (with both isoenzymes missing) cannot be generated through the breeding
of MAO A KO and MAO B KO mice due to the close proximity of these isoenzyme
genes on the X chromosomes. A mouse line with both these enzymes absent due to a
spontaneous mutation in MAO A exon 8, in a litter of MAO B KO mice was generated.
Previous studies have shown that these KO exhibit a unique biochemical, molecular, and
behavioral characteristics. The MAO A/B KO showed reduced body weight compared
with wild type mice. Brain levels of serotonin, norepinephrine, dopamine, and
phenylethylamine increased, and serotonin metabolite 5-hydroxyindoleacetic acid levels
decreased, to a much greater degree than in either MAO A or MAO B KO mice. These
mice have shown observed chase/escape and anxiety-like behavior. Further
characterization has shown that these KO mice have unaltered behavior in the acute
responses to different forms of mild environmental stress - including tail suspension in a
brightly lit environment, exposure to novel marbles in the cage, and startle reflex elicited
by 120-dB acoustic bursts.
The focus of the present study was to examine the emotional and motor learning in these
KO mice to establish a correlation between abnormal monoamine neurotransmitters and
126
cognitive aberrations. Emotional learning was tested using fear conditioning as the
behavioral paradigm while motor learning was analyzed using classical eyeblink
conditioning. Aymgdala, critically involved in fear learning, and cerebellum and
associated circuitry, playing the key role in eyeblink conditioning, are innervated with
monoaminergic inputs. Several studies have shown that (Roozendaal et al., 2006;
LaLumiere et al., 2003; Berlau and McGaugh 2006) these neurotransmitters play a
critical role in fear conditioning and also modulate synaptic transmittion in Purkinje cells
and other cerebellar cortical neurons, thereby affecting eyeblink conditioning (Cartford et
al., 2004).
Materials and methods
Animals
Fear conditioning was assessed in experimentally naïve male MAO A/B double knockout
(KO) mice (n = 8) and wild-type (WT) littermates (n = 8) at an age of 3 - 4 months. Mice
were individually housed in plastic and metal cages with ad libitum access to food and
water and maintained in a climate-controlled vivarium on a 12 hr light/dark cycle. The
background strain of the MAO A/B was that of the MAO B KO mice, which had been
originally generated in a C57-BL/6J/129Sv strain, whose males were subsequently
backcrossed over 25 generations with 129/SvEv females. All the experiments performed
were approved by the Institutional Animal Use and Care Committee. A different set of
naïve mice (n > 5) was used for eyeblink conditioning.
127
Pain test
To measure nociception, MAO A/B KO (n = 8) and WT (n = 8) mice were restrained in a
plastic cylinder (inner diameter, 4cm). The tail was put on a hot plate (56° C), and the
onset latency of tail flick was recorded three times with 1 min intervals. Nociception test
was done after the behavioral test to avoid any sensitization.
Auditory test
To assess hearing, animals were anesthetized (ketamine and xylazine) and placed in a
sound-attenuated chamber on an isothermal pad. Platinum subdermal needles were
inserted on the vertex of the skull and ventrolateral to pinna. Animals were presented
with a 2kHz tone (10 msec duration; 1 msec rise-fall;100 msec between tones), filtered
through a Krohn-Hite filter (model 3700; 1.5 kHz low cutoff; 20 kHz high cutoff; Krohn-
Hite, Brockton, MA). Each mouse was tested with 2000 tone-on (85 dB) trials and
followed by 2000 tone-off (0 dB) control trials.
The auditory brainstem response (ABR) is widely used to test for hearing in rodents
(Steel and Hardisty, 2000). The signal was amplified (10,000gain) and filtered (300 Hz
low cutoff; 5kHz high cutoff) through a differential AC amplifier (model 1800; A-M
Systems, Carlsborg, WA) into the sound card. The sound card averaged the ABR signal
recorded from 2000 trials and calculated the average amplitude (peak-to-peak microvolt)
of the ABR during the presentation of the tone.
128
Fear conditioning
All behavior tests were performed and analyzed with the experimenter blind to the
condition, WT or KO, of the animals. Animal behavior was recorded with a video camera
that was linked to a TV monitor and VCR for online observation and recording. On day
one, mice were individually placed in a 10.5 × 12 × 12″ conditioning chamber with
electrifiable flooring (Coulbourn Instruments). To enhance the salience of the chamber,
three walls had broad, 1 inch, red and white diagonal stripes at 45° while one wall of the
chamber was Plexiglas to allow video recording. One milliliter of vanilla extract was
placed in a small Petri dish below the cage flooring and out of the animals reach, also to
enhance the salience of the context.
Mice were allowed to explore the chamber for 3 mins. During this time, activity was
scored to control for possible locomotor differences between WT and KO mice by
counting the number of crossings through the midline of the chamber and the number of
rearings performed. After 3 mins, an 80 dB tone (2 kHz) was played for 20 sec. During
the last second of the tone, a mild electric current (0.70 mA) was passed through the
flooring with a 1 sec duration. Tone and footshock deliveries were controlled by software
(Lab Linc Operant Control Software; Coulbourne Instruments). Following a 1-min
interval, the tone was played again for 20 sec accompanied by a 1 sec current stimulus
overlapping the last second of the tone. After another 1-min interval, the tone-shock
sequence was repeated one last time for a total of 3 pairings, and the mice were left in the
chamber for another 30 sec.
129
The chamber was cleaned with 70% ethanol between individual sessions. One day later,
on day 2, mice were evaluated for fear conditioning to context by re-exposing them to the
conditioning chamber for 8 mins without tone or footshock and freezing behavior was
scored (referred to as context test). Freezing behavior is defined here as the lack of all
body movement except for that associated with respiration. Animals were observed every
5 sec at which time the animal was either considered freezing or not. The number of
observations during which the animal was freezing was divided by the total number of
observations made to determine the percentage of freezing. 48 hours after the initial
conditioning (day 3) mice were evaluated for cued fear conditioning to the tone (tone
test). Mice were placed in a novel chamber (12 × 8 × 5 inches) that was cleaned between
individual sessions with 70% isopropyl alcohol. Following 3 mins of exploration, an
80 dB, 2 Hz tone was played for 8 mins and behavior was observed and recorded every
5 sec for the duration of the tone.
Delay eyeblink conditioning
Surgeries: Under deep anesthesia by intraperitoneal (i.p.) injection with ketamine
(100mg/kg i.p.) and xylazine (25 mg/kg i.p.), a 4 pin head stage (DIGI-KEY) was
cemented to the skull the mouse with dental acrylic. The connector has four Teflon-
coated stainless steel wires and one bare stainless steel wire (0.003” bare and 0.0055”
coated, A-M Systems, Inc.). The bare wire is attached via a gold pin (Time Electronics)
to the head stage. The coated wires were implanted s.c. in the orbicularis oculi of the left
upper eyelid (Tseng et al., 2004). All animals were placed on a warm isothermal pad after
130
surgery to recover for 30 mins. Following the surgery, mice were individually housed,
provided with ad libitum access to food and water, and were maintained on a 12 hour
light/dark cycle.
Training paradigm: During the training, mice were placed within Plexiglas cylinders in a
sound-attenuated chamber and received a single habituation session for 1 day without
tone and shock and the spontaneous eyeblink activity was recorded. Following
habituation mice were trained with delay eyeblink conditioning for a period of 8 days
(A1-A8, the acquisition period) with a 352 msec tone (2kHz, 80-85 dB SPL) as the
conditioned stimulus (CS), co-terminating with a 100 msec shock (100 Hz, biphasic) as
the unconditioned stimulus (US). The US level was adjusted daily for each mouse to give
a small head turn before each day of training. The inter-stimulus interval (ISI) was kept at
252 ms and the inter-trial interval was randomized between 20 and 40 sec (average 30
sec). Every training session consisted of 100 trials in 10 blocks. Each block consisted of 1
tone-alone (1
st
) trial and 9 paired trials. Following 7 days of acquisition mice were
exposed to 5 days of extinction. In this phase mice were given tone alone trials (100
each) for 5 days and their rate of extinction was determined.
Data collection and analysis: EMG activity during each trial was collected, rectified, and
integrated using specially designed Labview routines on a computer (Tseng et al., 2004;
Ohno et al., 2005). The eyeblink electromyographic (EMG) activity was amplified 104
times, filtered at 100-5000k Hz. The EMG data was then analyzed by using a custom-
made program described by Lee and Kim (2004. Briefly, the conditioned responses (CRs)
131
were determined in all the trials when the baseline was lower than 0.1 units, without
unstable activity or an unconditioned startle response (see…for further details). The % of
CR is defined as the ratio of the number of CRs in valid trials in both CS/US paired trials
and CS-only trials.
Statistical analysis
Data were analyzed using a one-way ANOVA followed by Neuman–Keuls post hoc
analysis. Data displayed in graphs were reported as mean ± SEM or fold change ± SEM.
P-values of < 0.05 were considered to be significant regardless of the statistical test used.
Results
MAO A/B KO mice showed no detectable difference in their sensory perceptions
Because many transgenic mice show developmental deficits that can affect their
performance in behavioral tasks, we first assessed MAO A/B KO mice for sensory
impairments that may affect performance in the fear-conditioning paradigm. Because
tone is one of the contional stimuli in our fear-conditioning paradigm and eyeblink
conditioning, we first examined MAO A/B KO mice with auditory brainstem recordings.
Results showed that MAO A/B KO mice have comparable hearing to their WT
littermates. No significant difference in brainstem auditory responses was observed
between MAO A/B KO and WT mice (data not shown), suggesting that these mutant
mice have normal perceptual ability for the tone cue. During the initial training sessions
involving tone-footshock pairings, MAO A/B KO mice jumped and vocalized in response
to the electric footshock to a similar degree as the WT, suggesting normal pain perception
132
in these mutant mice. Additonally, both MAO A/B KO and WT mice exhibited
characteristic tail-flick responses in the heat-induced tail-flick responses and no
significant difference was observed in the average latencies to tail flick between the two
genotypes, indicating normal pain and heat perception in MAO A/B KO mice (data not
shown).
MAO A/B KO mice showed equal exploration and baseline freezing before the tone-
shock paired training.
Basic motor activity patterns were compared between MAO A/B KO and WT mice by
measuring the number of cage crossings within the initial three minutes after placing
mice in the fear conditioning chamber. No significant difference was observed between
the MAO A/B KO and WT mice (F
(1, 27)
= 0.133; p > 0.05) (fig. 17)
133
Figure 17: Crossing behavior of wild-type and MAO A/B KO mice. Basic exploratory
behavior was measured by the number of center line crossings in the conditioning chamber prior
to conditioning and was not different between the wild-type and MAO A/B KO mice (p > 0.05).
Data is plotted as Average ± SEM (n > 10).
MAO A/B KO mice showed elevated freezing during training and context test.
As shown in fig. 18, fear conditioning (percent freezing) was significantly elevated in the
MAO A/B KO mice, in comparison to the wild-type mice, during training [immediate
postshock freezing during minutes 4, 5 and 6 (F
(1, 79)
= 6.609; p < 0.01).
Mice were exposed to the context that was used during the paired training after 24 hrs.
MAO A/B KO mice displayed significantly enhanced contextual learning as compared to
the WT mice. The starting levels of % freezing was identical for both the genotypes (F
(1,
134
25)
= 2.298; p > 0.05). However, their responses to the exposure to the context were very
different. The change of freezing for the two genotypes from the 1
st
to the 2
nd
min. was
significantly different. The % of time spent in freezing was significantly different in the
2
nd
minute of exposure to the context (F
(1, 25)
= 9.501; p < 0.01) amongst the two
genotypes. The freezing of WT mice increased gradually to a maximum level during the
5
th
minute of exposure and then decreased significantly, equal in levels to the baseline at
the start of the contextual test. The % freezing between the 1
st
and the 5
th
min. for WT
was significantly different (F
(1, 28)
= 24.715; p < 0.0001). Apart from this significant
increase in freezing of the WT, there was also a significant decrease in their freezing
from 5
th
min. to the 8
th
min. (F
(1, 28)
= 9.839; p < 0.01) and their freezing during the 8
th
and final min. was not significantly different from that during the 1
st
min. (F
(1, 28)
= 1.482;
p > 0.05). These observations suggest that WT mice processed the contextual information
in a gradual manner, with the feared behavior being elicited at a slower/normal rate. On
the other hand, MAO A/B KO mice were very robust in their feared behavior. Their
freezing increased significantly from the 1
st
to the 2
nd
min. and it persisted for the
remaining duration of the context test. The final level of freezing was significantly higher
than the baseline freezing at the onset of the context test. Also MAO A/B KO mice
showed significantly higher freezing than WT mice during the 8
th
and the final min. of
the context exposure (F
(1, 25)
= 6.964; p < 0.01), indicative of impaired extinction.
135
Figure 18: MAO A/B KO mice showed enhanced freezing during training and context test.
MAO A/B KO mice displayed enhanced freezing during the training as compared to the wild-
type mice (p < 0.01). MAO A/B KO mice also showed significantly higher retention of the
contextual learning on day 2. Moreover, MAO A/B KO mice did not show any extinction and
showed significantly higher freezing at the end of the context exposure as compared to the wild-
type mice (p < 0.01). * p < 0.05; ** p < 0.01. Data is plotted as Average ± SEM (n > 10).
MAO A/B KO mice showed significantly decreased exploration in a novel
environment after fear conditioning.
One of the most important observation from the current study was MAO A/B KO mice
stopped exploring a novel environment. As discussed before, the baseline freezing was
identical for MAO A/B KO and WT mice. On day 3, MAO A/B KO and WT mice were
136
exposed to a novel environment for 3 mins. and were tested for cue (tone) – associated
fear learning for 8 mins. As expected WT mice did not show high baseline freezing to the
novel environment used and explored the new context. However, MAO A/B KO mice
displayed significantly elevated levels of freezing to the new context used (fig. 19; the
first 3 mins.). Without the hint of any threat or threat related cue, the freezing of MAO
A/B KO mice kept on increasing. The freezing levels between MAO A/B KO and WT
mice were significantly different during the whole duration of exposure before the onset
of the tone (F
(1, 18)
= 6.273; p < 0.05 for the 1
st
min., F
(1, 18)
= 13.580; p < 0.01 for the 2
nd
min. and F
(1, 18)
= 23.686; p < 0.0001 for the 3
rd
min.)
MAO A/B KO mice significantly enhanced freezing in response to the tone.
Following 3 mins. of exposure to a novel environment, the tone that was used for paired
training was played for the next 8 mins. During the entire 8 mins. of the tone, MAO A/B
KO mice showed significantly higher freezing levels as compared to the WT mice (fig.
19). Since MAO A/B KO mice showed significantly high freezing during the baseline,
we compared the change of % freezing from 3
rd
min. to the 4
th
min., i.e. during the first
min. of tone exposure. As was observed during the context test, MAO A/B KO mice
exhibited a more robust response to the tone as compared to the WT mice. During the
first min. of the tone, the freezing of MAO A/B KO mice increased significantly as
compared to that during the 3
rd
min. of the baseline (F
(1, 16)
= 27.809; p < 0.0001) and
persisted after that for the entire duration of the tone, whereas compared to the 3
rd
min. of
the baseline, the freezing of WT mice in response to the tone increased significantly only
during the 2
nd
min. of tone exposure (F
(1, 20)
= 7.082; p < 0.01) and persisted after that.
137
The levels of freezing during the 8
th
and final min. of tone exposure were significantly
different (F
(1, 18)
= 24.277; p < 0.0001).
Figure 19: MAO A/B KO mice showed less exploration in a novel chamber and higher
freezing in response to the tone. MAO A/B KO mice did not explore a novel environment that
was used for tone testing on day 3. Mice were allowed to explore the new environment for the
first 3 mins., following which the tone was played for 8 mins. MAO A/B KO mice showed
significantly higher freezing during the first 3 mins and also during the next 8 mins as compared
to the wild-type mice. The rate of change in the % freezing in MAO A/B KO mice from min. 3 to
min. 4 was significantly high as compared to that for the wild-types. * p < 0.05, ** p < 0.01, † p
< 0.0001, †† p < 0.00001. Data is plotted as Average ± SEM (n > 9).
138
MAO A/B KO mice never exhibited exploration of a novel environment after fear
conditioning.
Different types of contexts were used to study the exploration of MAO A/B KO and WT
mice for over 6 weeks. MAO A/B KO mice never explored a new environment after the
fear conditioning (data not shown).
MAO A/B KO mice exhibited higher learning in delay eyeblink conditioning.
A different set of MAO A/B KO (n = 7) and WT (n = 7) mice were used to study motor
learning in these mice using the delay eyeblink conditioning paradigm. 2 of the MAO
A/B KO mice died 1 day after the surgeries. Our observations have shown that MAO A/B
KO mice have different sensitivity to ketamine that is normally used as an anesthesia
during the surgeries for implanting the headstage and the electrodes. During the
acquisition phase, MAO A/B KO mice were a little slower in learning (though not
significant) during the first 3 days but started performing better than WT mice after that
till the end of the training with reaching significantly higher levels during the last three
days of acquisition (F
(1, 35)
= 7.52; p < 0.01) (fig. 20A). Following the acquisition phase,
mice underwent 5 days of extinction using the tone alone trials. Throughout the
extinction phase, MAO A/B KO mice performed worse than the WT mice (F
(1, 59)
= 5.63;
p < 0.02) (fig. 20A). The performance in extinction phase was proportional to the
learning levels in the acquisition phase and does not indicate that MAO A/B KO mice
were impaired in extinction of the learned association.
139
As opposed to these differences in the acquisition and extinction phase, no difference was
observed in the US intensities between the two genotypes during the entire acquisition
phase (fig. 20B).
Figure 20: Delay eyeblink conditioning in MAO A/B KO and wild-type mice. A. Mean (±
SEM) percentage of conditioned responsed exhibited by MAO A/B KO and wild-type mice
during 8 days of acquisition and 5 days of extinction. Results show that MAO A/B KO mice
showed enhanced learning as compared to the wild-type mice. However, the extinction phase was
proportional to the learning levels and comparable to the wild-type mice at the end. B. Mean (±
SEM) periorbital shock (US) intensity used for eyeblink conditioning in MAO A/B KO and wild-
type mice during the 8 days of acquisition. The US intensity, the minimal voltage required to
elicit an eyeblink/head turn response, was adjusted daily for each animal. No significant
difference was observed between the two genotypes.
140
Discussion
In the present study, we have characterized the emotional and motor learning in MAO
A/B KO mice. MAO A/B KO mice used in our study arose serendipitiously in a colony
of MAO B knockout mice. Previous study on these mice has shown that MAO A/B KO
mice display anxiety-like behaviors and have significantly elevated levels of the
monoamines 5-HT, NE, DA and PEA (Chen et al., 2004). Monoaminergic
neurotransmitters have been implicated in emotional learning in many previous stuies,
Naiive MAO KOs and wild-type mice were used in fear conditioning. None of the
genotype displayed any freezing prior to the conditioning, which indicates no difference
in their basic explorative behavior. Inspite of having anxiety-like traits, MAO A/B KO
mice performed similar to the wild-types in terms of their baseline freezing and
exploration. In contrast, MAO A/B KO mice displayed enhanced freezing responses
during training (immediate postshock periods), context test (24hrs later) and tone test (48
hrs later) in comparison to normal mice. Additionally, MAO A/B KO mice exhibited
significant elevated freezing in a novel environment. The enhancement of fear learning
observed in MAO A/B KO mice may be due to elevated catecholamine levels because
injections of catecholamines into the brains of mice enhances fear memory formation
(Haycock et al., 1977; Stein et al., 1975), whereas drugs that lower the levels of
catecholamines impair it (Lavond et al., 1993). Since the amygdala is critically involved
in fear conditioning it is possible that the alterations of catecholamine levels in the
amygdala is responsible for the enhancement of fear learning observed in the MAO A/B
KO mice. Previous studies have shown that pharmacological treatments that increase the
141
noradrenergic transmission in the amygdala (e.g. infusions of NE into the amygdala)
enhance fear learning (McGaugh et al., 1990) , whereas treatments that decrease
noradrenergic transmission impair fear learning (e.g. infusions of noradrenergic
antagonists). It is conceivable then that the enhancement of emotional learning exhibited
by the MAO A/B KO mice is due to elevation of NE in the amygdala. Previous study on
these KO mice has shown that NE level was elevated in the brain structures innervated by
the locus coeruleus, such as the frontal cortex, the hippocampus, and the cerebellum Chen
et al., 2004). Additonally, these mice have elevated levels of serotonins (5-HT),
dopamine and phenylethylamine and these neurotransmitters could also be the
contributing factors in the abnormal fear learning.
In contrast to our previous study using MAO A-deficient mice we found out that MAO
A/B KO mice have an enhanced motor learning in the delay eyeblink conditioning
paradigm. Monoaminergic afferents constitute one of the three major afferent systems in
the cerebellum (the others are mossy fiber and climbing fiber afferents) (Ito 1984). These
monoaminergic inputs include well-defined noradrenergic and serotonergic afferents
from the locus coeruleus and raphe nucleus, respectively. There is evidence that these
monoaminergic afferents modulate synaptic transmission in Purkinje cells and other
cerebellar cortical neurons. For instance, iontophoretic application of NE to Purkinje cells
results in enhancement of both excitatory and inhibitory responses of Purkinje cells, both
to mossy fiber and climbing fiber inputs. Since the levels of 5-HT and NE are elevated
much more than was seen in MAO A KO mice, it could be possible that the enhancement
seen in MAO A/B KO mice and no effect seen in MAO A KO mice is because of the
142
differential levels of these neurotransmitters. Hence these observations further suggest
the involvement of monoamine systems in the normal eyeblink conditioning situation.
Interestingly, MAO A/B KO mice exhibited no significant alterations in comparison to
wild-type mice in the acute responses to different forms of mild environmental stress
including the tail suspension in a brightly lit environment, exposure to novel marbles in
the cage, and startle reflex elicited by 120-dB acoustic bursts. A Previous study has
shown that MAO A/B KO mice have a deficit in SVZ neurogenesis, although no studies
have been done to investigate the SGZ neurogenesis. Our results also show that MAO
A/B KO mice have different sensitivity to ketamine, an NMDA antagonist. MAO A/B
KO mice also lack the barrel cortex in the primary somatosensory cortex. Since NMDA
receptors play a significant role in SVZ neurogenesis, the action of ketamine, the
formation of barrel cortex during development, the possibility of an abnormal NMDA
receptor expression could not be ignored and could be one of the contributing factors in
the enhanced fear and motor learning.
Overall our results suggest that MAO A/B KO mouse is a very interesting system to
study the developmental and behavioral abnormalities related to the monoaminergic
neurotransmitters.
143
CHAPTER SIX
Conclusions and Future directions
This thesis focused on the memory abnormalities in Alzheimer‟s disease (AD) and
Anxiety models. Specifically, we have studied how different types of memories are
affected in these two disorders.
As has been shown in previous studies, trace eyeblink conditioning is affected more in
AD as compard to normal aging. A possible reason for this is that trace eyeblink
conditioning requires an intact hippocampus along with the cerebellum and other
forebrain structures. Since hippocampus is one of the first structures that are affected in
AD, it causes a significant deficit in behavioral tasks that require it. A similar trend has
been seen in SGZ neurogenesis. Recent studies have shown that early neurogenic deficits
in AD could be one of the contributing factors for the observed cognitive deficits.
Additionally, a very recent study has shown that adult-born hippocampal dentate granule
cells undergoing maturation modulate learning and memory in the brain. We
hypothesized that if SGZ can be stimulated by an exogenous agent, then at a suitable
time, the new born neurons will affect the hippocampal dependent learning. To this
effect, we found out that APα, a metabolite of progesterone, stimulates the endogenous
neural progenitor cells in the hippocampus of 3xTgAD mice – both in the absence of
detectable pathology and in the presence to some extent. These newborn neurons induced
by APα showed a high correlation to the increase in learning and memory. In addition,
the effect of APα on learning was tested after one week, a time at which the maturing
144
newborn neurons in the hippocampus play a role in learning and memory. Since APα has
multiple targets, there is a possibility of change in the GABA receptor subunits because
of this treatment, which can later on affect the learning. Previous studies have shown that
APα affects the subunit composition of GABA receptors only if given chronically. No
chronic treatment was done in the current study. Besides, the half-life of APα is around
30-45 mins so it does not persist for a long time after administration and cannot affect the
learning after one week.
APα also showed significant effects on learning and memory in 15 months old non-Tg
mice, which indicates that even in the course of normal aging, one of the contributing
factors for cognitive impairment is the decline in hippocampal neurogenesis and can be
reversed by APα. Whether a threshold concentration of APα is the reason for it to exert
its effect early in AD and later in normal aging needs more studies to confirm. Looking at
the endogenous concentration and metabolism of APα in an age-dependent manner in
3xTgAD and non-Tg mice would be of great interest. The time between APα
administration and its effect on learning could also be varied to further confirm that
enhancement in learning is due to the immature neuron in their maturation phase.
Recent studies have shown that high levels of Aβ cause an imbalance between the
GABAergic and glutamatergic transmission impairs adult neurogenesis in an animal
model of AD. This effect was not seen when Aβ was present in lower concentration,
suggesting that at a certain stage of AD, Aβ starts exerting its influence on the maturation
of the newborn neurons.
145
More than 68% of all AD patients are females and hence studying the efficiency of any
potential therapeutics on females becomes very important. We have done a few sets of
experiments (not reported here) on the efficacy of female 3xTgAD and non-Tg mice. Our
results were not conclusive and we are still working on these mice. The problem in
working with female mice is the presence of APα as an endogenous neurosteroids, which
keeps on varying depending upon the phase of their menstrual cycle. To counter this, we
ovariectomized the female mice and studied the efficiency of APα on neurogenesis and
learning and memory. We observed that APα enhances the proliferation of neural
progenitor cells in the hippocampus only after 6 weeks of ovariectomy. We conducted
behavioral assays using this time frame but did not see any effect of APα on the learning
and memory. Our current hypothesis is that following the procedure of ovariectomy, the
concentration of cortisol is very high and it interferes with the survival of new born
neurons and hence they are not able to survive for long and affect the learning in these
mice. Further tests are needed to validate this hypothesis and experiments are underway.
As a further control we have seen that APα does affect a much simple version of eyeblink
conditioning – delay eyeblink conditioning that depends only on the cerebellum and its
associated circuitry. The absence of any difference between this learning in the two
genotypes and a significant difference in trace eyeblink conditioning suggests that our
previous observations, that showed a reversal of cognitive impairment in 3xTgAD mice,
was indeed because of the hippocampal involvement. The enhancement in delay eyeblink
conditioning seen in both 3xTgAD and non-Tg mice as a result of APα also suggests the
possibility of the existence of neural stem/progenitor cells in the cerebellum of mice. A
146
recent report has shown the presence of such cells in the rabbit cerebellum. Hence it will
be interesting to find out BrdU positive cells in the cerebellum of mice, with and without
treatment with APα
Similar to the observations in SGZ, SVZ neurogenesis is also impaired in 3xTgAD mice.
For future studies, it will be very interesting to study the effect of APα on SVZ
neurogenesis and investigate the functional role of the newborn neurons in SVZ. There
are a few studies that have shown the significance of SVZ neurogenesis in odor learning
and memory. Loss of smell is one of the first few symptoms that of AD patients and this
could reflect a deficit in SVZ neurogenesis. Moreover, new neurons in the SVZ migrate
over a longer distance as compared to SGZ so studying the effect of APα-induced
neurons to migrate to other parts of the cortex will be very interesting. This might turn
out to be an interesting therapeutic for traumatic brain injury, where APα-induced new
neurons could be targeted to the sites of injury. In fact a pilot study has shown that APα
increased the proliferation of SVZ progenitor cells significantly in 3xTgAD mice.
The ultimate challenge for APα will be to work in a similar fashion when given
chronically. A recent study has shown that chronic treatment (Chen et al., 2010) with
APα significanly decreases the neuropathology in 3xTgAD mice. Studying learning and
memory in a similar fashion will be quite interesting for future work. Similarly, it will be
of interest to study the effect of APα on other behavioral paradigms. Site specific
injection of APα and electrophysiological studies of the hippocampus following APα
injection could also shed light on the exact mechanism of action of this neurosteroid.
In contrast to the impairments in memory in AD, memories in MAO A/B KO mice were
147
significantly enhanced. Specifically, emotional memories, in the form of fear
conditioning, were found to be significantly enhanced. MAO A/B KO mice have been
shown to have anxiety-like characteristics. The interaction between anxiety and fear
learning has been extensively studied. In our study, we have found that the absence of
monoamine oxidase A and B isoenzymes led to a significantly elevated level of
catecholamine and indolamines. As with other neurotransmitters, rapid removal and
degradation of brain monoamines is essential for the correct functioning of synaptic
neurotranismission. As has been discussed before, monoaminergic signaling plays a very
critical role in the modulation of mood and emotion, as well as the control of motor,
perceptual and cognitive functions. Any abnormality in their levels due to excess
production or less degradation (due to genetics or environmental factors like prolonged
stress) can give rise to unstable moods and other psychiatric disorders. MAO inhibitors
are the first category of antidepressants and show high mood-enhancing efficacy. Since
the monoamines 5-HT, NE, DA, and PEA are all elevated in the MAO A/B KO mice, it
is difficult to pinpoint which monoamines is primarily responsible for the observed
behavior, particularly since all these amines have anxiogenic properties. Furthermore,
developmental adaptations during brain maturation in knock-out mice may result in a
phenotype that is paradoxically different from that elicited by acute pharmacologic
intervention in an adult wild type animal.
Anxiety disorder is a big problem in the present world. Estimates show that more than 4
million Americans suffer from Generalized Anxiety Disorder (GAD), definded as a
chronic disorder characterized by excessive, long-lasting anxiety and worry about
148
nonspecific life event, objects and situation. Abnormal monoamine levels are one of the
most important factors that cause anxiety disorders. MAO A/B KO mice having elevated
monoamine levels will be a very important use for future pharmacological studies to find
out and test new drugs for these disorders. At present, we are testing L-NAME to
decrease the anxiety levels in these mice and estimate the correlation between decreased
anxiety and fear acquisition. This model also suggests that some people are genetically
pre-disposed to developing anxiety and PTSD because of their abnormal neurotransmitter
levels. Additionally, we also found a significant enhancement in motor learning in these
mice. As discussed, monoaminergic afferents in the cerebellum plays a modulatory role
in motor learning. We corroborated these findings using MAO A/B KO mice with
elevated levels of these neurotransmitters in the cerebellum. Other studies from our
collaborators have shown that these mice have a deficit in SVZ neurogenesis and they
also lack the barrel cortex. Future studies could also be directed towards investigating
SGZ neurogenesis and hippocampus dependent learning. Moreover, since our studies
have shown that MAO A/B KO mice displayed enhanced stress behavior, their cortisol
levels should be studied before and after fear conditioning. Since amygdala and
hippocampus are involved in making fear memories, electrophysiological studies are in
progress in our lab to find out the abnormalities, if any, in synaptic transmission. Absence
of barrel cortex, deficit in SVZ neurogenesis, less sensistivity to ketamine – all indicate
that the NMDA receptors might be having an altered expression and studies are in
progress to test this possibility.
149
On a whole, our studies using MAO A/B KO mice have shown that these mice are a very
interesting model for future studies correlating monoamines and morphological,
electrophysiological and behavioral characteristics.
150
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Abstract (if available)
Abstract
This dissertation seeks to investigate the memory abnormalities that result from Alzheimer's disease and anxiety disorders. In particular, this dissertation will focus on investigating if neurogenic deficits in the hippocampus contribute to the etiology of Alzheimer's disease by studying a hippocampal-dependent associative behavior test. Additionally, we will study the potential of allopregnanolone (APα), a metabolite of progesterone, to increase neurogenesis in the hippocampus of mice and to determine the functional significance of these new neurons. Our data has demonstrated that APα reverses the neurogenic and cognitive deficits of Alzheimer's disease mice to levels of age-matched normal mice. This study suggests that APα could serve as a regenerative therapeutic to prevent or delay neurogenic and cognitive deficits associated with mild cognitive impairment and Alzheimer's disease. As a second part of this thesis, we have investigated memory abnormalities in a mouse model of anxiety. This mouse model does not have any monoamine oxidase A and B and hence, has significantly elevated levels of monoaminergic neurotransmitters. Previous study has shown that MAO A/B KO mice have anxiety-like behavior. As a further characterization of these mice, we have found that there is a significant enhancement of emotional memories in these mutants and point to these mice as an interesting animal model to study the role of monoamines in fear-related behaviors and post-traumatic stress disorder. Furthermore, MAO A/B KO mice have shown enhanced level of learning in cerebellar-dependent delay eyeblink conditioning, further shedding light on the possible role of monoaminergic afferents in the modulation of motor learning in the cerebellum.
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Creator
Singh, Chanpreet
(author)
Core Title
Memory abnormalities in Alzheimer's disease and anxiety models
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Neuroscience
Degree Conferral Date
2010-05
Publication Date
05/07/2010
Defense Date
03/22/2010
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University of Southern California
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allopregnanolone,Alzheimer's disease,anxiety,eyeblink conditioning,learning and memory,neurogenesis,OAI-PMH Harvest
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), Baudry, Michel (
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), Brinton, Roberta Diaz. (
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), Madigan, Stephen A. (
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), Swanson, Larry W. (
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Tags
allopregnanolone
Alzheimer's disease
anxiety
eyeblink conditioning
learning and memory
neurogenesis