i 
 
 
 
 
 
ABSTRACT 
 
 
 
 
Title of Thesis: CHARACTERIZING rTg4510 MICE MODEL 
OF ALZHEIMER’S DISEASE ON ADULT 
NEUROGENESIS AND COGNITIVE 
FUNCTION  
  
 Laurel A. Gordon, Alice Yanhong Lu, Melinda 
M. Matos, Jennie Se Yeon Seo, Lisa Yi Yeon 
Seo 
  
Thesis Directed By: Dr. Ricardo C. Araneda, Ph. D 
Associate Professor and Director BISI-PSYS 
Graduate Program 
University of Maryland, Department of Biology 
 
 
Alzheimer’s Disease (AD) is a debilitating illness that affects millions of 
Americans each year. While there is not one definitive hypothesis that exists 
regarding how the disease develops, some hypotheses include the cholinergic model, 
the amyloid beta hypothesis, and the tau pathology model. The rTg4510 (Tg) mouse 
is a model of AD that over-expresses inducible human mutant tau (P301L), a 
pathological hallmark of AD. This study characterizes the Tg mouse through analysis 
of adult neurogenesis in the olfactory system, due to the fact that the olfactory system 
is one of the first sensory systems of the brain to be affected by AD. For this, we 
examined the behavioral changes in different age groups, specifically 2 and 7 months. 
Adult neurogenesis within the granule cell layers and glomerular layers in the main 
  
ii 
and accessory olfactory bulb was analyzed by methods of immunohistochemistry 
using appropriate antibodies and cell counting with confocal microscopy. Several 
behavioral paradigms, such as habituation/dishabituation, odor detection threshold, 
and novel object recognition, were executed to assess cognitive function of these 
mice, especially in relation to markers of olfactory behavior.  Our data suggest that 
there is an age-dependent cognitive impairment but an age-dependent increase in 
neurogenesis. Further study is required to identify the effects of inducing the mutant 
tau on neurogenesis in the olfactory bulb (OB) and whether the overexpression of tau 
directly impacts cognitive decline. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
iii 
 
 
 
 
 
CHARACTERIZING rTg4510 MICE MODEL OF ALZHEIMER’S DISEASE ON 
ADULT NEUROGENESIS AND COGNITIVE FUNCTION    
 
 
 
by 
 
 
Team Dream 
Laurel A. Gordon, Alice Yanhong Lu, Melinda M. Matos, Jennie Se Yeon Seo, 
Lisa Yi Yeon Seo 
 
 
 
 
 
Thesis submitted in partial fulfillment of the Gemstone Program 
University of Maryland 
2017 
 
 
 
 
 
 
 
 
 
Advisory Committee: 
Dr. Ricardo C. Araneda, Faculty Mentor 
Dr. Kara Duffy 
Dr. Quentin Gaudry 
Dr. Erica Glasper 
Dr. Hadiya Woodham 
Dr. David Yager 
 
 
 
 
 
  
iv 
 
 
 
 
 
 
 
© Copyright by 
Laurel A. Gordon, Alice Yanhong Lu, Melinda M.  Matos, Jennie Se Yeon Seo, 
Lisa Yi Yeon Seo 
 
Dr. Ricardo C. Araneda 
 
2017 
 
 
 
  
v 
 
Acknowledgements 
We would like to thank Dr. Ricardo Araneda, our mentor, for providing us 
with continuous guidance, insight, and expertise as well as lab space, necessary 
equipment, and materials. We would also like to give special thanks to Krista Krahe, 
the lab’s research assistant, for her support and guidance in the lab as well as a 
semester as our mentor. Thank you to all the LaunchUMD donors, especially Mr. 
Shaun McDuffee of the Stop Alzheimer’s Now Foundation who has gifted us with an 
incredibly generous donation. We would like to thanks all the lab members who have 
given us assistance in times of need throughout our project. Thank you to Dr. Kristan 
Skendall, Dr. Frank Coale, and the Gemstone staff for their support on team building. 
Lastly, thank you to Dr. Kara Duffy, Dr. Quentin Gaudry, Dr. Erica Glasper, Dr. 
Hadiya Woodham, and Dr. David Yager for swiftly agreeing to be our discussants 
during our Thesis Conference. 
  
  
vi 
Table of Contents 
 
 
Acknowledgements v 
Table of Contents vi 
Chapter 1: Introduction 1 
1.1 Neurodegenerative Diseases 1 
1.2 Alzheimer’s Disease 1 
1.2.1 Definition 1 
1.2.2 Risk Factors 3 
1.2.3 Characterization of Alzheimer’s 3 
1.3 Alzheimer’s Disease and Olfaction 4 
1.4 Adult Neurogenesis 5 
1.4.1 Overview of Neurogenesis 5 
1.4.2 Mouse Models of Neurogenesis 6 
1.5 Inspiration for Project/ Experimental Approach 6 
1.6 Research Questions and Hypothesis 7 
Chapter 2: Literature Review 9 
2.1 Pathology of Alzheimer’s Disease 9 
2.1.1 Tau Pathology 9 
2.1.2 Amyloid Beta Plaques 10 
2.1.3 Neurofibrillary Tangles 11 
2.2 Neurogenesis in the Olfactory Bulb 12 
2.3 Therapeutic Approaches for Neurodegenerative Diseases 15 
2.4 Recent Research on Neurodegenerative Diseases Using Mice Models 16 
2.5 Selection of a Mouse Model 17 
Chapter 3: Methodology 20 
3.1 Research Design 20 
3.1.1 Animals 20 
3.1.2 Environmental Conditions 20 
3.1.3 Treatment Groups 21 
3.1.4 Behavioral Tests 22 
3.1.5 BrdU Injections 25 
3.1.6 Perfusion 25 
  
vii 
3.1.7 Tissue Preservation 25 
3.1.8 Cryostat and Slicing 26 
3.1.9 Immunohistochemistry 26 
3.1.10 Confocal Microscopy 27 
3.1.11 Statistical Analysis 28 
Chapter 4: Results 30 
4.1 Behavioral Data 30 
4.1.1 Habituation/Dishabituation 30 
4.1.2 Odor Detection Threshold 31 
4.1.3 Novel Object Recognition 34 
4.2 Confocal Data 36 
Chapter 5:  Discussion 38 
5.1 Behavioral Data 38 
5.1.1 Habituation/Dishabituation 38 
5.1.2 Odor Detection Threshold 39 
5.1.3 Novel Object Recognition 40 
5.2 Confocal Data 41 
Chapter 6:  Limitations 43 
6.1 Behavioral Limitations 43 
6.1.1 Cube Recognition 43 
6.1.2 Defining Wildtype Mice 43 
6.1.3 Sample Size Limitations 44 
6.1.4 Use of the rTg4510 Mice Model 44 
Chapter 7:  Conclusion 45 
Chapter 8:  Future Directions 45 
Glossary 47 
Bibliography 52 
 
 
	
 
 
  
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Chapter 1: Introduction 
1.1 Neurodegenerative Diseases 
Neurodegenerative diseases are a group of conditions in which there is significant 
degeneration of the central or peripheral nervous system in individuals (“Neurodegenerative 
Diseases”). Neurodegenerative diseases affect millions of Americans each year and lead to 
devastating changes in the individual’s sense of self and ability to perform typical tasks. 
Neurodegenerative diseases often result in the degeneration or the death of nerve cells, leading to 
issues with mental functioning, otherwise known as dementia (“What Is Neurodegenerative 
Disease?”). In this way, dementias form a broad category of neurodegenerative diseases, and can 
be described as a type of cognitive decline that affects individuals’ daily lives. Dementia is the 
sixth leading cause of death in America and affects 1 in 9 elderly Americans. Dementia not only 
affects the individual’s own life but also comes to affect the patient’s family through depression, 
stress, and financial burdens. Each year, about $200 billion is spent on healthcare costs related to 
dementia, half of which. About half of this is accounted to Alzheimer’s disease (AD), the most 
common form of dementia. Eventually, society as a whole is affected by the neurodegenerative 
decline that occurs in AD (Alzheimer’s Association, 2017). 
1.2 Alzheimer’s Disease 
1.2.1 Definition 
 Scientifically, AD can be described as “a progressive disease that destroys memory and 
other important mental functions” (Mayo Clinic Staff Print, 2015).  It is estimated that between 
11 million and 16 million Americans will have the disease by 2050, and that this will lead to a 
  
2 
proportional increase in the cost of providing care to patients, leading to a huge economic issue 
in healthcare (Alzheimer’s Association, 2017).  Evidently, AD is not only an issue in regards to 
the impacts on the lifestyles of patients and those around them, but also in regards to the 
economy and healthcare as a whole.   
AD is a progressive disorder with no set timeline or rate of cognitive decline. Landmarks 
may be different for each individual. Phases include the asymptomatic phase, the symptomatic 
predementia phase, during which there may be changes in cognitive function, and finally the 
dementia phase. According to the National Institute on Aging and the Alzheimer’s Association, 
the dementia phase usually marks the diagnosis of AD. It is more difficult to definitively 
diagnose AD in the earlier phases of the disease (Albert et al., 2011).  
During the symptomatic predementia phase, a decline in cognitive ability may signal AD. 
Observations include a decline in episodic memory (ability to learn new skills) and functional 
memory. Cognitive assessment tests like the Rey Auditory Verbal Learning Test and Wechsler 
Memory Scale Revised are used to diagnose impairment during this phase. There may also be 
informal methods of testing if formal testing is not feasible. Genetics may also play a role in the 
diagnosis. If there are mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1), and 
presenilin 2 (PSEN2), which are autosomal dominant genes linked to AD, the patient is most 
likely prone to early-onset familial AD.  Additionally, medical specialists may look for the 
presence of an E4 allele in the apolipoprotein E (APOE gene), which helps to identify patients 
that may be prone to late-onset AD. Other biomarkers involved in AD diagnosis include amyloid 
beta plaques, tau accumulation in neurofibrillary tangles, neuronal damage, tau in cerebrospinal 
fluid, oxidative damage, cell death, and hippocampal atrophy. Visualization techniques using 
fMRI, PET and MRI studies may be involved in identifying biomarkers (Albert et al., 2011). 
  
3 
1.2.2 Risk Factors 
 According to the Alzheimer’s Association (2017), the most important risk factors of AD 
are age, family history, and heredity. The greatest known risk factor is age, as nearly 1 in 3 
people age 85 or older have the disease. However, more research is needed to fully understand 
the mechanisms responsible for rising AD risks with advancing age. People with family history 
of the disease are also more likely to develop the disease, which may be due to hereditary genes 
associated with AD, such as risk genes and deterministic genes. Risk genes are genes that 
“increase the likelihood of developing a disease,” such as APOE-e4 is for AD (Alzheimer's 
Association, 2017).  About 25 percent of AD cases are associated with this gene. More severely, 
deterministic genes “directly cause a disease”; there are a small number of these genes that cause 
AD in a few hundred extended families in the world. About 5 percent of people with familial 
early-onset AD are affected by these deterministic genes (Alzheimer's Association, 2017). 
1.2.3 Characterization of Alzheimer’s 
 One way in which AD can be differentiated is by the age of onset, specifically whether a 
case is early-onset or late-onset. Early-onset AD, in which patients display symptoms before age 
65, is the less common type. Early-onset patients will often suffer more severe symptoms and 
undergo a faster cognitive decline, as early-onset is a more aggressive form of AD. Neurological 
differences, such as increased gray matter atrophy in the parietal and temporal lobe, are 
associated with early-onset AD. On the other hand, late-onset AD patients will suffer more 
damage to the hippocampus, and will undergo slower cognitive decline (Canu et al., 2012). 
Another categorization is familial vs. sporadic AD. Most cases of AD are sporadic, 
showing up in individuals with no specified familial basis. Sporadic AD is also generally 
  
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associated with aging, and thus is more likely to occur with late-onset AD. While there has only 
been one specific gene, APOE, consistently associated with sporadic AD, several susceptibility 
genes have been linked to the familial form of the disease. Familial AD occurs in individuals 
with strong familial links to the disease, and is associated with both EO and LO. The genes 
PSEN1, PSEN2, and APP are correlated with familial AD, and are often used in genetic testing 
when evaluating individuals’ risk for AD (Alzheimer Society of Canada, 2017).   
1.3 Alzheimer’s Disease and Olfaction 
 One of the areas affected in AD are the olfactory bulb (OB), which is the neuronal 
structure primarily responsible for olfaction or sense of smell, and the hippocampus, which is 
involved in cognitive functions and plays a key role in memory (Zou, Lu, Liu, Zhang, & Zhou, 
2016). Therefore, a better understanding of the alteration of neurogenesis and cognitive function 
within these brain structures in models of AD may be useful in further establishing preventative 
measures and treatment for AD, alongside other neurodegenerative diseases. Loss of smell is not 
only evident in Alzheimer’s Disease, but also in other neurodegenerative diseases such as 
Parkinson’s Disease (Doty, 2012). The importance of olfaction in various diseases is commonly 
overlooked and has resulted in several studies that utilize quantitative smell tests.  
One of the earliest symptoms of AD is the loss of smell. Olfactory dysfunction may be 
related to the presence of neurofibrillary tangles and amyloid plaques in the OB. Studies have 
shown that OB tract atrophy increases over the progression of the disease (Thomann et al., 
2009). Thus, knowledge regarding the detection of sensory loss in the OB would be helpful in 
developments towards earlier recognition of AD, and it is relevant to study the OB in an attempt 
to better understand the pathology behind AD and its development as the disease progresses 
  
5 
(Wesson, Levy, Nixon, & Wilson, 2010). 
1.4 Adult Neurogenesis 
1.4.1 Overview of Neurogenesis 
 The OB is also one of the primary final destinations of neurons that are generated 
from the process of adult neurogenesis. Neurogenesis is the process of forming new neurons. 
Neural stem cells (NSCs) initially have the ability to differentiate into neurons, astrocytes, and 
oligodendrocytes. When dividing, a NSC produces one NSC and one neuron. NSC proliferation 
and differentiation occurs in the adult subventricular zone (SVZ), the rostral migratory streams 
(RMS), the granular dentate gyrus (DG), and the OB (Seki, 2011). The final destinations of 
NSCs are the granule cell layer (GCL) and glomerular layer (GL) of the OB. AD ultimately 
results in a loss of neurons and synaptic contacts due to impaired neurogenesis in the 
hippocampus and OB. This loss of neurons, or decrease in neurogenesis, is believed to be age-
dependent in AD, with older individuals having greater decreases. There are many proposed 
reasons for the age-dependent decline in neurogenesis (Rodríguez & Verkhratsky, 2011). 
 
Figure 1. Neurogenesis (Duman, Malberg & Nakagawa, 2001). 
  
6 
The degeneration of neurons in the areas of brain that are generally regulated by adult 
neurogenesis contribute to the decline in memory that occurs in AD (Cutuli et al., 2014). In order 
to better understand these mechanisms behind neurodegeneration in AD and ways to reverse its 
detrimental effects, researchers have explored a variety of different directions, many of which 
involve non-human models of AD.  
1.4.2 Mouse Models of Neurogenesis 
 Many recent studies investigating neurogenesis in both the OB and the hippocampus have 
used mouse models. Mouse models allow researchers to perform both behavioral studies to 
assess memory and to quantify cells in neurogenic areas of the brain. Additionally, these models, 
many of which have mutations in genes related to the pathological hallmarks of AD, allow for 
studying differential brain functioning for individuals with the disease; such models are useful 
for understanding adult neurogenesis in humans (Guerin, Sacquet, Mandairon, Jourdan, & 
Didier, 2009). It is for these reasons that we selected the mouse as our model organism in 
studying adult neurogenesis in the OB. 
1.5 Inspiration for Project/ Experimental Approach 
 In the brain, the OB is one of the sensory systems affected by changes in neurogenesis. In 
previous studies, the OB and hippocampus have both served as models for studies in 
neurogenesis. However, it is believed that the neurodegeneration in AD may occur up to 10 years 
prior to the onset of loss-of-memory symptoms in patients, and thus it is more promising in 
identifying AD patients earlier through looking at behaviors that are mediated by neurogenesis, 
such as olfactory behaviors (Dhilla Albers et al., 2016). Through better understanding the 
relationship between loss of neurogenesis in the olfactory bulb and olfactory function as 
  
7 
measured through olfactory tests in AD models, better methods of identifying AD patients earlier 
may be established.  Thus, this study will focus on the OB in modeling AD. In order to replicate 
the effects of one of the most prominent pathologies of AD (i.e., the accumulation of tau), 
transgenic mice models will be used.  These transgenic models have a transgene, which is a gene 
that is isolated from another organism, in this case humans, and is introduced into another 
organism (i.e., mice), resulting in their production of the protein coded by the transgene. 
Through the use of mice models, we will be able to make more realistic observations and 
conclusions in terms of the effects of the transgene on AD in humans, as opposed to other 
potential methods such as cell cultures. Thus, we propose this study to characterize the 
transgenic mice model, rTg4510 (Tg), in terms of the level of cognitive decline expressed as 
measured through olfactory behavioral tests, and in their levels of neurogenesis within the OB at 
various ages in the mouse’s life. This research will allow us to evaluate the effectiveness in the 
Tg model in serving as a representative model for AD. 
1.6 Research Questions and Hypothesis 
 This research aims to characterize Tg mice and determine whether it is an appropriate 
transgenic mice model for further study of AD pathology. Previous research studies, such as 
Scott et al. (2016), have focused on the hippocampus of Tg mice, but little work has been done 
regarding effects on the OB in this model, leading us to focus on this sensory system for our 
research. Specifically, this study will concentrate on quantifying neurogenesis in the OB, 
measured by using bromodeoxyuridine, or BrdU, a nucleoside that can be incorporated in newly 
replicated DNA and detected using antibodies. Behavioral tests to assess function of the OB and 
the ability to discriminate scents will also be carried out in this study.  Ultimately, our research 
  
8 
will seek to answer the following questions:  (1) Are there olfactory changes that occur in AD? 
(2) What are these changes (behavior/neurogenesis)? (3) How early do these deficits occur? We 
speculate that the Tg mouse model will show significantly greater age-dependent decline 
regarding cognitive function and neurogenesis in the OB compared to the WT mice due to the 
effect of the transgene that is meant to induce tau pathology that is characteristic of AD.  
  
  
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Chapter 2: Literature Review 
2.1 Pathology of Alzheimer’s Disease 
2.1.1 Tau Pathology 
 Tau and amyloid beta are the two main proteins involved in AD. Amyloid beta (Aβ) is 
cleaved from APP by α, β and γ-secretases and causes the formation of amyloid plaques in the 
brain. While amyloid beta protein is found extraneuronally, tau is found intraneuronally, and is 
primarily expressed in the gray matter of the neocortex. In its natural state, tau is a soluble and an 
unfolded microtubule-associated protein. However, it can become insoluble and build up as 
hyperphosphorylated filamentous aggregations. Such aggregations have been linked to changes 
in axonal transport, as hyperphosphorylation renders tau incapable of microtubule interactions. 
While the mechanism of tau hyperphosphorylation is not entirely understood, some of the 
kinases believed to be involved are glycogen synthase kinase 3 (GSK3) and casein kinase 2 
(CK2) (Demars, Hu, Gadadhar, & Lazarov, 2010).  Tau pathology is not restricted to AD, as tau 
has been implicated in other neurodegenerative diseases.  
Six isoforms of tau are found in humans due to differential splicing on the microtubule-
associated protein tau (MAPT) gene, and all six have been found in AD brain samples. 
Tauopathies are not limited only to AD but also found in a variety of neurodegenerative diseases 
including Down syndrome, encephalopathy, various dementias and brain iron accumulation. 
Increased levels of tau in cerebrospinal fluid (CSF) has been found up to 15 years before any 
signs of AD; however, the link has not been clarified (Spillantini & Goedert, 2013). Previous 
tau-based treatment directions have included inhibiting tau aggregation and phosphorylation, 
reducing general levels of tau, stabilizing microtubules, and using tau antibodies (Spillantini & 
  
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Goedert, 2013). 
Tau hyperphosphorylation has previously been linked to many different problems within 
neurons that contribute to a decrease in neurogenesis. There is evidence suggesting that tau 
hyperphosphorylation can lead to chromosome aberrations, which include missing, extra, or 
irregular portions of chromosomes, and that this can prevent mitosis from occurring.  In addition 
to affecting mitosis, hyperphosphorylation of tau may also interfere with the transport of 
molecules across axons and the maturation of neurons.  Specifically, Demars et al. (2010) found 
that in mice with mutations linked to AD, which showed decreased proliferation of neural 
progenitor cells and neural progenitor cell differentiation, there were greater levels of 
hyperphosphorylated tau.  Such results suggest that the decreased proliferation of progenitor 
cells may be due to the intracellular hyperphosphorylation of tau, and that NFTs formed by this 
hyperphosphorylation are a partial contributor to decreased neurogenesis (Demars et al., 2010). 
2.1.2 Amyloid Beta Plaques 
 As discussed above, the presence of abnormal Aβ peptide plaques is one of several 
important pathological features of AD. Aβ peptides are naturally produced as metabolic 
products. They originate from proteolysis of the APP by the beta-site amyloid precursor protein-
cleaving enzyme 1 (BACE-1), a β-secretase, and γ-secretase. Greater rates of production than 
that of clearance, as well as the aggregation of the peptides, result in the accumulation of Aβ 
plaques. Previous research indicates Aβ plaques as an implicating factor of AD (Querfurth & 
LaFeria, 2010). 
Aβ self-aggregates into different forms, one of which is oligomers that coalesce into 
intermediate assemblies. It is these oligomers and intermediate assemblies which are known to 
  
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be the most neurotoxic forms of Aβ and their numbers are associated with the severity of the 
cognitive defect in AD. High levels of Aβ at synapses dampen excitatory transmission and 
prevent neuronal hyperactivity. A reduction in both neprilysin, an endopeptidase that degrades 
Aβ, and insulin-degrading enzyme, a thiol metalloendopeptidase, causes Aβ accumulation 
(Querfurth & LaFeria, 2010). Various pathological hallmarks are triggered by Amyloid Beta 
species such as neuronal death, delayed timing in neuron death, dysfunctional synapses, and 
instant neuronal death. These pathological hallmarks require the cytoplasmic, tau pathology. The 
amyloid Beta proteins and tau enhance each other’s toxicity overall in the form of a feedback 
loop.  
2.1.3 Neurofibrillary Tangles 
 Mutation of the tau gene can lead to formation of insoluble filaments of 
hyperphosphorylated tau aggregates called neurofibrillary tangles (NFTs), which are considered 
to be another pathological hallmark of AD. NFTs are not restricted to AD and are also implicated 
in other neurodegenerative diseases. These mutations cause microtubule destabilization, decrease 
tau affinity to microtubules, and result in tau aggregation (Rocher et al., 2009). Despite positive 
correlations between NFTs and cognitive deficits, the direct relationship between them still 
remains unclear (Santa Cruz et al., 2005). Several previous studies have indicated that NFTs may 
not be linked to the progression of neurodegeneration in AD and may even play a protective role. 
One study concluded that structural and functional neuronal changes from expression of mutated 
tau were independent of NFT formation in mutant tau overexpressing mice models (Rocher et 
al., 2009). Another study observed NFT formation and extensive cell death in aged mice 
expressing non-mutant human tau, suggesting that cell death may occur independently of NFT 
  
12 
formation (Andorfer et al., 2005). Although the relationship between NFTs and tau mutations 
remains elusive, there is wide evidence for NFT formation contributing to the progression of AD.  
 
Figure 2. Pathological Hallmarks of AD (Boston University School of Public Health, 2013). 
 
Figure 3. Cascade of factors involved in the development of AD (Giacobini & Gold, 2013) 
2.2 Neurogenesis in the Olfactory Bulb 
 The OB is multi-layered. The source of newborn neurons is the subventricular zone 
(SVZ) of lateral ventricles and they migrate through the rostral migratory stream (RMS) to the 
  
13 
OB (Lazarini & Lledo, 2011). In the OB, the new neurons will encounter multiple cell layers in 
between the olfactory tract: GCL (closest to olfactory tract), internal plexiform layer, mitral cell 
layer, external plexiform layer, glomerular layer (GL), and olfactory nerve layer (Doty, 2012). 
This spatial migration process occurs during the period of six days (Alonso et al., 2006). 
 As the young neurons migrate from the SVZ to the OB, they differentiate into granule 
cells (GCs) and periglomerular cells (PCs). A majority of the GCs in the OB will be generated 
postnatally and continue generation into adulthood. Due to apoptosis occurring in the layers 
where young neurons are placed, these newly generated GCs replace older cells. The GCs begin 
to form into sheets for the GCL. Short neurites extend into deeper sections of the GCL and a 
large dendrite extends in the direction of the external plexiform layer (EPL). The role of GCs is 
to monitor the mitral and tufted cells to optimize olfactory function. They do so by decreasing 
possible overlap for odor representations executed by mitral and tufted cells. The tufted cells 
receive input from receptor cells in the olfactory epithelium (found in the nose) to aid in the 
sense of smell. Mitral cells’ response to odor is shaped by GCs in the form of activity-dependent 
mechanisms which leads to an increase in olfactory discrimination (Petreanu & Alvarez-Buylla, 
2002).  
 There are five stages in the development of newborn neurons. The classifications are: 1) 
tangentially migrating neuroblasts (days 2-7), 2) radially migrating young neurons (days 5-7), 3) 
GCs with a simple unbranched dendrite (days 9-13), 4) GCs with a non-spiny branched dendrite 
in the EPL (days 11-22), and 5) mature GCs (days 15-30), and periglomerular interneurons. 
After 15 to 22 days after birth, newly-generated GCs will display a mature morphology 
(Petreanu & Alvarez-Buylla, 2002).  
Stimulation of adult neurogenesis has been seen to improve long-term memory and 
  
14 
olfactory learning.  For example, Lepousez et al. (2014) found that increase in olfactory learning 
leads to higher spine density of adult-born GCs Olfactory learning also stimulates restoration of 
excitatory and inhibitory inputs in the dendritic domain of adult-born neurons. Both associative 
(classical and operant conditioning) and nonassociative (habituation and sensitization) forms of 
olfactory learning require adult neurogenesis. In turn, adult-generated neurons are needed to 
shape olfactory memory circuits. Odor deprivation generally decreases the survival and 
excitability of adult-generated OB neurons as well as olfactory memory performance (Alonso et 
al., 2006; Singer et al., 2009).  
The olfactory epithelium, specialized sensory tissue inside the nose, provides chemical 
information to the OB circuit. Sensory neurons transform this chemical signaling into electrical 
patterns (Lazarini & Lledo, 2011). Centrifugal fibers found in the OB contribute to context-
dependent modulation of circuit activity, such as attention, reward, learning, and memory 
(Lazarini & Lledo, 2011). Before a certain critical period, odor exposure does not affect the 
survival of newborn neurons, suggesting that the lack of activity does not have an effect on 
generation, migration, and early differentiation of GCs. However, when the GCs mature and 
presumably become synaptically connected, their survival depends on the level of activity. The 
dependence of mature GCs on odor exposure may allow construction of specific OB circuits and 
replacements (Petreanu & Alvarez-Buylla, 2002). 
  
15 
 
Figure 4. The neuronal circuit of the OB (Aimone et al., 2014). 
2.3 Therapeutic Approaches for Neurodegenerative Diseases 
 During the early stages of neurodegenerative diseases, changes in neurogenesis are 
observed. This suggests that interventions preserving neurogenesis could be helpful for 
presenting therapeutic strategies to slow down cognitive decline with the diseases (Rodríguez & 
Verkhratsky, 2011).  
The main focus of brain repair is to increase the amount of new neurons into the brain to 
fix any possible brain lesion damages. In addition to understanding neurogenesis and brain 
repair, research concluded that progenitors of new neurons are present in the subventricular and 
subgranular zones of the hippocampal DG. The generation of neuroblasts in the SVZ travel to 
  
16 
reach the adult OB and become neurons. Through this, certain types of grafted neurons are able 
to travel through the brain (Lois & Kelsch, 2014). 
2.4 Recent Research on Neurodegenerative Diseases Using Mice Models 
 Many studies target the Aβ hypothesis of AD, either by attempting to decrease initial 
production or increase eventual clearance. A 2014 study used the Tg2576 familial AD mouse 
model. After undergoing immunotherapy by intraperitoneal antibody injection to target specific 
oligomers in Aβ plaques, plaques were not reduced although synapse loss was alleviated. The 
study hypothesized that cognitive functions, as a result, may increase (Dorostkar et al., 2014).  
 Another theory of treatment focuses on the behavioral aspect of AD, focusing on the idea 
that increased cognitive stimulation might delay or alleviate symptoms of AD. A 2015 study of 
3xTg-AD mice with both APP and tau301L transgenes indicated that, if training began at p3mo, 
postnatal three months, mice were able to perform better on hippocampus-dependent spatial 
tests. This training was episodic and continued throughout the aging process. These tests 
included the Morris Water Maze and the novel object recognition memory task (Martinez-Coria 
et al., 2015).  
 In addition, a 2014 study investigated the relationship between memory deficits upon tau 
expression in the hippocampus using the rTg4510 model expressing the tau301L transgene. 
Behavioral tests such as hippocampal-dependent incremental repeated acquisition (IRA) and 
MWM were conducted to identify memory deficits. Tetracycline transactivator (TTA) allowed a 
conditional model of tau P301L expression; tau is expressed when tetracycline is absent. They 
found the presence of tau pathology after 3 months of tau expression in the hippocampus. The 
behavioral test results aligned with these findings as the Tg mice showed greater memory deficits 
  
17 
with tau expression (Hunsberger et al., 2014). 
2.5 Selection of a Mouse Model  
 We took many considerations when deciding which mouse model would be used for our 
study. Primarily, it was important to have a model that was in fact representative of AD through 
its pathology. 
  There has been a great deal of evidence that there is a loss of synaptic connections in AD, 
which partially contribute to the cognitive decline. While it was previously thought that the main 
protein responsible for cognitive decline in AD was strictly Aβ, there is more support due to 
recent studies for an equally important role of tau (Pooler, Noble, & Hanger, 2014). Specifically, 
in AD, tau becomes hyperphosphorylated, and it is no longer able to interact correctly with 
microtubules. Additionally, the soluble form of tau is thought to be neurotoxic and lead to 
synaptic generation, though the mechanism is not yet fully understood (Pooler, Noble, & Hanger, 
2014). 
 
 
Figure 5. Tau Function in Healthy vs. Diseased Neurons (Brunden, Trojanowski, & Lee, 2009)  
 
  
18 
 Although Aβ is usually considered the main pathological hallmark of AD, tau is equally 
important due to its role in the formation of neurofibrillary tangles. While there are several 
mouse models that are created to represent the Aβ hypothesis, specifically those that have 
mutations in APP or the presenilin genes, these models do not allow for the study of the effects 
of NFTs as produced by hyperphosphorylated tau (Chin, 2011). Thus it is important to choose a 
model that allows for the isolation of the effect of hyperphosphorylated tau pathology without 
the presence of Aβ aggregates.   
 It is currently not known why mice that are Tg in the APP gene (responsible for the 
formation of amyloid-beta aggregates) do not always develop NFTs, nor are the interactions 
between Aβ and tau pathologies understood very well (Chin, 2011). If models can be used that 
contain only transgenes involved in the formation of hyperphosphorylated tau and tau 
aggregates, the role of tau in cognitive impairment and neurodegeneration can be better 
understood.  
For our experiments, we selected the Tg4510 mouse model, which express a repressible 
form of the MAPT P301L mutation in the genome, responsible for the dementia pathology 
(SantaCruz et al., 2005; Hunsberger et al., 2014). The expression of this transgene leads to 
impairments in a wide range of areas, including spatial reference memory, forebrain atrophy, and 
pathologies of neurofibrillary tangles (Ramsden et al., 2005). The transgene was confirmed to be 
strongly expressed in structures present in the forebrain, specifically the neocortex and the 
hippocampus, and thus it is a model relevant to studying diseases that involve dysfunctional tau 
protein, such as AD and frontotemporal dementia with parkinsonism (Ramsden et al., 2005). 
Importantly, the expression of this transgene is suppressible, meaning that it can be turned off 
with the administration of doxycycline (Ramsden et al., 2005).  
  
19 
Following the generation of this mouse model, several experiments have been performed to 
study the effects of the expression of the P301L transgene on the Tg mouse model. One 
particular study looked at the neuronal network activity in the hippocampus of Tg mice at 
various ages. The study found that there was an age-dependent formation of the NFTs and loss of 
neurons in the hippocampus, as well as an impaired memory function, which was tested with the 
MWM, beginning as early as 2.5 months of age (Scott, Kiss, Kawabe, & Hajos, 2016). A similar 
study found that compared to WT littermates, Tg mice had significant cognitive impairments at 
the age of 4 months, in addition to a loss of neurons in the hippocampus (Ramsden et al., 2005). 
In this mouse model, pre-tangles were also found in the cortex and the basal ganglia by the age 
of 6.5 months (Lewis et al., 2000).  
 
Figure 6. Timeline of the Neurodegeneration of the Tg mouse model (Crimins, Rocher, & Leubke, 2012; 
Menkes-Caspi et al., 2015; Santa Cruz et al., 2005; Spires et al., 2006). 
 
These studies collectively suggest that this mouse model is a useful model for 
comparison with human tauopathy and that it may be established as a standard method of 
studying the buildup of tau for application to human neurodegenerative diseases including AD. 
  
20 
Chapter 3: Methodology 
3.1 Research Design 
This study aims to characterize the olfactory decline of Tg tau pathology mouse model 
through behavioral tests and neurogenesis analysis. 
3.1.1 Animals 
All of the animal procedures and experiments for this study were performed in 
accordance to the guidelines of the Institutional Animal Care and Use Committee of the 
University of Maryland. All experiments including the behavioral, perfusion, and 
immunohistochemistry were performed rTg4510 female and male mice which were littermates 
(1-7 months old). This strain of mice was obtained from the Jackson Laboratory (stock 
#015815). Since cortical cell loss occurs around 8 months in this strain, we performed 
experiments within the strain from the time of 60 days postnatal (PD60) through the age of 7 
months (Spires et al., 2006).  Because the Tg strain have a transgene inserted that causes the 
development of AD related pathology, we referred to these mice as the “AD group,” or our 
experimental group, and the same observations and injections were conducted in parallel for the 
non-positive littermates. Both female and male mice were used in experimental groups, with 
roughly equal numbers of each sex per trial group. 
3.1.2 Environmental Conditions 
It is important that mice have the same environmental conditions to prevent confounding 
factors. Animals were kept on a cycle with 12 light/12 dark cycle. All behavioral experiments 
were performed during the dark cycle of the day for the mice, during which time the mice are 
  
21 
known to be more active. Three to five mice were housed per cage. The mice were housed at 72 
degrees Fahrenheit and the humidity was controlled to 60 plus or minus 5%, in accordance with 
standards of mice studies. The mice were monitored daily to ensure healthy mass and food 
intake. 
3.1.3 Treatment Groups 
All experiments were performed on four treatment groups, each consisting of three to 
seven mice. The groups are composed of WT or Tg strain at 2 and 7 months. These mice are 
referred to as “Young” and “Old,” respectively. Some behavioral testing was performed on 
various younger mice groups (1 and 3 months) based on availability at the time of the 
experiments.  
 WT Tg 
Young p60 WT p60 Tg 
Old p7mo WT p7mo Tg 
 
Figure 7. Treatment groups of WT and Tg mouse models. 
  
22 
3.1.4 Behavioral Tests 
Several behavioral tests were performed in order to determine cognitive learning in the 
mice.  
Habituation/dishabituation. The habituation dishabituation protocol is used to analyze 
the ability to discriminate between odors. A 1:1000 dilution of the primary odor is presented on a 
small wooden cube and placed within the mouse’s cage after a thirty minute period of 
habituation to the cage with a plain cube. The mouse is presented with a repeated odor for 
several trials. The amount of time in two minutes that the mice spend investigating the face of 
the cube with the odor is recorded. The inter-trial interval approximately 30 seconds. On the 
seventh trial, this odor is replaced by a novel odor. If the mouse is able to discriminate between 
odors, it will demonstrate dishabituation. Ideally, the mice should have decreased investigation 
time as trial six approaches, and an increased investigation time in the seventh trial. That would 
indicate the mouse’s ability to distinguish between the two isomers. The 
habituation/dishabituation test serves as a general control, ensuring that the mice used in later 
behavioral experiments have normally functioning olfactory systems. It observes olfactory 
memory and learning by using two isomers, L-carvone and D-carvone.  A two-tailed t-test was 
used for data analysis.  
 
Figure 8. Habituation Dishabituation Protocol. Primary odor, L-carvone; Novel odor, D-carvone 
  
23 
Odor detection threshold. The Odor Detection Threshold (ODT) was determined by 
testing the investigation time of four groups of mice at different concentrations of L-carvone. 
The purpose of the test was to identify the lowest concentration at which the mice could detect 
different odors. The groups consisted of 1 month WT, 1 month Tg, 7 months WT, and 7 months 
Tg. Each mouse was placed into a clean cage with a small, wooden cube placed into the center 
for 30 minutes to allow it to become habituated to the cube. For the first trial, a new cube with 
100 microliters of water placed on one surface of it was inserted into the cage. The investigation 
time of the mice was recorded with a timer for 1 minute and this is influenced by the amount of 
time the mice spent investigating the face of the cube with the water present. This procedure with 
the water is repeated until the seventh trial. On the seventh trial, 100 microliters of serial 
dilutions of either 1:100,000, 1:150,000, 1:200,000, 1:200,000, 1:250,000, or 1:300,000 of two 
isomers, L-carvone or D-carvone.  
 
Figure 9. Odor Detection Threshold Protocol. Odor, either L-carvone or D-carvone. 
Novel object recognition. The Novel Object Recognition (NOR) task was adopted from 
the protocol used by Bevins and Besheer (Bevins & Besheer, 2006). The paradigm was carried 
out for four different groups of mice, including 2 month WT and Tg, as well as 7 month WT and 
Tg. The day of the testing, we placed the mice in an unfamiliar cage (the cage in which the 
  
24 
experiment would take place) for an acclimation period of 10 minutes. After 10 minutes, the 
mouse was removed and temporarily placed in a cup while we placed the training objects, which 
were two blue marbles, on opposite sides at the back of the cage. These marbles were taped to 
the bottom of the cage so that they could not be moved by the mice. The mice had a training 
period of 10 minutes during which time they were placed in the cage with the marbles, and the 
amount of time spent investigating each marble was recorded with the use of a stopwatch. The 
marbles were then removed and the mouse remained in the empty cage for a 45 minute period. 
We then placed the trained object, the blue marble, in the back corner of one side of the cage, 
and introduced the novel object, a yellow wooden cube of similar size, in the opposite corner. 
The side of the cage in which the novel object was placed was randomized. The mouse then 
underwent a 5 minute testing period with the trained object and the normal object, and the time 
spent investigating each object was again recorded. During the testing interval, both objects were 
taped down in the cage to prevent them from being moved. The NOR serves as a control for 
general neurogenesis and cognitive ability in the mice. 
 
 
Figure 10. Depiction of phases for novel object recognition test. 
  
25 
3.1.5 BrdU Injections 
Cell tracer 5’-bromo-2’-deoxyuridine (BrdU) is a thymidine analog that becomes 
integrated into the cell during cell division and is subsequently passed down to daughter cells 
(Wojtowicz & Kee, 2006). BrdU marks cells undergoing cell division and newly formed neurons 
at stages of proliferation, differentiation and maturation, and can thus be used to trace 
neurogenesis from proliferation to maturation. BrdU prepared in phosphate-buffered saline (PBS, 
10 mg/mL) was injected intraperitoneally at 100m/kg per injection for a total of three times, 
every two hours to 1 month old mice (Nunez-Parra, Pugh & Araneda 2010). BrdU was used to 
visualize newly born adult neurons. 
3.1.6 Perfusion 
T as previous studies have indicated that newly born neurons have differentiated into 
mature GCs and periglomerular cells (PG) approximately 30 days after their formation (Nunez-
Parra, Pugh & Araneda 2010). Mice were anesthetized with isoflurane and the brains were fixed 
by transcardial perfusion with about 40mL of cold PBS until the fluid was clear of blood when 
leaving the body of the mouse. Cold 4% paraformaldehyde solution (PFA, Electron Microscopy 
Science) was prepared in PBS in 1:4 ratio. After perfusion, mice brains were carefully dissected 
to preserve whole, intact OB, and post fixed in 4% PFA overnight at 4°C in order to minimize 
damage to the OB (Nunez-Parra, Pugh & Araneda 2010).   
3.1.7 Tissue Preservation 
After perfusion fixation, the mice brains were removed and cryoprotected overnight with 
a solution of 30% sucrose in PBS until they were in equilibrium with the solution and sunk to the 
bottom. These brains were then sectioned into right and left OBs and hippocampi. All four 
  
26 
sections were embedded in Tissue-Tek O.C.T. (Electron Microscopy Science) and cryopreserved 
in -80°C. The hippocampal sections were preserved for further reference due to time constraints 
of the experiment (Nunez-Parra, Pugh & Araneda 2010). 
3.1.8 Cryostat and Slicing 
Randomly chosen OB sections of each preserved brains were sliced in 20µm thick 
sagittal sections using a Leica CM1850 cryostat. Four sections were mounted on each of six 
frozen tissue slides (Fisher) and stored at −20 °C until use (Nunez-Parra, Pugh & Araneda 2010). 
3.1.9 Immunohistochemistry 
The brain slices containing the accessory and main OBs (AOB and MOB, respectively) 
were prepared for histology under the confocal microscope using immunohistochemical staining 
methods. These slices were rinsed with PBS twice and double distilled water once after being 
warmed to room temperature. Then, they were placed in hydrochloric acid solution at 37
 for 
one hour to denature the DNA and facilitate staining by antibody binding. After rinsing three 
times with sodium tetraborate buffer for neutralization, the slices were washed twice with PBS 
and once with PBS-T (PBS solution with Triton-x-100). 10% donkey serum in PBS-T was used 
to block nonspecific sites of the brain slices for one hour. Next, they were incubated with rat 
anti-BrdU to label newly born cells and mouse anti-NeuN to label all neurons diluted in PBS-T 
and donkey serum overnight at room temperature. The slices were rinsed four times with PBS-T 
the next day and incubated with secondary antibodies donkey anti-Rat Alexa-594 and donkey 
anti-Mouse Alexa-488 diluted in donkey serum and PBS-T for two hours at room temperature in 
the dark. Finally, they were rinsed with PBS-T once, PBS three times, mounted with 
Vectashield, sealed coverslips with clear nail polish, and stored in a dark place (Nunez-Parra, 
  
27 
Pugh & Araneda 2010). 
3.1.10 Confocal Microscopy 
Three out of the four sagittal sections per animal containing the AOB and MOB GL and 
GCL layers were used for quantification. We used the Leica SP5X confocal microscope provided 
by Imaging Core, with white light laser (470-670 nm in 1 nm increments) at 70%, xyz scanning, 
and z-Galvo configurations. We used 63x oil immersion objective at 3µm optical steps. In order 
to focus and navigate, we also visualized the specimen through the eyepiece under filter sets GFP 
and RFP. For each sample, we only imaged if one or more neurons could be visualized in filter 
set GFP and RFP. We used double labeling with BrdU, indicating newly generated neurons in 
red, and NeuN, indicating all neurons in green. Cells which expressed both indicated the 
presence of neurogenesis (Wojtowicz & Kee, 2006).    
  
28 
  
Figure 11. Confocal images of sagittal sections contained AOB and MOB GL and GCL layers. RFP is 
used to visualize BrdU (newborn neurons), and GFP is used to visualize NeuN (all neurons). When there 
is colocalization between red and green, cell is positive for both BrdU and NeuN (Nunez-Parra, Pugh & 
Araneda, 2010). 
 
3.1.11 Statistical Analysis 
A total of twenty-four sagittal brain slices were produced of either right or left brain 
hemispheres for each mouse. The twenty-four serial sagittal sections were divided into six slides, 
four sections per slide, and one out of the six slides were used for quantification. Only three 
  
29 
sections out of the four on the randomly chose slide were used for confocal analysis. For each 
section, BrdU positive cells were quantified in a total of fourteen regions, including the GCL and 
GL of the AOB and MOB, 2 to 4 Z-stack images, respectively. Data was expressed as the mean 
number of BrdU positive cells normalized to the area of the Z-stack in mm3 (BrdU positive 
cells/mm3) ± standard error for each region (Nunez-Parra, Pugh & Araneda 2010). Statistical 
significance was determined using two-tailed t-tests on excel. Graphs were produced using the 
Igor software. 
 
  
  
30 
Chapter 4: Results 
4.1 Behavioral Data 
4.1.1 Habituation/Dishabituation 
The habituation/dishabituation paradigm was conducted for six different groups of mice, 
including 2 month WT and Tg, 3 month WT and Tg, and 7 month WT and Tg. 3 month Tg mice 
were compared by gender and the female Tg mice generally showed lower investigation times in 
all seven trials than male Tg mice did (Fig. 12A). Behavioral data by gender can be further tested 
in future studies to determine significance of gender in habituation and dishabituation. Overall, 
the investigation time of the novel odor used in the seventh trial increased for all six groups as 
compared to that of the odor used in the sixth trial (Fig. 12B). Of the six groups, 2 month Tg, 3 
month WT, 3 month Tg, and 7 month Tg mice showed statistically significant increase between 
the two trials and 2 month WT and 7 month WT did not show any significance (Fig. 12B). 
 
Figure 12. Habituation/Dishabituation results. (A) Investigation time in seconds across 7 trials of 3 
month Tg mice for gender comparison. Female Tg mice have lower investigation time in general when 
compared to male Tg mice. (B) Investigation time in seconds across three age groups (2 months, 3 
months, 7 months), WT vs. Tg. Light gray indicates investigation time for trial 6, L-carvone, the same 
odor used for trials 1-5. Dark gray indicates the novel odor, D-carvone. There should be a significant 
increase in investigation time. Significance is indicated with an asterisk (t-test;*, p<0.05). 
 
 
  
31 
Trial 6 (sec) Trial 7 (sec) Number of mice p-value 
4.6 ± 0.8 12.6 ± 1.4 8 <0.05* 
 
Table 1: Figure 12A data 
 
Age/Genotype Trial 6 (sec) Trial 7 (sec) Number of 
mice 
p-value 
2mo WT 1.7 ± 0.5 4.3 ± 1.5 4 0.19 
2mo Tg 2.8 ± 0.4 7.0 ± 1.1 12 <0.05* 
3mo WT 5.1 ± 1.5 16.8 ± 0.8 4 <0.05* 
3mo Tg 4.6 ± 0.8 12.6 ± 0.8 8 <0.05* 
7mo WT 0.6 ± 0.6 2.2 ± 1.1 2 0.24 
7mo Tg 0.0 ± 0.0 1.8 ± 0.6 4 <0.05* 
 
Table 2: Figure 12B data 
4.1.2 Odor Detection Threshold 
Previous studies show a correlation between olfactory dysfunction and neurodegenerative 
diseases (Albers, Tabert & Devanand, 2006; Enwere, 2004). The groups were compared in four 
different combinations and the data showed a general increase in the investigation time 
difference between the sixth and seventh trials as the concentration of L-carvone increased (Fig. 
13). Significance was shown in Fig. 13B comparing 1 month and 7 month Tg mice at 1:150,000 
concentration and in Fig. 13C comparing 1 month WT and Tg mice at 1:200,000 concentration . 
No significance was seen in Fig. 13A comparing 1 month and 7 month WT mice at all 
  
32 
concentrations , Fig. 13B at 1:100,000 and 1:200,000 concentrations , Fig. 13C at 1:100,000 and 
1:150,000 concentrations , and Fig. 13D comparing 7 month WT and Tg mice at all 
concentrations . 
 
Figure 13. Odor Detection Threshold of different age and genotypic groups. (A) Investigation time in 
seconds of mice at  trial 6 and 7, where the odor is changed, recorded of five different concentration of L-
carvone comparing 1 month and 7 month WT mice (B). Comparing 1 month and 7 month Tg mice at five 
different concentrations. (C). Comparing 1 month WT and 1 month Tg mice at three different 
concentrations. (D). Comparing 7 month WT and 7 month Tg mice at three different concentrations (t-
test; *, p<0.05). Time difference in trial 6 and 7 increases as concentration increases in all mice but 1 
month WT mice generally have higher time differences than older Tg mice. 
 
 
 
 
 
 
  
33 
 
Concentration 1mo WT 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
7mo WT 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
p-value 
1:200,000 1.5 ± 0.7 2 0.9 ± 0.4 4 0.48 
1:150,000 4.3 ± 1.2 2 1.1 ± 0.8 4 0.18 
1:100,000 5.2 ± 1.2 2 1.9 ± 0.7 4 0.21 
 
Table 3: Figure 13A data 
 
Concentration 1mo Tg Δ 
Trial 6 & 
7 (sec) 
Number of 
mice 
7mo Tg 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
p-value 
1:200,000 1.5 ± 0.7 2 1.8 ± 0.5 3 0.08 
1:150,000 2.6 ± 1.5 8 1.6 ± 0.4 4 0.02* 
1:100,000 3.6 ± 1.0 6 2.8 ± 0.8 4 0.31 
 
Table 4: Figure 13B data 
 
Concentration 1mo WT 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
1mo Tg 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
p-value 
1:200,000 1.5 ± 0.7 2 3.5 ± 1.6 8 0.03* 
1:150,000 4.3 ± 1.2 2 2.6 ± 1.5 8 0.25 
1:100,000 5.2 ± 1.2 2 3.6 ± 1.0 6 0.41 
 
Table 5: Figure 13C data 
  
34 
 
Concentration 7mo WT 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
7mo Tg 
Δ Trial 6 
& 7 (sec) 
Number of 
mice 
p-value 
1:300,000 0.5 ± 0.3 4 1.2 ± 0.8 3 0.14 
1:250,000 1.3 ± 0.8 4 0.7 ± 0.4 3 0.37 
1:200,000 0.9 ± 0.4 4 1.8 ± 0.5 3 0.12 
1:150,000 1.1 ± 0.8 4 1.6 ± 0.4 4 0.19 
1:100,000 1.9 ± 0.7 4 2.8 ± 0.8 4 0.06 
 
Table 6: Figure 13D data 
4.1.3 Novel Object Recognition 
The results of the novel object recognition test showed that there were significant 
differences in investigation times between the trained and novel objects for the 2 month WT, 2 
month Tg, and 7 month WT mice (Fig. 14). No statistical significance was shown in 7 month Tg 
mice (Fig. 14). Despite the statistical significance, all mice groups had higher exploration time of 
the novel object as compared to that of the trained object. Discrimination index was calculated 
using a formula taken from a paper by Antunes and Biala in order to confirm that the mice could 
successfully discriminate the trained and novel objects. The formula used was DI  =  (Novel 
Object Exploration Time - Trained Object Exploration Time)/Total Exploration Time (Antunes 
& Biala, 2012). A positive score indicated more time spent with the novel object whereas a 
negative score indicated more time spent with the familiar object. A zero score indicated a null 
preference (Antunes & Biala, 2012). Calculations were all positive with the highest DI for 2 
  
35 
month WT and lowest DI for 7 months Tg mice (2 month WT, 0.786; 2 month Tg, 0.623; 7 
month WT, 0.576; 7 month Tg, 0.382; not shown in figure). 
 
Figure 14. Novel Object Recognition Test of 2 and 7 month old WT and Tg mice (t-test;*, p<0.05, 2mo 
WT, n = 6, 2mo Tg, n = 4, 7mo WT, n = 6, 7mo Tg, n = 3). Longer exploration time in seconds of novel 
object in all mice groups. 
 
Age/Genotype Exp time of 
TO (sec) 
Exp time of 
NO (sec) 
Number of 
mice 
p-value 
2mo WT 1.2 ± 0.4 10.0 ± 1.8 6 0.003* 
2mo Tg 2.0 ± 0.2 8.6 ± 1.9 4 0.038* 
7mo WT 2.1 ± 0.5 7.8 ± 0.6 6 0.00003* 
7mo Tg 5.5 ± 1.4 12.3 ± 2.7 3 0.108 
 
Table 7: Figure 14 data 
  
36 
4.2 Confocal Data 
 The confocal imaging data consisted of sections of the AOB and MOB to examine the 
correlation between age, genotype, and number of BrdU+ cells. No statistical significance was 
observed in all four groups in the AOB and the MOB (Fig. 15).   
 
 
Figure 15. Confocal data visualized under Leica SP5X (confocal microscope). BrdU+ cells/mm³ 
represents the number of cells that stained positive for BrdU and NeuN, or the number of newly 
differentiated and colocalized neurons. (A) The AOB exhibited a surprising increase in BrdU+ cells from 
2 to 7 months of age, although WT mice had greater neurogenesis than Tg mice did at 7 months of age. 
(B) The main OB neurogenesis count increased for WT mice, while it decreased for Tg mice, over age. 
No statistical significance was observed (t-test, *, p<0.05).  
 
Age/Genotype BrdU+ cells/mm3 Number of mice p-value 
2mo WT 1369 ± 157 3 0.996 
2mo Tg 1365 ± 6 3 
7mo WT 2195 ± 248 3 0.692 
7mo Tg 1647 ± 222 3 
 
Table 8: Figure 15A data 
 
  
37 
Age/Genotype BrdU+ cells/mm3 Number of mice p-value 
2mo WT 2855 ± 148 3 0.583 
2mo Tg 2213 ± 17 3 
7mo WT 3733 ± 228 3 0.167 
7mo Tg 1650 ± 104 3 
 
Table 9: Figure 15B data 
  
  
38 
Chapter 5:  Discussion 
5.1 Behavioral Data 
 Multiple behavioral tests were conducted to analyze the cognitive deficits in the Tg mice 
when compared to the control. 
5.1.1 Habituation/Dishabituation 
 The habituation/dishabituation protocol was chosen as a standard baseline test that 
controls for the mouse’s ability to discriminate between odors. If the mouse has this ability, it 
demonstrates dishabituation upon the seventh trial’s introduction of the novel odor. Neurogenesis 
continues in the OB, where neurons mature and integrate. This indicates that the mice have 
functional olfactory processing systems, as well as olfactory learning and memory, and can be 
accurately used for behavioral paradigms such as ODT and NOR. 
The habituation/dishabituation protocol analyzed the ability to distinguish odors at 2, 3, 
and 7 months of age. 2 month Tg mice actually had higher investigation times than 7 month WT 
mice. At 2 months, it is likely that the effects of neurodegeneration have not yet materialized in 
the mouse model. Mice are not finished with the developmental phase at this age. In many other 
mouse models for AD, the effects of neurodegenerative disease do not become apparent in 
behavioral testing until around 3 or 4 months of age. At 3 months, there is a markedly significant 
difference. WT mice investigated the novel odor for a longer time than did the Tg mice group. 
Overall, the 3 month group investigated the novel odor for a longer time than the 2 month age 
group did; this may be attributed to brain and OB maturation. At 7 months, both WT and Tg 
mice groups show a marked decline in NOR. The WT group has a slightly higher investigation 
time. The results of the habituation/dishabituation paradigm seem to support our hypothesis that 
  
39 
the age-dependent cognitive decline and behavioral test performance due to development of tau 
introduced by the transgene.  
5.1.2 Odor Detection Threshold 
 Odor detection protocol observed the ability of the mice to detect minute difference in 
odor and to determine the ODT. The 1 month WT mice group showed significant increased 
levels of investigation time with the different concentration of L-carvone compared to the 7 
month WT group who showed only a small increase in investigation time for the different 
concentration of L-carvone. The 1 month Tg mice showed a higher increase in investigation time 
as concentrations increased compared to the 7 month Tg mice which may be due to the fact that 
the 7 month mice are older and may show a decline in odor recognition. The 1 month WT group 
of mice showed a higher investigation time as the concentrations of the odor solution increased 
compared to the decreased and increased investigation time by the 1 month Tg group as the 
concentration of the L-carvone odor solution increased. There was only a slight difference 
between the investigation time as the concentrations increased and the 7 month Tg group actually 
showed a slightly higher investigation time compared to the WT group.  
These results may indicate that aging in both mice types are correlated to decreased 
investigation time in detecting the change in odor concentration of the L-carvone solution. This 
is in relation to the hypothesis of observable behavioral changes between 1 month and 7 month 
old mice. WT mice have also shown a significantly higher amount of investigation time to the 
increased concentration of the odor solution which may support our hypothesis discussing the 
differences between WT and Tg mice at each age related to the age-dependent cognitive decline 
and decrease in neurogenesis due to the introduction of the tau gene. A decrease in GCs due to 
  
40 
decreased neurogenesis from AD can be contributing to these results. A decrease in GCs can lead 
to a decrease in odor discrimination abilities in mice due to their role in coding odorant 
information (Gheusi et al., 2000). 
5.1.3 Novel Object Recognition 
 NOR protocol observed the ability of the mice to detect differences of objects and 
therefore indicates their ability to explore the environment and detect changes (Bevins & 
Besheer, 2006). This task is meant to be reflective of neural processes that are involved in 
memory storage, and thus serves as a reflection of the activity of the hippocampus, the main 
brain structure associated with object recognition. 
The NOR task was selected for our research due to its ability to promote the natural 
tendency of mice to explore their environment and new objects, and because it is in no way 
aversive to the animals. In this way, we are more confidently able to attribute the results directly 
to the cognitive function of the mice, rather than to any effects of fear or stress in the mice. 
Because the mice of the 2 month WT, 2 month Tg, and 7 month WT were able to significantly 
distinguish the novel object in their environment, this suggests that the mice do not have 
impaired functioning of the hippocampus, or at least not to an extent that results in an inability to 
distinguish between objects. Also, 2 month WT mice showed the highest positive DI, meaning 
this mice group spent more time investigating the novel object compared to the trained object, 
hence showing discrimination. Although our sample size was small, if we had a large sample 
size and less deviation, the fact that the 7 month Tg mice were unable to distinguish the two 
objects while the 2 month Tg did would suggest that there is cognitive impairment in the 
hippocampus and memory storage functioning that develops within the Tg mice sometime 
  
41 
between these two age groups, while no such deficits develop for WT mice. The 7 month Tg 
mice also had the lowest positive DI indicating that it spent the least amount of time 
investigating the novel object as opposed to the trained object compared to other mice groups, 
hence they were worse at discriminating the objects. Such findings support the results of similar 
studies that found Tg mice to have reduced dentate gyrus spike amplitude and worse 
performance on memory tasks such as the MWM (Scott, Kiss, Kawabe, & Hajos, 2016; Roof et 
al., 2010). 
As the NOR serves to supplement our characterization of the Tg mouse model in addition 
to the behavioral tests and cell quantification directly related to the functioning and neurogenesis 
of the OB, these results suggest that if we were to perform neurogenesis quantifications within 
the hippocampus of these mice, we would perhaps find significant differences in neurogenesis 
between 2 and 7 month old Tg mice. Such an experiment could be carried out within the dentate 
gyrus, the location of neurogenesis within the hippocampus, in order to determine whether this 
decline in hippocampal function is due to the lack of new neuron formation within the region or 
whether there are other neuronal processes with which tau is interfering. Ultimately, our NOR 
results support our hypothesis that there are significant differences in cognitive function between 
2 month and 7 month Tg mice, and that the effect of tau is cumulative. 
5.2 Confocal Data 
Confocal data sought to analyze the effects of tau development in Tg mice on 
neurogenesis, as indicated by the density of BrdU+ cells/mm³ in sagittal sections of the OB. The 
AOB showed a lower-than-expected value of neurogenesis with 1000-1500 BrdU+ cells/mm³ at 
2 months for both WT and Tg mouse models. In conclusion, there was a small increase in 
  
42 
neurogenesis from 2 to 7 months in the AOB, although the WT mice had the characteristically 
higher neurogenesis rate at 7 month. The MOB displayed a 2000-3000 BrdU+ cells/mm³ count 
for 2 months. This value increased to between 3000-4000 BrdU+ cells/mm³ count for WT mice, 
and decreased to about 1500 BrdU+ cells/mm³ count for Tg. Our results were not significant and 
not consistent with results from previous studies (Nunez-Parra, Pugh & Araneda, 2010).  
The results of quantification of neurogenesis was not exactly as expected, as it was 
hypothesized that the Tg mouse model would show a significant decline in neurogenesis for both 
WT and Tg groups (Nunez-Parra, Pugh & Araneda, 2010). This marked decline in neurogenesis 
occurs only for the Tg MOB from 2 to 7 months. To conclude, there was an increase in 
neurogenesis in the WT from 2 to 7 months in both the AOB and MOB. Although exact 
explanations remain unclear, this could be due to a defect in the WT mice group at 2 months 
since the WT are littermates of the Tg mice. The littermates may have expressed transgenes that 
resulted in the low amount of new neurons in the OB.  
An explanation for the increase in neurogenesis of Tg in the AOB could be due to the 
onset of tau expression. Based on the 2014 study, behavioral deficits were observed before 
neuronal loss was readily detectable in the hippocampus of adult-onset P301L tau expression 
(Hunsberger et al., 2014). Although it is unclear how adult-onset tau expression affects 
neurogenesis in the OB in the Tg model, we can relate the findings of the 2014 study to speculate 
that early-onset of tau expressed our Tg mice could have resulted in observation of behavioral 
deficits before neuronal loss was detected. 
 
  
43 
Chapter 6:  Limitations 
6.1 Behavioral Limitations 
There were several limitations to this study that may potentially explain the concluding 
results.  
6.1.1 Cube Recognition 
In additional experiments performed in Dr. Araneda’s lab to study any interesting 
behaviors of the Tg mice, mice were presented with several trials of a cube with water, similar to 
what is done in the odor discrimination test. However, in these experiments in which no diluted 
odors were actually presented (as only water was spread on the cube each trial), it was found that 
the mice displayed an increased investigation time towards the cube each time a new cube was 
placed in the cage, rather than the same cube that had been previously presented in a former trial. 
Thus, it is possible that in our odor detection threshold and habituation/dishabituation 
experiments, Tg mice had an increased investigation time due to both the presence of the new 
cube and the presence of a new odor, not strictly the presence of a new odor itself. As a result, it 
is not definitively conclusive to say that an increased investigation time in response to a cube 
with an odor, whether it be a new or diluted odor, is strictly due to the presence of that odor and 
thus that the mice were able to differentiate these odors. 
6.1.2 Defining Wildtype Mice 
Although exact explanations remain unclear, one plausible explanation for the increase in 
neurogenesis from 2 to 7 months in both the MOB and AOB of the WT mice was that our WT 
mice were littermates of the Tg mice which were not positive for the transgene. Since the Tg 
  
44 
mice were heterozygous and the WT mice were homozygous for the transgene, the genotype of 
the WT mice may have been affected. Further experiments should include the use of a more 
well-established WT mice such as the C57 mouse model that includes mice that are not 
littermates of the positive Tg mouse in order to confirm the validity of our results. 
6.1.3 Sample Size Limitations 
In order to gather data that adequately represents the characterization of the Tg mice, we 
should increase the sample size of each treatment groups, and observe whether there are any 
deviations in the effects we observed in a smaller sample. A 2011 study that quantified the 
olfactory neurogenesis in the C57/BL6 mouse models used around 3 to 6 mice per treatment 
group. Since our experiments were also analyzing olfactory neurogenesis, an appropriate sample 
size would be in this range (Nunez-Parra, Pugh & Araneda, 2011).  
6.1.4 Use of the rTg4510 Mice Model 
The Tg mice was chosen in order to observe the effects of isolated tauopathies and NFTs 
in mice with the aim of better understanding the interactions of tau in the OB on a cellular level 
as well as understand its cognitive repercussions. It is important to characterize different strains 
of AD mice since each has differences regarding baseline learning ability and activity; thus, 
other models of tauopathy in AD that exist, such as the JNPL3, Htau, and TAPP models, may be 
important to use in order to verify our results and observe the cognitive and neurogenesis effects 
of tau in mice of different learning capabilities. 
  
  
45 
Chapter 7:  Conclusion 
 Characterization of Tg mice models to be used specifically for AD research in the OB is 
still in its infancy. Experimental results from this study are inconclusive as to whether Tg mice 
models are ideal for OB research and can serve as a representative model of the olfactory 
changes that occur in AD. However, behavioral results are promising and therefore more 
histological research should be performed in order to fully understand the mice models’ 
characterization and potential. Future research in addition to this study may be beneficial and 
necessary to ascertain the current results.  
 
Chapter 8:  Future Directions 
This study aimed to characterize a relatively new AD Tg mice at 2 and 7 months for 
behavioral and neurogenesis qualities. Since this study used both genders of the mice and 
differences in sex have not been addressed particularly, more research should be conducted that 
compares the behavioral and neurogenesis differences between the sexes of the mice. Since our 
results showed that neurogenesis increased in older mice in general, which is not explained by 
any other studies done on AD Tg mice models to our knowledge, more trials of the experiments 
should be conducted with a larger sample size. In addition, in order to gain more insight into 
whether the WT littermates were affected by the transgene, we can conduct more experiments 
using a more well-established WT model such as C57/BL6 as the control. Furthermore, more 
experiments using mice at different ages can be conducted to characterize the onset of cognitive 
deficits and neuronal loss in the olfactory bulb. Lastly, Tg mice have been previously tested 
using doxycycline to suppress the Tg tau and observed to see whether there were behavioral, 
  
46 
biochemical, and histological changes in these mice (SantaCruz, 2005). Doxycycline treatments 
can be used to test for changes in neurogenesis in different age groups in the OB as well as the 
hippocampus, including treatments based on the onset of tau expression.  
 
  
  
47 
Glossary 
AMPA receptor: a transmembrane receptor for glutamate that is involved in fast synaptic 
transmission in the nervous system. 
Amyloid-β: peptides of 36-43 amino that serve as the main component of amyloid plaques found 
in Alzheimer’s patients’ brains. 
Apoptosis: the process of programmed cell death observed in multicellular organisms. 
APP (Amyloid Precursor Protein): gene involved in familial AD 
Assay: an investigative procedure used to determine the amount of a certain activity in a target 
area. 
Associative learning: any learning process in which a new response becomes associated with a 
particular stimulus. 
Autoradiograph: recorded on a photographic film, an image produced by emitted radiation 
from a specimen, such as a section of tissue, treated with a radioactively labeled isotope. 
BACE-1: B-site amyloid precursor protein (APP) cleaving enzyme; results in amyloid plaques  
Bromodeoxyuridine (BrdU): a reagent often used for cell proliferation assays incorporated into 
DNA during the S-phase of mitosis. 
Cognition: the process of gaining knowledge through thought and experience. 
Confocal microscopy: an optical imaging technique for increasing optical resolution and 
contrast of a micrograph. 
Cryopreserve: to maintain viability of cells, tissue, organs, etc. by storing them at very low 
temperature. 
Dentate gyrus (DG): a structure in the brain that is part of the hippocampus and is known to 
have high rates of neurogenesis. 
  
48 
Deterministic genes: genes that directly cause the disease; inheritance of the genes guarantees 
one will develop the disease 
Dopamine: a natural hormone and neurotransmitter responsible for cognitive alertness. 
Early onset (EO): display of symptoms of AD before age 65 
External plexiform layer: the layer of cells in the olfactory bulb between the glomerular layer 
and mitral cell layer. Contains mostly dendrites of mitral cells and GABAergic GCs. 
GABA: gamma-aminobutyric acid. The main inhibitory neurotransmitter in the mammalian 
nervous system. 
Glomerular layer: the region of the olfactory bulb where the synapses form between the 
terminals of the olfactory nerves and dendrites of mitral, periglomerular and tufted cells. 
Granular layer: thin layer of cells in the epidermis. 
Habituation: the diminishing of a physiological or emotional response to a frequently repeated 
stimulus. 
Hippocampus: section of brain known as the center of emotion, memory, and autonomic 
nervous system. 
Immunohistochemistry: the process of detecting antigens in cells of a tissue by utilizing 
antibodies that bind specifically to said antigens. 
Internal plexiform layer: layer of cells in the olfactory bulb between the mitral cell layer and 
the GCL. Consists mostly of GC dendrites, but also includes some axons and dendritic process of 
short-axon cells. 
Late onset (LO): display of AD symptoms after age 65 
MAPT (microtubule-associated protein tau): gene involved in development of tau isoforms 
Mitral cell layer: a narrow band of cells in the olfactory bulb that sends it primary dendrites to 
  
49 
the glomerular layer where it forms a tuft within the layer and synapses with primary olfactory 
nerve axons. 
Neurodegenerative disease: a condition primarily associated with death of neurons in the brain. 
Neurons: a cell in the brain that can process and transmit information both chemically and 
electrically. 
Neuroplasticity: changes in neural pathways and synapses due to factors like the environment, 
bodily injury, or behavior. 
Neurotrophins: proteins that influence the survival, development, and function of neurons. 
Non-associative learning: progressive diminution of behavioral response probability with 
repetition stimulus. 
Noradrenaline: a hormone produced naturally by the body that also serves as a neurotransmitter 
and is associated with strong concentration. 
Nucleoside: contains the nitrogenous base and ribose/deoxyribose but not the phosphate group 
that a nucleotide would have. 
Olfactory bulb: a section of the brain located on the inferior area and is primarily responsible 
for the sense of smell.  
Olfactory epithelium (OE): specialized epithelial tissue inside the nasal cavity that is involved 
in smell. 
Olfactory nerve layer: the layer of neuron cells in the olfactory epithelium that receive direct 
input and relay it into the olfactory bulb. 
Operant conditioning: a type of learning in which an individual's behavior is modified by its 
antecedents and consequences. 
Peritoneum: a thin membrane lining the inside of the abdominal cavity 
  
50 
PSEN1 (presenilin-1): gene on chromosome 14 that enables the cleaving action of gamma 
secretase; implicated in familial AD 
PSEN2 (presenilin-2): gene on chromosome 14 that enables the cleaving action of gamma 
secretase; implicated in familial AD 
Psychotropic: affecting the mental state. 
REM Behavior Disorder (RBD): a disorder characterized by loss of body paralysis during 
REM sleep. 
Risk genes: genes that increase the likelihood of developing the disease; does not guarantee one 
will develop the disease 
Rostral migratory stream: extends from the subventricular/subependymal zone of the anterior 
horn of the lateral ventricle along the ventricular extension into the olfactory bulb. 
Rostro-caudal: between head and tail. 
Sagittal: relating to suture on top of the skull that runs between the parietal bones in a front to 
back direction. 
Saturation point: the point at which there is no longer any room to hold any more of a certain 
liquid, object, or physical material 
Sensitization: repeated administrations of a stimulus results in the progressive amplification of a 
response. 
Serotonin: a neurotransmitter found in the central nervous system of humans commonly 
associated with maintaining mood balance. 
Sliding microtome: a microtome in which the object to be cut is fixed and the knife is carried 
obliquely across it 
Subventricular zone (SVZ): a layered brain structure that is known to have high rates of adult 
  
51 
neurogenesis and contains a large amount of proliferating cells. 
Synucleinopathies: a group of neurodegenerative diseases characterized by neuronal or glial 
inclusions of alpha-synuclein. 
Tauopathies: neuronal inclusions or neurofibrillary tangles in AD made of microtubule 
associated protein tau. 
Tg2576 mice (APP Tg mice): Tg mice with a mutation that causes overproduction of amyloid in 
the brain which is also associated with impaired learning, loss of working memory, and changes 
in synaptic plasticity.  
Tryptophan hydroxylase (TPH): a rate-limiting enzyme that is affects the rate of serotonin 
synthesis in the brain. 
  
  
52 
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