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The impact of reduced Src kinase activity on memory-related oscillatory activity in adulthood and memory function in age in a mouse model of schizophrenia
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The impact of reduced Src kinase activity on memory-related oscillatory activity in adulthood and memory function in age in a mouse model of schizophrenia
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Copyright 2022 Krishna Parekh
The impact of reduced Src kinase activity on memory-related oscillatory activity in adulthood
and memory function in age in a mouse model of schizophrenia
by
Krishna Parekh
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MEDICAL PHYSIOLOGY)
December 2022
ii
Acknowledgments
I would like to acknowledge and thank my mentor, Dr. Steven Siegel for making this possible. I
would also like to give special thanks to Dr. Robert Featherstone and Dr. Lindsey Crown for
providing mentorship. Their guidance and support carried me throughout every stage of this
project and my own personal growth. I would also like to thank my committee members, Dr.
Marcelo Coba and Dr. Li Zhang for taking the time to sit on my panel and reading my thesis. I
would like to acknowledge Dr. Darrin Lee and his lab for allowing me to work with their
equipment and receive guidance.
I would like to give thanks to Dr. Harvey Kaslow who has provided me with learning
opportunities and support throughout my time in the Medical Physiology program.
Finally, I would like to give thanks to my family and friends. With the overwhelming support,
love, and understanding of my parents, sisters, brother-in-law, nephew, and friends I was able to
get through the rough times.
iii
Table of Contents
Acknowledgments ........................................................................................................................... ii
List of Figures ................................................................................................................................ iv
Abstract ........................................................................................................................................... v
Chapter 1: Introduction ................................................................................................................... 1
Characteristics of Schizophrenia ................................................................................................. 1
Memory and Learning ................................................................................................................. 1
Electrophysiology ....................................................................................................................... 3
NMDARs and Src Kinase in Schizophrenia ............................................................................... 4
Aging in Schizophrenia ............................................................................................................... 6
Current Study .............................................................................................................................. 6
Chapter 2: Materials and Methods .................................................................................................. 8
Animals ....................................................................................................................................... 8
Experiments ................................................................................................................................ 8
Experiment 1 ........................................................................................................................... 8
Experiment 2: .......................................................................................................................... 9
Surgery ........................................................................................................................................ 9
Behavior .................................................................................................................................... 10
Novel Object Recognition Task ............................................................................................ 10
T-Maze .................................................................................................................................. 12
Elevated Plus Maze ............................................................................................................... 13
Statistical Analysis .................................................................................................................... 14
Chapter 3: Results ......................................................................................................................... 15
Novel Object Recognition ......................................................................................................... 15
T-Maze ...................................................................................................................................... 23
Elevated Plus Maze ................................................................................................................... 23
Chapter 4: Discussion ................................................................................................................... 25
Chapter 5: References ................................................................................................................... 28
iv
List of Figures
Figure 1: Trisynaptic pathway in the mouse hippocampus. ........................................................... 3
Figure 2: NMDAR Activation and Src Kinase upregulation. ......................................................... 6
Figure 3: Experimental Paradigm for Experiment 1 and 2. ............................................................ 8
Figure 4: Electrode placements in the mouse brain. ..................................................................... 10
Figure 5: NOR behavioral schema over the course of 2 days. ...................................................... 12
Figure 6: Discrete spontaneous alternation T-maze task conducted over the course of 3 days. ... 13
Figure 7: Elevated plus maze consists of the 2 open arms and 2 closed arms. ............................. 14
Figure 8: Novel Object Recognition Discrimination Index. ......................................................... 15
Figure 9: Electrophysiological measurements taken during the NOR task training phase. .......... 16
Figure 10: Coherogram of the CA3-dCA1 circuit during the training phase. .............................. 20
Figure 11: Coherogram of the vCA1-mPFC circuit during the training phase. ............................ 20
Figure 12: erogram of the vCA1-mPFC circuit during the testing phase. .................................... 21
Figure 13: erogram of the CA3-dCA1 circuit during the training phase. ..................................... 21
Figure 14: Comparison of coherence to discrimination index. .................................................... 22
Figure 15: Behavioral task results for the NOR, T-maze, and EPM tasks. .................................. 24
v
Abstract
Memory and learning are interrelated components of cognition that are impaired in aging and a
variety of neuropsychiatric illnesses such as schizophrenia. One of the leading hypotheses that
has been implicated in cognitive impairments for schizophrenia is the N-Methyl-D-Aspartate
Receptor (NMDAR) hypofunction hypothesis. Much evidence has been found that there are
various molecular pathways that are associated with the pathophysiology of schizophrenia, and
which alter the activity of the NMDAR receptor. A common convergent point where the
molecular pathways meet is Src Kinase. In a previous study done by our lab, Src deficient mice
showed deficits in behavior related to cognition and electrophysiological measures. There have
been numerous studies that have examined the relationship between schizophrenia and aging. As
such, a prominent hypothesis known as the accelerated aging hypothesis was developed and
states that physiological changes associated with age happens at an earlier age in patients with
schizophrenia. Despite the growing evidence related to aging in schizophrenia, not much is
known about the relationship between Src deficiency induced NMDAR hypofunction and aging.
Therefore, the current study examined the impact of reduced Src kinase activity on memory -
related oscillatory activity in adulthood and memory function in age in Src (+/-) heterozygous
mice in different behavioral paradigms. Src (+/-) mice demonstrated increased low gamma
coherence in CA3-CA1 circuitry during encoding and recall in learning and episodic memory
compared to their WT littermates. This evidence is consistent with what was found in studies
related to NMDAR models of schizophrenia involving NMDAR knock-out and NMDAR
antagonist models. This suggests that the abnormal level of coherence between structures could
be indicative of hyper-connectivity or cellular hyper-excitability and there may be impaired
communication between the CA3 and CA1 which may contribute to the underlying memory
vi
deficits associated with NMDA hypofunction. Furthermore, Src (+/-) mice and their WT
littermates showed no differences observed in anxiety levels and spatial and episodic memory in
both aged and adult mice. This suggests that Src Kinase may not have an impact on memory and
aging in schizophrenia.
1
Chapter 1: Introduction
Characteristics of Schizophrenia
Schizophrenia is a complex, debilitating, neuropsychiatric illness with poor prognosis and low
rates of remission. It is characterized by three domains of symptoms: positive, negative, and
cognitive (Crow 1980, Elvevag & Goldberg, 2000). Positive symptoms are defined as features
that are abnormally present and include hallucinations, delusions, and disorganized thoughts and
behaviors (Crow, 1980). Negative symptoms are defined by features that are abnormally absent
and include avolition and anhedonia (Crow, 1980; Makinen 2008; Jablensky, 2010). Prominent
cognitive deficits include impairments in areas such as memory (both long-term episodic
memory and short-term working memory), learning, and attention (Carbon and Correll, 2014).
Cognitive impairments are a particularly important target for treatment as they appear prior to the
onset of positive and negative symptoms (Simon et al., 2007). They are especially important as
cognitive deficits are related to poor functional outcome (i.e., inability to maintain social
relationships, jobs, and care for themselves) (Keefe and Harvey, 2012; Lepage et al., 2014; Fu et
al, 2017). Furthermore, treatment options are severely limited as the standardized treatment
options are relatively ineffective in treating cognitive dysfunction (Kane and Correll, 2010;
Carbon and Correll, 2014; McCutcheon et al., 2020).
Memory and Learning
Memory and learning are interrelated components of cognition. There are multiple types of
memory processes. For example, learning a skill is a form of motor memory often termed
“implicit” because it cannot often be described. Explicit memory, on the other hand, is which a
2
person could theoretically name specifically, they can be for events and for facts (Squire, 2009).
The types of memory that is the most impacted by schizophrenia are explicit memory and
working memory. Working memory can be explicit in nature but is generally considered to be
short-term and involves the manipulation of information within the brain of an animal. Episodic
memory is a subset of explicit memory and includes memories for events and places. It is the
most impacted by age-related cognitive decline and psychiatric disorders such as schizophrenia
(Glisky, 2007). While present thesis will focus on episodic memory, it should be noted that
working memory deficits are also a major characteristic of the disease.
To form and use episodic memories, one must store sensory information about the environment
(encoding), integrate this information with previous experience and knowledge about the world
(consolidation), and retrieve this information at the appropriate time (recall) (Roediger and
Karpicke, 2005; Brem et al., 2013). What is less known are which parts of the learning and
memory process are the most disrupted in schizophrenia.
There are various direct and indirect pathways and structures related to the process of memory
formation and retrieval. Regarding episodic memory, the most implicated structure is the
hippocampus. There are 3 different monosynaptic pathways that are associated with how
information is processed in the hippocampus that together form the trisynaptic loop (Figure 1).
The first is the perforant pathway which involves projections from the entorhinal cortex (EC) to
the dentate gyrus (DG) and is thought to facilitate the initial encoding of information. The second
pathway is the mossy fiber pathway that projects from the DG to the CA3 of the hippocampus
(Rolls and Treves, 1994). From the CA3, through the third pathway, Schaffer collateral path,
there are indirect and direct projections onto the CA1 (Rolls and Treves, 1994). From the CA1
information is output to different areas of the brain for consolidation and later retrieval.
3
Figure 1: Trisynaptic pathway in the mouse hippocampus.
Created Using BioRender.
Another monosynaptic pathway that is often studied in the context of episodic memory, often in
rodents, is that from the ventral hippocampus to the medial prefrontal cortex (mPFC). This
pathway has been hypothesized to play a crucial role in object memory, as test for which in
rodents in the novel object recognition task.
Electrophysiology
Neural oscillations reflect rhythmic electrical activity as a response to stimuli in regions of the
CNS and can be recorded via the electroencephalogram (EEG), electrocorticography (ECoG), or
local field potential activity (LFP). Oscillatory activity can be analyzed in either the time-
domain, as a series of voltage values across time (as is typical in event-related potential
analysis), or in the frequency-domain, in which a signal is composed of multiple sinusoids of
4
different frequencies. Commonly studied frequencies in cognitive neuroscience include delta (<4
Hz), theta (5-10), alpha (8-12 Hz), beta (12-30 Hz), and gamma (30-100 Hz) (Moran et al., 2011,
Sun et al, 2011). Each band is associated with specific neural generators and, as such, are
thought to be associated with specific functions and activities within the brain. Regarding
learning and memory, theta and gamma oscillatory activity has been the most studied (Abhang et
al., 2016).
Abnormal neural oscillations are associated with schizophrenia (Ulhaas and Singer, 2010, Moran
and Hong, 2011). Specifically, theta power is reduced in patients and gamma power may be
increased or reduced depending on the frequency or the cognitive task studied (Amann et al.,
2010).
Despite the extensive research, the true mechanism for the pathophysiology of schizophrenia and
abnormal oscillatory activity are unclear however there are genetic (susceptibility genes),
molecular, and environmental factors that likely play a role in the development of the disease.
NMDARs and Src Kinase in Schizophrenia
N-methyl-D-aspartate receptors (NMDARs) are excitatory glutamate receptors crucial to
synaptic plasticity, memory formation, and learning (Li & Tsien, 2009). Each NMDAR
comprises an obligatory NR1subunit paired with the NR2, and/or NR3 subunits (Paoletti et al.,
2007; Lee and Zhou, 2019). The NR2 and NR3 subunits have different subtypes. For this study,
NR2 subunits will be the focus for this part of the discussion. NR2a and/or NR2b subunits are
found in the forebrain, especially in the hippocampus and prefrontal cortex (two areas that are
commonly implicated in schizophrenia) (Lee and Zhou, 2019; Paoletti et al., 2007).
5
When the cell is in a resting phase, the NMDAR has a Mg2+ molecule blocking the pore (Figure
2). The Mg2+ molecule is released when glycine and glutamate co-agonists bind to the NR2
subunit on the receptor (Chen and Roche, 2007; Rajani et al., 2020). Once the Mg2+ molecule is
released the channel pore opens, allowing Ca2+ to flow through the pore and depolarization of
the cell (Chen and Roche, 2007; Rajani et al., 2020).
When the postsynaptic cell is depolarized, the current of the depolarization is regulated by
sarcoma tyrosine kinase (Src). Src upregulates NMDAR function by phosphorylating the NR2a
and NR2b subunits in response to receptor activation, thereby allowing for enhanced NMDAR
synaptic currents (Figure 2) (Ali and Salter, 2001). Research by Hahn and colleagues identified
Src as a common convergent pathway of multiple schizophrenia susceptibility genes (Hahn,
2011, Pitcher et al., 2011, Banerjee et al., 2014). The susceptibility genes (i.e., neuregulin-1,
RTPa, ErbB4, and PSD-95) have been found to be involved in Src hypoactivity which eventually
leads to decreased NMDAR phosphorylation and activity (Kalia et al., 2006, Pitcher et al., 2011,
Banerjee et al., 2014). A previous study also found evidence of reduced Src activity in post-
mortem brain tissue (in the dorsolateral prefrontal cortex (DLPFC)) of patients with
schizophrenia (Banerjee et al., 2014). Src downregulation ultimately leads to NMDA receptor
hypofunction. A growing body of literature supports the notion that NMDAR hypofunction may
be a critical mediator of the pathophysiology and etiology of the symptoms of schizophrenia.
The hypothesized relationship between NMDAR hypofunction and schizophrenia is based on
postmortem studies, pharmacological models and transgenic mouse models which have shown
behavioral deficits related to the development of the symptoms of schizophrenia (Mohn et al.,
1999, Banerjee et al., 2014; Milenkovic et al., 2014, Lee and Zhou, 2019). As a result, it has
made Src kinases an important therapeutic target.
6
Figure 2: NMDAR Activation and Src Kinase upregulation.
Created Using BioRender.
Aging in Schizophrenia
In the normal aging process, cognitive functions such as memory, learning, and attention decline
(Li et al., 2001). However, there have been numerous studies that have examined the impact of
schizophrenia on the aging process. One hypothesis that has been proposed is the accelerated
aging hypothesis which postulates that the physiological changes associated with the normal
aging process occurs at an earlier age in patients with schizophrenia (Okusaga, 2013). There are
different rationales for accelerated aging which include abnormal brain development in late
adolescence/early adulthood, comorbidities, and other physiological changes associated with the
progress of the disease.
Current Study
Previous work done by our group has demonstrated deficits in cognition, behavior, and
oscillatory activity related to schizophrenia in Src heterozygous mice (Src (+/-)) (Ward et al.,
2019). Specifically, Src (+/-) mice showed decreased working memory (T-maze), sociability
(social interaction task), P20 event related potential, mismatch negativity, and evoked gamma
7
power (Ward et al., 2019). The evidence presented in the study has provided evidence that Src is
sufficient in producing schizophrenia like changes in EEG and cognition.
To assess the relationship between NMDAR hypofunction and aging in Src (+/-) mice, the
current study assessed factors such as recognition memory (novel object recognition), anxiety
(elevated plus maze), and spatial working memory (spontaneous alternation T-maze).
Furthermore, the coherence between dCA1 and CA3 as well as vCA1 and mPFC were assessed
through electrophysiological modes of measurement to compare the involvement of these two
memory pathways.
This led to the formation of the following hypotheses:
1. Src (+/-) mice will exhibit impaired object recognition memory as well as
abnormalities in theta and gamma oscillatory activity during the novel object
recognition task. Furthermore, the mice will exhibit normal anxiety levels during
the elevated plus maze task and decreased spatial working memory during the
spontaneous alternation T-maze task.
2. Src (+/-) will show NMDA receptor accelerated age related decline in cognitive
tasks related to episodic memory and spatial working memory, as well as tasks
related to anxiety.
8
Chapter 2: Materials and Methods
Animals
Src (+/-) heterozygous mice and wild-type (WT) littermates were obtained from an established
colony under similar conditions as used previously (Ward et al., 2019).
The mice were created with a C57BL/6 background and bred to have pups of Src (+/-) and WT
genotypes (Ward et al., 2019). Homozygous mice are not used due to developmental
abnormalities such as reduced body size, bone abnormalities, and other health issues which
results in decreased lifespan (Soriano et al., 1991, Takeshita et al., 2018). The animals were
housed in a 12-hour light/ dark cycle (6:00 am – 6:00 pm) and were fed and provided with water
ad libitum.
All animals went through the same behavioral testing paradigm and schedule (Figure 3). All
testing and procedures were performed in accordance with the University of Southern
California’s Institute of Animal Care and Use Committee.
Figure 3: Experimental Paradigm for Experiment 1 and 2.
Surgery and behavioral testing schedule used for mice in Experiment 1. Experiment 2 mice followed the same schedule starting
at week 1 for handling. Created Using BioRender.
Experiments
Experiment 1
17 Src (+/-) mice (n = 8; 4M, 4F) and WT littermates (n= 9, 4M, 5F) were designated to undergo
surgery and collect electrophysiological data. The animals were aged 5-7 months at the time of
9
surgery and testing. 1 week prior to surgery, animals were handled in the behavioral testing room
to optimize behavioral outcomes and reduce anxiety levels.
Experiment 2:
19 Src (+/-) mice (n = 10; 3M, 7F) and WT littermates (n= 9, 7M, 2F) between the ages of 20-22
months were designated to be a part of the aged cohort.
14 Src (+/-) mice (n = 5; 5F) and WT littermates (n= 9, 5M, 4F) between the ages of 5-6 months
of age were designated as the adult group.
Surgery
Prior to the start of surgery, electrodes (Plastics 1, Roanoke, Virginia) were soldered to wire (A-
M Systems, Sequim, Washington) and attached to 16-channel Electrode Interface Boards (EIB-
16-QC-PEM; Neuralynx, Boseman, Montana). Electrodes were also sterilized prior to
implantation. Animals were anesthetized with isoflurane and placed in a stereotaxic instrument.
Electrodes were implanted in the medial prefrontal cortex (mPFC; AP: 2.15, ML: 0.35, DV: -
1.6), ventral CA1 (vCA1; AP: -3.16, ML: -3.0, DV: -4.0), dorsal CA1 (dCA1; AP: -2.54, ML:
1.5, DV: -1.25), and CA3 (AP: -1.7, ML: 2.0, DV: -1.6) (Figure 4). All electrodes were
implanted relative to bregma. A ground screw was placed dorsally to bregma with an additional
3-6 skull screws placed into the skull to ensure the acrylic headcap would be secure for the
duration of the behavior. Acrylic was used to create a headcap to secure the board and protect the
skull and electrodes. The animals were given 1-week post-operative rest and care before the start
of behavior. They were also individually housed to prevent fighting and potential injury.
10
Figure 4: Electrode placements in the mouse brain.
All electrodes implanted in the mPFC, dCA1, CA3, and vCA1 were placed relative to bregma. Created Using BioRender.
Behavior
After the 1-week post-operative care, Experiment 1 animals were handled once again and
reacclimated to the behavior room to reduce anxiety. Experiment 2 animals were handled 3-5
days prior to the start of testing to reduce animal anxiety. Behavior was completed over the
course of 2 weeks during the light cycle. Before every task, animals were acclimated to the
behavior room for 30 minutes. All behavior was recorded with the Topscan behavior analysis
system (CleverSys, Reston, Virginia).
Novel Object Recognition Task
Novel Object Recognition (NOR) was used to measure episodic memory. All behavior related to
the task was completed over the course of 2 days in a low-lit room with a white noise machine to
11
reduce outside noises and distractions. The behavior was carried out in a 16 in x 16 in x12 in
white box which was surrounded by a faraday cage.
The first day of the task was a 5-minute habituation where the animal was allowed to explore the
box freely (Figure 5). The second day of the task included the habituation, training, and testing.
During the training phase (encoding phase), 2 identical objects were placed in the box and the
animals were allowed to explore freely for 5 minutes. During the testing phase (encoding phase),
one of the objects was replaced with a novel object and the animal was allowed to explore the
box freely for 5 minutes. Between every part of the task, the box was thoroughly wiped with
70% ethanol to remove additional cues prior to the start of each phase.
Experiment 1 animals were attached to a tether connected to a Digital Lynx SX (Neuralynx,
Boseman, Montana) recording system for the duration of each part of each 5-minute phase.
Electrophysiological data was collected using Cheetah (Neuralynx, Boseman, Montana)
recording software. Behavior was analyzed with Topscan and recordings were read and analyzed
with MATLAB.
The discrimination index created to analyze behavior was calculated by taking the ratio of time
spent exploring the novel object to the time spent exploring both objects.
Animals that spent less than 10 seconds on both the novel and familiar objects combined were
excluded from the data.
12
Figure 5: NOR behavioral schema over the course of 2 days.
Created Using BioRender.
T-Maze
The discrete spontaneous alternation T-maze task is used to assess spatial working memory. The
protocol was obtained and modified (d’Isa et al., 2021). The task was carried out using the same
behavioral room conditions as the NOR task. The task was performed over the course of 3 days
and the Experiment 1 mice were given the opportunity to alternate 9 while Experiment 2 mice
were given the opportunity to alternate 13 times.
Each alternation was defined by the animal correctly alternating between each goal arm. The
animal was placed in the starting arm of the maze and given the opportunity to choose between
goal arms (Figure 6). Once the decision was made, they were locked in their decision arm by
sliding a guillotine door down for 30 seconds. After the 30 second time frame, the animal was
placed back into the starting arm and was allowed to choose a goal arm once again and was
locked in the chosen goal arm for 30 seconds.
A correct trial is when the animal can alternate between the goal arms for each trial. An incorrect
alternation is when the animal re-entered a goal arm during the trial. Behavior was analyzed by
calculating the ratio of the total correct alternations over the course of the 3 days to the total
number of alternations. Mice that completed less than 3 alternations in a day, had that day
excluded from the data set.
13
Figure 6: Discrete spontaneous alternation T-maze task conducted over the course of 3 days.
A correct alternation is when the animal can enter the opposite goal arm than what was chosen previously. Incorrect alternations
are when the mouse chooses to enter the same arm as previously. Created Using BioRender.
Elevated Plus Maze
The elevated plus maze is a standard rodent model of anxiety. It is completed over a period of 5
minutes. The task was carried out in the same environmental condition as previously used (Walf
and Frye, 2007). The task was conducted in a brightly lit room with a white noise machine. The
behavior was performed in an elevated plus maze apparatus. The apparatus has 2 closed arms
covered by walls and 2 open arms (Figure 6). The maze was elevated above the ground at 18
inches. The animal was placed in the center of the maze and allowed to explore all parts of the
maze for 5 minutes. The percentage of time spent in the open arms was calculated by looking at
the ratio of time spent in the open arms to the total time in all the arms.
Exclusion criteria removed animals which fell off the maze (Walf and Frye, 2007).
14
Figure 7: Elevated plus maze consists of the 2 open arms and 2 closed arms.
Created Using BioRender.
Statistical Analysis
Statistical analysis was carried out using a 2-way ANOVA and T-test.
15
Chapter 3: Results
Novel Object Recognition
Experiment 1:
It was revealed that there was a significant difference in novel object exploration between
the Experiment 1 WT and Src (+/-) animals during the testing phase (Src (+/-): n=3, WT: n=8;
t=3.398, p=0.0079) (Figure 8). Subsequent t-tests were performed to assess if the mice had better
than chance performance. Neither the WT nor Src (+/-) mice showed better than chance
performance (Src (+/-): t=3.130, p=.0887; WT: t=1.909, p=0.0979).
Figure 8: Novel Object Recognition Discrimination Index.
Src (+/-) animals spent significantly less time on the novel object compared to the WT.
In terms of the electrophysiological measurements taken during the NOR training part of
the task, there were no differences between the powers of low gamma, high gamma, and theta
for dCA1, mPFC, and CA3 (Figures (9 a-c). There was a significant increase found in the power
of vCA1 theta for Src animals (Figure 9d). In examining the CA3 to CA1 coherence in the WT
and Src (+/-) mice, it was found that Src (+/-) animals had increased low gamma coherence
16
(Figure 10). However, vCA1-mPFC coherence for both the WT and Src (+/-) animals, had no
significant difference (Figure 11).
Figure 9: Electrophysiological measurements taken during the NOR task training phase.
a-c.) There were no significant differences in the theta, low gamma, and high gamma powers in the Src (+/-) and WT mPFC,
CA#, and dCA1. d) There was a significant difference in the theta power for Src animals in the vCA1.
a.
17
b.
18
c.
19
d.
20
Figure 10: Coherogram of the CA3-dCA1 circuit during the training phase.
Src (+/-) animals showed a significant increase in low gamma coherence compared to the WT mice in the CA3-dCA1 circuit.
Figure 11: Coherogram of the vCA1-mPFC circuit during the training phase.
There were no differences in coherence detected in the vCA1-mPFC for either group.
During the testing phase of the task, there was no significant difference between low gamma
coherence in the vCA1-mPFC pathway for both Src (+/-) and WT mice (Figure 12). The CA3-
CA1 pathway showed a significant increase in low gamma coherence for the Src (+/-) compared
to the WT mice (p=0.0450, d=-1.400) (Figure 13). Furthermore, there was no significance
between NOR performance and theta and gamma coherence compared to the discrimination
index for both groups in the vCA1-mPFC pathway (Figure 14a-e). There was a significant
21
relationship between dCA1-CA3 theta coherence when compared to the discrimination index for
WT animals but not Src (+/-) (Src R= 0.51, p=0.30; WT= r=0.88 p=0.046) (Figure 13f).
Figure 12: Coherogram of the vCA1-mPFC circuit during the testing phase.
Src (+/-) and WT animals showed no differences in vCA1-mPFC low gamma coherence.
Figure 13: Coherogram of the CA3-dCA1 circuit during the training phase.
There was a significant increase in low gamma coherence for the Src (+/-) mice.
22
Figure 14: Comparison of coherence to discrimination index.
a-e) No differences when comparing vCA1-mPFC coherence at all frequencies compared to the discrimination index as well as
in the dCA1-mPFC gamma frequencies for both groups. f) There was a significant dCA1-mPFC relationship with the coherence
and discrimination index in WT mice.
23
Experiment 2:
In the aged cohort, there was no significant difference in the novel object exploration between
Src (+/-) and WT mice as well as the aged mice compared to the adult mice (Figure 15a).
Furthermore, the interaction between age and Src (+/-) was not significant. The performance of
the task was also assessed if the mice performed better than chance. The aged Src (+/-) mice and
WT mice performed significantly better than chance (aged Src (+/-): n=10; t=2.949, p= 0.0163;
aged WT: n=9; t=3.949, p=0.0082) as well as the adult Src (+/-) mice (n=5; t= 3.762, p= 0.0197).
The adult WT mice did not perform better than chance (n=9; t=1.690, p=0.1295).
T-Maze
The number of correct alternations was calculated by taking the number of correct alternations
divided by the total number of alternations completed over the 3 days of the task.
Experiment 1:
It was found that there was no significant difference in the correct alternations made between the
WT and Src (+/-) mice (Src (+/-): n=6, WT: n= 4; t=.6107, p=0.5583) (Figure 15b).
Experiment 2:
It was found that the mice had no significant difference in correct alternations among all the
groups (aged Src (+/-): n=10, aged WT: n= 9, adult Src (+/-): n=5, adult WT: n= 9; F (1, 29) =
0.5859, p=0.4502) (Figure 15c).
Elevated Plus Maze
Anxiety measures were calculated by using the ratio of the amount of time spent in the open
arms compared to the time spent in all the arms. There was no significant difference in the ratio
of amount of time spent on the open arm between the WT and Src (+/-) mice (t=0.3160, p=
24
0.7563) (Figure 15d). Furthermore, there were no significant difference between the aged and
adult groups (aged Src (+/-): n=8, aged WT: n= 8, adult Src (+/-): n=4, adult WT: n= 7; F (1, 23)
= 0.5023, p=0.4956) (Figure 15e).
Figure 15: Behavioral task results for the NOR, T-maze, and EPM tasks.
Behavior from NOR, T-Maze and EPM: a) No significant differences detected in the discrimination index between all
Experiment 2 groups. b-c) No significant differences noted between groups in Experiment 1 and Experiment 2 for T-maze. d-e)
No difference noted between groups both Experiment 1 and 2 for EPM.
25
Chapter 4: Discussion
The current study investigated the effects of reduced Src kinase activity on memory-
associated oscillatory activity in adult mice and compared memory and anxiety measures in adult
and aged Src and WT mice. The results of the study showed that there was a difference in
encoding of information during the training phase of object recognition between the Src
heterozygous and WT mice. The Src heterozygous mice demonstrated increased low gamma
coherence CA3-CA1 circuitry coherence during encoding and recall in learning and episodic
memory. However, there were no differences in the CA3 or CA1 low gamma, high gamma, or
theta power in both groups. This suggests that there may be impaired communication between
the CA3 and CA1 which may contribute to the underlying memory deficits associated with
NMDA hypofunction.
During the recognition and spatial working memory tasks tested in aged and adult mice,
there were no differences observed in anxiety nor abnormalities observed in spatial memory and
episodic memory between the Src (+/-) and WT mice. This may suggest that the Src (+/-)
heterozygous mice may not experience accelerated aging due to NMDAR hypofunction and may
have the normal aging process.
Experiment 1:
Src (+/-) mice displayed impairments in recall and recognition memory. This is consistent
with what was found in studies related to NMDAR models of schizophrenia involving NMDAR
knock-out and NMDAR antagonist models (i.e., phencyclidine and ketamine) (Hashimoto, 2005;
Nilsson et al., 2007; Duffy et al., 2010). In their electrophysiological recordings, Src (+/-) mice
exhibited increased low gamma coherence between CA3 to CA1 while their WT littermates did
26
not. This suggests that the abnormal level of coherence between structures could be indicative of
hyper-connectivity or cellular hyper-excitability as seen in a rodent model of autism spectrum
disorder (ASD) (Mouchati et al., 2019). A previous study found increased NR2B containing
NMDARs in the CA3 as well as increased levels of PSD-95 protein, which was associated with
increased excitatory signaling in the CA3 (Li et al., 2015). This evidence in combination with the
observed hyperexcitability may explain the increased coherence and associated memory deficits
observed in the Src (+/-) mice.
Src (+/-) mice behaved similarly to their WT littermates during the discrete spontaneous
alternation task. It was found that our behavioral results relating to this task were like the results
of a study done on the ablation of parvalbumin interneurons during the discrete spontaneous
alternation task (Billingslea et al., 2014). However, during the continuous alternation task done
previously, the WT mice performed better than their Src (+/-) littermates (Ward et al., 2019). As
for the anxiety levels that were observed in the elevated plus maze task for the current study,
there were no noted differences among each cohort, which was consistent with what was
previously found (Ward et al., 2019). Based on the results and the previous study related to
NMDAR hypofunction, it can be concluded that reduced Src activity may be associated with
select cognitive deficits and may contribute to other deficits related other symptom domains
which would need further study.
Experiment 2:
It was shown that Src (+/-) and WT mice in both the aged and adult groups did not show
differences in anxiety measures, recall during the novel object recognition nor differences in
spatial working memory. This implies that Src (+/-) mice go through the same aging process as
27
their WT counterparts and there may be a different connection between NMDAR hypofunction
and aging. Furthermore, there may be more contributing components upstream, downstream, and
in the NMDAR complex that contribute to accelerated aging. In previous studies, it was found
that factors such as oxidative stress, translational modifications, different levels of subunit
expression in certain brain regions and receptor localization may have a role in NMDAR
hypofunction and aging (Liu, 2008, Bodhinathan, 2010; Kumar, 2015).
Limitations and Future Directions
One of the major limitations of this study was that all the cohort sizes for each
experiment were small which did not allow for major conclusions to be drawn between males
and females among the Src (+/-) and WT mice. To combat this small sample size, both male and
female groups belonging to their respective genotype were combined for the current study.
Therefore, a larger population size for each genotype and sex is needed to increase the power of
the study and detect any other potential differences between groups.
From this study, more behavioral testing that is related to the different characteristics of
schizophrenia as well as the collection of electrophysiological data in aged mice could further
define the relationship between NMDAR hypofunction due to reduced Src activity and age in
mice. Furthermore, the use of genome wide studies in this mouse model could be useful in
understanding the upstream and downstream components that play a role in the development of
the different endophenotypes of schizophrenia.
28
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Abstract (if available)
Abstract
Memory and learning are interrelated components of cognition that are impaired in aging and a variety of neuropsychiatric illnesses such as schizophrenia. One of the leading hypotheses that has been implicated in cognitive impairments for schizophrenia is the N-Methyl-D-Aspartate Receptor (NMDAR) hypofunction hypothesis. Much evidence has been found that there are various molecular pathways that are associated with the pathophysiology of schizophrenia, and which alter the activity of the NMDAR receptor. A common convergent point where the molecular pathways meet is Src Kinase. In a previous study done by our lab, Src deficient mice showed deficits in behavior related to cognition and electrophysiological measures. There have been numerous studies that have examined the relationship between schizophrenia and aging. As such, a prominent hypothesis known as the accelerated aging hypothesis was developed and states that physiological changes associated with age happens at an earlier age in patients with schizophrenia. Despite the growing evidence related to aging in schizophrenia, not much is known about the relationship between Src deficiency induced NMDAR hypofunction and aging. Therefore, the current study examined the impact of reduced Src kinase activity on memory -related oscillatory activity in adulthood and memory function in age in Src (+/-) heterozygous mice in different behavioral paradigms. Src (+/-) mice demonstrated increased low gamma coherence in CA3-CA1 circuitry during encoding and recall in learning and episodic memory compared to their WT littermates. This evidence is consistent with what was found in studies related to NMDAR models of schizophrenia involving NMDAR knock-out and NMDAR antagonist models. This suggests that the abnormal level of coherence between structures could be indicative of hyper-connectivity or cellular hyper-excitability and there may be impaired communication between the CA3 and CA1 which may contribute to the underlying memory deficits associated with NMDA hypofunction. Furthermore, Src (+/-) mice and their WT littermates showed no differences observed in anxiety levels and spatial and episodic memory in both aged and adult mice. This suggests that Src Kinase may not have an impact on memory and aging in schizophrenia.
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The impact of reduced Src kinase activity on memory-related oscillatory activity in adulthood and memory function in age in a mouse model of schizophrenia
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