Etiopathogenesis of obsessive compulsive disorder [autosaved]
Joelle_Petrei_Finished_Grant_2016
1. Mice with Deficient NMDA Receptors
The effects of glutamate NMDA receptor deficiencies in producing and
treating schizophrenia-like symptoms in mice.
Joelle M. Petrei
25 March 2016
General Physiology
Elizabethtown College
BIO324LA: Monday Afternoon Lab
2. Mice with Deficient NMDA Receptors 1
Abstract
A complex disorder, schizophrenia is a severe psychiatric disorder which causes difficulties in
thinking, socializing, and separating delusions from reality. Usually manifesting during puberty, extensive
studies have discovered only a few pieces of the puzzle underlying the disorder from a genetic standpoint
and have proposed two main theories based on imbalances of neurotransmitters: the very heavily tested
dopamine theory and the less understood glutamate theory. Utilizing a genetically modified brood of
NR1neo gene mice, experiments on both schizophrenic symptoms in mice and the various glutamate
medications which can be used to improve symptoms of this severe debilitating condition while potentially
improving the understanding of other psychiatric disorders as well. Highly specific medications which
affect only the synaptic spines of neurons will also be examined, as it is predicted there will be additional
improvements in the reduction of schizophrenic symptoms. These aspects will be measured using
cognitive testing while alive, genetic testing at birth and post-mortem, and full-scale post-mortem analysis
of the deceased brain all compared through ANOVA alongside the usual observational recordings.
Introduction
Schizophrenia
Schizophrenia was a term first introduced in 1908 by Eugen Bleuler, a Swiss psychiatrist who
created the term from schiz (split) and phren (mind) as a way to describe the condition which he believed
to be similar to dementia praecox (McNally, 2011). This definition then led to the description of the
disease as one which causes a splitting of functionality within the mind and personality in psychotic
outbursts. By 1940, schizophrenia was listed as a broad variety of symptoms from societal withdraw,
delusional thought, paranoia, or even catatonia (McNally, 2011). Although included in DSM-I and DSM-II,
the term schizophrenia was more generalized catch all term to describe psychosis symptoms. It wasn’t
until 1980 in DSM-III, schizophrenia was finally classified as its own series of disorders with a variety of
symptoms (McNally, 2011).
The newest version of the DSM series, DSM-5, has schizophrenia listed as a spectrum of
psychotic disorders which range in symptom severity (Pagsberg, 2013). Analysis is now performed based
on a 0-4 point scale for severity where zero is the absence of symptoms while four is the presence of
3. Mice with Deficient NMDA Receptors 2
highly disruptive or extreme symptoms; an example of this scale can be seen in Table 1 (Pagsberg,
2013). Due to the newest changes, the definitions and symptoms which define the disorder will now
exclude approximately 2% of people previously diagnosed with schizophrenia as of DSM-IV but will
include ten different classifications of schizophrenia including schizotypal personality disorder, brief
psychotic disorder, schizophreniform disorder, schizophrenia, and psychotic or catatonic disorders
(Pagsberg, 2013). The differences between the two editions can be seen in Appendix A under Table 3.
Though altered from the previous version in some ways, DSM-5 states early onset schizophrenia (EOS)
as the most severe and rarest form as it occurs in under 4% of schizophrenia cases; the reason for this
severity is earlier onset of the disorder leads to increased negative symptoms, thought disorders,
developmental delays, higher prevalence of familial diagnoses, and poorer life outcomes than those who
have later onset schizophrenia (Pagsberg, 2013).
With this psychiatric disorder, there are three types of symptoms present: positive symptoms,
negative symptoms, and cognitive symptoms. Positive symptoms are the most commonly thought of
characteristics of schizophrenics, including delusions, hallucinations, paranoia, psychosis, or rarely
catatonia (Mohn et al., 1999). Negative symptoms however, are often persistent and disruptive to social
lives as they include flattened affect or social withdraw while cognitive symptoms disrupt the ability to pay
attention and focus on the world around them (Mohn et al., 1999). Although portrayed otherwise in media,
schizophrenics are very seldom dangerous to society and actually more likely to self-harm due to their
symptoms than to lash out at other individuals.
It has also been recorded through meta-analyses how schizophrenic brains display larger
ventricles and decreased brain size, especially in the medial temporal lobe (Harrison, 1999). It is
important to note these structural abnormalities are present prior to the onset of symptoms or treatment
and therefore, they are not the result of the disorder nor its treatment. High-risk individuals are often
found to have smaller left and right hippocampus-amygdala regions than normal or low-risk individuals;
the individuals with immediate family members suffering from the disorder, such as parents or siblings,
often display the same decreased or enlarged regions of the brain as the affected individual though to a
lesser extent (Harrison, 1999). This has been found true among families with several affected individuals,
where children of schizophrenics display less severe alterations in brain structure than their parents.
4. Mice with Deficient NMDA Receptors 3
There also has been a significant amount of experimentation on the various stages of
schizophrenia and the various alterations it has within the brain. Alongside with the expansion of the
ventricles, lessening of gray matter, and decreased medial temporal lobe, imaging with MRI studies have
closely examined the brains of untreated schizophrenics versus healthy subjects, the brains of
schizophrenics originally on first generation antipsychotics who were then switched to second generation
antipsychotics, and drug-naïve schizophrenics before and after antipsychotic treatment (Vita & de Peri,
2007). The results of first generation versus second generation antipsychotics revealed significantly
higher volumes of caudate nucleus and putamen for those treated with the first generation antipsychotics
(Vita & de Peri, 2007). Analysis through PET scans also revealed a difference in the basal ganglia where
the rates of metabolism increased alongside the subcortical metabolism while undergoing antipsychotic
treatments (Vita & de Peri, 2007). Due to symptoms and alterations, two theories have been proposed.
Dopamine Theory
As the foundation for understanding the workings of schizophrenia, the dopamine hypothesis
discusses schizophrenia from the framework of how the neurotransmitter’s elevation leads to the positive
symptoms of the disorder such as hallucinations and delusions (Steele et al., 2012). Hallucinations
consist of perceiving an object or something which is not present while delusions are beliefs or
impressions which contradict reality or rationality. For people without the disorder, they are common signs
of severe psychiatric disorders while schizophrenics find these to be a painful reminder of their horrifying
reality. However these abnormal behaviors are often associated with the dysfunction of the dopamine
system, and, given the more effective drugs treating these symptoms are medications blocking dopamine
receptors, it is consistently found that schizophrenia is associated with elevated levels of dopaminergic
neurotransmission (Mohn et al., 1999).
Many tests have proven dopamine imbalances cause schizophrenia-like symptoms such as
delusions or hallucinations. Drugs which alter the dopamine pathways have long since been seen as
hallucinogenic, the improper neurotransmission of dopamine is then corrected using antipsychotics which
lessen these positive symptoms to the point they generally no longer occur. Despite the long lasting effect
this theory has had on the understanding of schizophrenia, it falls short on explaining all of the symptoms
schizophrenics suffer from including those which cause an inability to focus.
5. Mice with Deficient NMDA Receptors 4
Glutamate Theory
After the discovery of the influence dopamine played in the display of positive symptoms, the
negative symptoms of the disorder were left a mystery. Even with second-generation antipsychotics
(SGAs) treating many of the problems which came with schizophrenia, the debilitating negative and
cognitive symptoms of the disorder could not be explained by improper dopamine functioning. At the time
of the glutamate theory’s origin, the dopamine theory was over fifty years old but could not explain
negative symptoms, cognitive symptoms, how dopamine receptors failed to relieve these symptoms, and
why even stabilization of dopamine in schizophrenics still resulted in the display of some symptoms
(Steele et al., 2012). All that was really known about glutamate during this time was how it was a major
mediator of excitatory processes in the central nervous system and imbalance of the neurotransmitter
could have serious consequences, such as apoptosis of neurons due to excitotoxicity when glutamate
was present at abnormally high levels (Fiorentino et al., 2015).
Finding a similarity between people high on phencyclidine (PCP) or ketamine (Special K) and
schizophrenic negative symptoms, the glutamate theory came into testing. The agents listed were
described as dissociative anesthetics and binders to the N-methyl-D-aspartate (NMDA) receptors of
glutamate; given their ability to cause the symptoms, it was proposed hyperdopamine and hypoglutamate
activity may be the full cause of schizophrenic symptoms in a combined neurotransmitter theory (Steele et
al., 2012). This was also stated in the article by Mohn et al., where it was mentioned glutamate and
dopamine receptors often have reciprocal actions at different subcortical structures (1999). The
combination theory states dopamine changes may be influenced first by the hypofunction of glutamate
causing inhibitory gamma aminobutyric acid (GABA) release in excess, leading to dopamine inhibition
through the mesolimbic pathway (Steele et al., 2012). Alongside the combined theory, a direct theory
states the mesocortical, nigrostriatal, and tuberoinfundibular dopamine pathways are directly excited by
dopamine neurons and therefore explain the negative as well as cognitive symptoms, the motor
difficulties, and hormone imbalance (Steele et al., 2012). As described by Bennett, the beauty of the
hypoglutamate theory rests in the fact it is able to integrate the failure of neural networks such as the
prefrontal cortex, nucleus accumbens, thalamus, and ventral tegmental area in terms of both negative
and positive symptoms while still explaining the changes with serotonin and dopamine (2009).
6. Mice with Deficient NMDA Receptors 5
Given the disruption of glutamate NMDA receptors, the influx of sodium and calcium trigger the
activation of the receptor. This receptor is unique, however, because it requires the simultaneous binding
of two agonist molecules through an obligate co-agonist, in this case glutamate and glycine (Steele et al.,
2012). Most receptors do not require the binding of two molecules in order to activate, hence this
glutamate receptor is very different from most other receptors. Without both present, the cell does not
depolarize its membrane properly, preventing the removal of magnesium which otherwise blocks the
voltage-gated ion channel (Steele et al., 2012). One study found patients treated with medications which
treated metabotropic receptor agonists had fewer or lessened positive as well as negative symptoms in
comparison to a placebo and olanzapine (Steele et al., 2012). Likewise an AMPAkine was used to treat
schizophrenics though no improvement in treatment was found in comparison to current medications
(Steele et al., 2012). This is an example of how schizophrenia is a specific disorder, something which
does not alter every process, rather it operates to disable or change a few. Although some studies have
dabbled in experimenting with drugs affecting glycine receptors, most medications have been found to
improve either very little, cause no change in symptoms, or to possibly even worsen symptoms present; in
contrast, drugs affecting reuptake of glycine have found improvement of symptoms generally occurs
when they are added to a pre-existing medication regimen though not by themselves (Steele et al., 2012).
A very specific article on NMDA receptor hypofunction due to neuregulin/ErbB4 and synapse
regression, glutamate’s NMDA receptor is most effective under the presence of the proper neuregulin
(NRG) and the ErbB4 receptor which goes with it; as such, the NRG binds to the ErbB receptors to create
heterodimers which activate intrinsic kinases that phosphorylate very specific areas of the cytoplasmic
region on the ErbB’s (Bennett, 2009). By utilization of proper docking sites for the kinases (Src family),
intracellular signaling monitors the NMDA receptor function thanks to several neuregulin genes such as
NRG-1, NRG-2, NRG-3, and NRG-4 which are ligand-based to promote ErbB4’s autophosphorylation
(Bennett, 2009). The interactions and complexity of the NMDA receptor can be seen thoroughly explained
by Figure 3 and Figure 4 of Appendix A. The main point of the article by Bennett is how based on current
understanding and research, enhancement of the activity of the NMDA receptor by neuregulin/ErbB4
improvements should help more schizophrenics by improving overall functionality while NMDA agonists
would only hinder progress by increasing excitotoxic cell death and therefore, increased calcium entry to
7. Mice with Deficient NMDA Receptors 6
other neurons (2009). Though a lot has been found out and more experimentation is still to come, there
are shortcomings of this theory as well. For example, the regression of synapses with resulting positive
symptoms such as hallucinations still cannot be explained through the NMDA hypofunction hypothesis
(Bennett, 2009). The importance of neuregulin-1 (NRG-1) is emphasized by O’Tuathaigh et al. by
explaining its risk factor for hypoglutamatergic function and schizophrenia onset (2010). Due to this,
recent experimentation has focused on the cytoskeletal system spines for answers as to why there are
still failures among the neural network.
Mice Studies
Given the difficulties of testing variations of genes within human subjects, animal model
substitutes with similar or nearly identical systems are able to be studied. Mice in particular are often
relatively cheap and easy to experiment on given both their life spans and the time it takes for them to
reach maturity. In previous studies with both mice and humans, NMDA receptors have gained interest
due to the unique pharmacological and electrophysiological properties these receptors have on the
synapses of cells as well as with their roles in synapse refinement, plasticity of neurons, and excitotoxicity
(Mohn et al., 1999). Mice have been generated lacking each of the known NMDA receptor subunits such
as NR2, like NR2A-NR2D (except NR2B), and NR3; however, mice lacking NR1 and NR2B proteins died
prenatally, displaying the importance of these two in proper life functions (Mohn et al., 1999). This ability
to manipulate NMDA receptors by producing mice with the various protein deficiencies allows for the
studying of several diseases which are harder to study in humans such as schizophrenia.
In the study performed on mice, genetically altered mice were used which expressed 5-10% of
the normal levels of NR1 and although they were deficient in NMDA receptors, the deficiencies showed
similarities to schizophrenic behaviors which are also alieved by the usage of antipsychotic medications
like haloperidol and clozapine (Mohn et al., 1999). A demonstration of the odd habits of these mutation-
bearing mice can be seen in Figure 2 of Appendix A. In the experiment performed by Fiorentino et al.,
mice were found to have a specialized transporter known as the glial high-affinity glutamate transporter
(GLT1) which is responsible for approximately 90% of the glutamate removal from synaptic clefts utilizing
astrocytes and oligodendrocytes (2015). Unlike in other studies, this study closely examined the
excitatory amino acid transporter 2 (EAAT2) under the impression dysregulation of EAAT2 caused
8. Mice with Deficient NMDA Receptors 7
psychiatric disorders such as depression, autism, and schizophrenia, but also several neurological
diseases like amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and epilepsy (Fiorentino et al.,
2015). Although EAAT2 alterations were non-conclusive, a few case-only missense variations were found
to increase the likelihood schizophrenia or bipolar disorder would develop. This has been found true for
other mutations as well where some results may be inconclusive or not yet replicated successfully, while
other similar alterations have had clear results. Additionally, another experiment was performed in mice
utilizing the NRG-1 gene which tested for schizophrenia-like symptoms.
In this experiment, both heterozygous and homozygous mice were generated and maintained
with little human intervention. The mice were genotyped using polymerase chain reaction (PCR) and kept
in consistent, stable environments with the following: a caged area with two-four other mice, a standard
12/12 hour light to dark cycle, and free access to food and water which was confirmed as acceptable
conditions for research (O’Tuathaigh et al., 2010). Using both genders of wild-type and mutant mice,
medications were administered subcutaneously and results of both behaviors as well as brain analysis
post-mortem were performed.
Genetic Components
With relatively high heritability, schizophrenia has been found as a common disorder which may
run in families (Kukshal et al., 2012). With its debilitating effects, many gene mapping studies have been
performed in order to locate genes responsible for a variety of disorders – some with better success than
others. A fraction of the heritability of schizophrenia has been discovered with many mutations on a
variety of genes. Despite some controversy over gene mapping, it is proposed the extensive amounts of
research being placed on gene mapping will lead to better and earlier diagnoses, enhanced
pharmacogenetics understanding, and personalized treatments for the varieties of the disorder.
With 23 pairs of chromosomes and nearly three billion base pairs, two individuals can potential
vary at over 10 million base pair sites (Kukshal et al, 2012). One of the most useful tools for gene-
mappers known as linkage analysis tests along family lines with similar diseases or disorders in order to
find regions of “chance occurrence” where there are similarities along a region of particular chromosomes
which may cause or lead to the disorders (Kukshal et al., 2012). By closer analysis known as fine
mapping, DNA variation is more closely examined to find problem regions of mutations which are
9. Mice with Deficient NMDA Receptors 8
common factors among people affected by the disorder. Unfortunately, given the difficulty of finding large
families with the condition, most research on schizophrenia inheritance have been performed using
association analysis which uses a population of people affected rather than family members which may
bear more genetic similarities; this makes the process of association still possible through a more indirect
method (Kukshal et al., 2012). After the Human Genome Project, the human DNA variants have been
made public through several sites which inspire genome-wide association studies – also known as GWAS
– which have led to a greater understanding of genetic components involved with the onset of
schizophrenia (Kukshal et al., 2012).
Current gene mapping studies of schizophrenia have produced several results. Both genetic
factors and gene-environment interactions contribute well over 80% of the probability for a person to
develop schizophrenia (Kukshal et al., 2012). Linkage studies of schizophrenics found two chromosomal
regions were most responsible for schizophrenia: 8p21-22 and 22q11-12 (Kukshal et al., 2012). The
following locations have also been determined to cause risk for the disorder: 1p13-q23, 1q23-31, 2p12-
q22, 2q22-q23, 3p25-p22, 5q23-q34, 6p22-p21, 6q15-23, 10pter-p14, 11q22-q24, 14pter-q13, 15q21-q26,
16p13-12, 17q21-q24, 18q22-qter, 20p20-p11, and 22pter-q12 (Kukshal et al., 2012). Of these risk
locations, the risks on chromosomes 1, 6, 8, and 22 were predicted by association analysis to be the
main problem areas. In association studies, the disorder was found within Neuregulin-1 (NRG1) of
Icelandic populations on chromosome 8 as well as among people with catechol-o-methyltransferase
(COMT), an enzyme which serves as part of dopamine metabolism (Kukshal et al., 2012). GWAS, a
cheaper and more accurate way for analysis of the disorder, pointed in the direction of the
histocompatibility complex region found at 6p22-p21 as well as several other new loci including those
found within Chinese and Japanese ancestry which are still currently undergoing research (Kukshal et al.,
2012). Studies of copy number variations resulted in the observance of chromosomal anomalies of
deletions on chromosome 22q11, a balanced through reciprocal translocation of 1q42 with 11q14, and
another anomaly on the X chromosome (Kukshal et al., 2012). More in depth work has been performed
recently within populations of India and can been seen in Appendix A on Table 2 for more information.
Along with increasing the risk of schizophrenia, many copy number variations (CNVs) have
suggested areas also create high relative risk factors for other psychiatric disorders such as bipolar
10. Mice with Deficient NMDA Receptors 9
disorder, autism, learning disability, ADHD, seizure disorders, obesity and dyslexia (Kukshal et al., 2012).
Like many CNV studies, the study by Fiorentino et al. was performed due to the belief the SLC1A2 gene
held the answers behind a variety of neurological conditions including schizophrenia. Although variants of
the gene were found to not be associated with only schizophrenia, the experiment was performed under
small population sizes and did find missense variations which were associated with the combination of
bipolar disorder and schizophrenia (Fiorentino et al., 2015).
Proposed Study
Mouse Schizophrenia
Upon the birth of the genetically modified mice, each mouse will be carefully examined for weight
and size then tagged as either wild-type class, heterozygous class, or homozygous mutation-bearing
class. These mice will then be separated according to their genetic compositions. Mice will then be
examined for any differences during growth and provided with proper amounts of care to allow for further
growth. Human interaction will be kept at a minimum, occurring only during feedings or maintenance of
the cage where they will be kept for the experimentation period. Any abnormal behaviors will be noted,
though otherwise the mice will be left alone to mature with their brood mates.
Near maturation, the mice will be more closely monitored. In human schizophrenia, the onset
generally occurs for most during puberty up until the age of 21 though onset may be later or earlier
depending on the individual and their disorder’s severity (ex. earlier onset is often associated with
increased severity of their psychiatric disorder). Given this fact, the maturing mice will be monitored and
interactions with their brood will be recorded to test the amount of time each of the three types of mice
spend exhibiting symptoms mimicking schizophrenia through head twitches or frantic running, avoiding
other mice, or socializing with the other mice. Any abnormal traits will be more closely monitored though
human interaction again will be kept strictly to feedings or cleaning of the cage.
Once maturation has ended, the mice will again be examined for the same behaviors or abnormal
traits prior to either letting them live out their lives until their natural deaths or inducing an early death
through lethal inhalation of carbon dioxide. The brains of each type of mice will be extracted for
placement on dry ice to preserve them and then the frozen specimen will be compared on the following
11. Mice with Deficient NMDA Receptors 10
terms: weight, ventricle size, abnormal brain structures, or other observations on the full-brain extraction.
After such, blot analyses will be used and DNA will be replicated to allow for testing which confirms the
presence of the mutation along with polymerase chain reaction (PCR). Dopamine and glutamate levels
will also be compared in the brains then tested for significance.
Medication Testing
Aforementioned, emphasis has recently been placed on the study of synaptic spine loss and its
effect on NMDA receptors as well as glutamate uptake. Reported by Bennett, the loss of synaptic spines
and their functionality at the synapse in regions of the brain are associated with hallucinations, delusions,
and cognitive decline (2009). Due to this, the mice will be tested using a variety of medications which
have been formulated to act as antipsychotics, glutamate enhancers, glutamate inhibitors, and
medications which are specialized to act particularly at synaptic spines in hopes of re-establishing neural
circuitry in mice with schizophrenia-like symptoms. Based on the results from the various medication
experiments, the effectiveness of glutamate versus dopamine medications will provide a starting ground
for calculating appropriate dosages. Similarly, an attempt to create synaptic spine medications will be
made in hopes of creating a specific drug which can be monitored as better than current medications, at
the same level as current medications, or worse than current medications in terms of treating the
condition. Considering glutamate has the potential to become excitotoxic, dosages must be handled with
caution in order to maintain and improve the life of the organism without harming it.
Aims of the Study
(1) To expand on current research of the glutamate theory of schizophrenia.
(2) To create a method for studying schizophrenia in a non-human model to allow for easier and
cheaper testing on ways to improve the condition.
(3) To lead to improved medications which better treat the symptoms of schizophrenia.
Experimental Design
Model Organism
Aforementioned, the usage of a cheaper and smaller organism with physiological similarities to
humans is beneficial to begin understanding a possible alternate pathway which schizophrenia symptoms
12. Mice with Deficient NMDA Receptors 11
may emerge from. By generating mice using homologous recombination in embryonic stem cells, the
hypomorphic allele of NR1 are able to be replicated (Mohn et al., 1999). In previous studies with NR1
mice, the animals are intercrossed to create NR1neo-/- mice which were recorded as being consistently
smaller than littermates from birth until adulthood although by adulthood, their weights were within normal
range (Mohn et al., 1999).
Raising Mice and Production of NR1neo Mutation
Preparation of mice with proper mutations are often difficult as the mutations must either be easy
to verify based on physical alterations or genetic testing. Along with methods used by Mohn et al.,
genomic clones can be prepared by isolation from a bacteriophage library such as 129/SvEv λ spanning
the NR1 locus and utilizing NR1 cDNA exons which are extracted using a probe (1999). The mutation is
then inserted into the 5’ portion of the DNA strand, along in this case the neo gene where it is linearized
with Pvul and electroporated into cells (Mohn et al., 1999). By doing so, the presence of the proper
mutation can be validated. The blastocysts were then used to generate chimeras, or organisms which are
composed of cells from different zygotes prior to breeding for the NR1neo -/- mice (Mohn et al., 1999).
Once the new NR1neo -/- mice were obtained, they were then crossed with wild-type mice to obtain the
NR1neo +/- mice which were used to create the breeding colony used for the rest of the experiment (Mohn
et al., 1999). By this method, the wild-type, heterozygous, and homozygous NR1 gene mutant mice could
be obtained from the same brood.
Validation of the Proper Mutation
After the mice were imbedded with the mutations, the targeted mutations were identified by
Southern analysis and injected into blastocysts as one method of validating the mutation (Mohn et al.,
1999). Mice will be examined for the schizophrenia-like symptoms, further confirming whether the
mutation achieved its proper functionality or dysfunctionality. Upon death, whole brain extraction and DNA
testing will expand upon the issue to again confirm the presence or absence of the mutation in the mice
with the help of PCR.
Mouse Care and Symptom Severity Measurement
As previously noted, NR1 mice were often found to be smaller during the growth process in
comparison to their non-deficient littermates. Often, mice and rats exhibit schizophrenia-like symptoms
13. Mice with Deficient NMDA Receptors 12
such as frantic running or incessant head-turning which many have proposed mimics psychosis of
humans (Wickelgren, 1998). Although mice do not display clear positive, cognitive, and negative
symptoms, severity of their schizophrenia-like symptoms will be determined based on the behaviors they
exhibit which mimic the disorder. These include but are not limited to: social withdraw, excessive head
turning, frantic movements, and inability to perform in cognitive tasks. It is also important to note, little
human intervention will occur during the raising of these mice as the conditions will mimic those used by
O’Tuathaigh et al., mentioned previously, to simply maintain their livelihood (2010).
Mouse Cognitive Capabilities
Food rewards have often been used to test the learning of mice. In an experiment explained by
Miss Wickelgren and tested by Moghaddam and Adams, rats were placed in a T-shaped maze to test
their working memory as one direction held a reward while the other held either a punishment or nothing;
rats with schizophrenia-like symptoms lacked the cognitive decision making and working memory, so they
were reported to make wrong decisions half the time (Wickelgren, 1998). The same study found that rats
treated with effective medications which altered the glutamate levels by pretreatment with LY354740
made wrong decisions less often, only 30-40% of the time which was in the range of normal rats
(Wickelgren, 1998). Similarly, the mice will be tested through this method in order to test for proper brain
functionality with signs of cognition so the impairment with decision-making can be recorded.
Post-Mortem Analysis
Upon the deaths of mice, either natural or induced by lethal inhalation of carbon dioxide, the
brains of the mice should be removed as a whole unit and placed on dry ice for preservation. As
mentioned previously, the brains of schizophrenics often display enlarged ventricles and a decreased
brain size, particularly in the medial temporal lobe (Harrison, 1999). Due to this factor, the removed brain
should carefully be examined for these two factors and careful examination of the medial temporal lobe
should be undergone. Abnormalities in this region or any other region should be made note of.
The expression of NR1 may then be examined in both wild-type mice, NR1neo+/-, and NR1neo-/-
mice in order to allow testing against blot analyses; in a previous study by Mohn et al., the results of a
Northern or Western blot analysis should indicate intronic insertion where the -/- mice held variations of
anywhere from 5.9-9.4% of normal levels (1999). Alongside these, liquid chromatography should be used
14. Mice with Deficient NMDA Receptors 13
to determine the amino acid peaks which would represent the retention of these substances in various
regions of the brain. With amino acid retention, the amount of neurotransmitters present in the areas of
the brain aforementioned should also be closely monitored. Areas more often associated with
schizophrenia should have abnormal dopamine levels particularly in the prefrontal cortex as well as
improper levels of glutamate elsewhere throughout the brain.
Statistical Testing & Data Analysis
Observational data should be recorded in seconds of types of behavior much like the article
published by Mohn et al., recording detailed evasive or socializing patterns the mice exhibit along with the
schizophrenia-like behavior of head-twitching and frantic running; in addition to these, any other odd
behavior should be noted (1999). These observations can then be used to determine both the similarities
to schizophrenia in human models as well as the significance of the mutation on the behavior. The
greater the similarity, the more the dysfunction of glutamate in mice may correspond to schizophrenia
found in human models.
Further analyses should be performed using ANOVA. As indicated previously, many
neurotransmitters are essential to proper functioning, however sometimes variations may lead to similar
functional properties. A recent article found that neuregulin-1 (NRG1) is a gene which is often associated
with increased risk of schizophrenia development, however several proteins and isoforms, similar proteins
which vary based on a few amino acids and are encoded by different genes or RNA transcripts by the
same gene with different removed exons, lead to a greater understanding of what delineations from
normality cause (O’Tuathaigh et al., 2010). By correlating the relevance of the genetic alterations to onset
of the condition or severity, genetic predisposition to schizophrenia can be more readily understood and
preventative measures can be taken to reduce the severity of the first symptom onset. Similarly, the
medication testing must be analyzed through ANOVA in order to confirm or deny the betterment of the
populations which were treated with each of the medications proposed. The results from the ANOVA will
therefore indicate not only which ones were significant based on a P-value less than 0.05, but it will also
in comparison between the medications show which medication was the most efficient at alleviating the
symptoms the mice were suffering from. A demonstration of an ANOVA calculated for stereotypy and
locomotor activity can be seen in Appendix A under Figure 1.
15. Mice with Deficient NMDA Receptors 14
Limitations of the Study
Many medications and drugs which are promising in animal models often fail short when used in
human models. By the time of the article by Wickelgren was published, fears were that PCP-induced
symptoms of rats and mice were not accurate reflections of human schizophrenia and therefore
treatments via medications would need to be further modified for human usage (1999). Likewise, the
usage of the model organism is not a direct measurement of what are classified as cognitive, positive,
and negative schizophrenia symptoms – instead, it is an indirect assumption of behaviors on cognitive
processes which mimic the symptoms, hence they are classified as “schizophrenia-like” behaviors. If
evidence were found in the genetically altered mice, the same may not be said for human schizophrenics
as the disorder is very complex, involving many different areas of the brain and therefore, the process of
onset may differ.
Aside from the difficulties with the animal model, there are two main difficulties with the
medication aspect of the experiment: the dosages and the synthesis of a synaptic spine specific drug. As
mentioned previously, excessive glutamate activity may lead to excitotoxicity which causes neuron death
which in turn, ultimately leads to death. However, inhibition of glutamate is equally dangerous as it is the
major excitatory neurotransmitter found in humans and some degree of activity is necessary, therefore
with the loss of glutamate function, the risk of death may increase. Similarly an issue, the creation of
highly specific medications is very difficult, thus the creation of a drug which focuses solely on improving
the synaptic spines will be arduous and require many, many trials. Given the hypothesized importance of
this area on the understanding of schizophrenia and the explanation of more components of the disorder,
it is something which should be closely looked into despite the time and patience it may require.
From a non-clinical perspective, there is still discrepancy on whether genetic testing and
preventative actions should be taken. Many approve, finding it as a valid option to allow people to make a
more informed decision to have children, but many still see this as a major issue. Though some see the
good in gene testing, many still see it as a way to classify people based on the presence of “inferior
genetics” which may lead to harder lives for people at high-risk for things like heart and mental disorders.
Still, many see it as a way to offer better, more personalized treatment while providing parents with a
better understanding as to how they should raise their children.
16. Mice with Deficient NMDA Receptors 15
Appendix A – Tables & Figures
Table 1: A copy of the 0-4 point scale used in grading the severity of the symptoms of schizophrenic
patients found within the article by Pagsberg (2013).
Table 2: Genes, SNP, and sample references based on studies within populations of India as found in the
paper by Kukshal et al. in 2012.
17. Mice with Deficient NMDA Receptors 16
Table 3: Comparison of DSM-IV and DSM-V Schizophrenia requirements as found in the article by
Pagsberg (2013).
18. Mice with Deficient NMDA Receptors 17
Figure 1: The display of mice with the NR1neo-/- mutation and their various activities such as (a) locomotor
activity, (b) stereotypy of untreated mice [open symbols] and wild-type littermates [filled symbols] with the
two-way ANOVA of p < 0.0001, F = 159.7, (c) locomotor activity when treated with 3 mg/kg of PCP, (d)
stereotypy of untreated mice and wild-type mice with the same treatment with two-way ANOVA of p =
0.0912, and then (e & f) which show the same conditions only with 0.2 mg/kg MK-801 which resulted in p
= 0.9521. Note: n= 8 for all groups and presented as found in the Mohn et al. article (1999).
19. Mice with Deficient NMDA Receptors 18
Figure 2: A demonstration of: (a) the behaviors exhibited by wild-type mice (left panels) and NR1neo -/-
mice (right panels) over the period of two hours (pictures from every half hour), (b) the time spent by the
resident male over a 6 minute period in wild-type, NR1neo -/-, and NR1neo -/- mice treated with clozapine,
and (c) the escape behaviors of the same three groups. Note all data was found to be statistically relevant
(p < 0.0005 and p < 0.005 respectively). Found in the Mohn et al. article (1999).
20. Mice with Deficient NMDA Receptors 19
Figure 3: Published in the article by Bennett in 2009, the image displays several the complexity of a
neuron through several different neurotransmitter receptors found on a single neuron including glutamate
and dopamine. The figurehead from the article remains intact, again to show the complexity of uptake for
a neurotransmitter.
21. Mice with Deficient NMDA Receptors 20
Figure 4: As published by Bennett in 2009, the two side by side images below show the ErbB
hyperphosphorylation hypothesis for schizophrenia by displaying heterodimerization on activation by
neuregulin through NMDA for uptake at several sites (left) as well as the failure on several sites due to
the hyperphosphorylation (right) as speculated within the experiment performed.
22. Mice with Deficient NMDA Receptors 21
Appendix B – References
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hypofunction hypothesis, neuregulin/ErbB4 and synapse regression. Australian and New Zealand
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Fiorentino, A., Sharp, S. I., & McQuillin, A. (2015). Association of rare variation in the
glutamate receptor gene SLC1A2 with susceptibility to bipolar disorder and
schizophrenia. European Journal of Human Genetics, 23(9), 1200–1206.
http://doi.org/10.1038/ejhg.2014.261
Harrison, P. J. (1999). Brains at risk of schizophrenia. The Lancet, 353(9146), 3-4. Retrieved
March 11, 2016.
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Weissman, M, Hultman, C., Landén, M., Levinson, D., Kendler, K., Smoller, J., Wray., N., & Lee,
S. (2015). Joint Analysis of Psychiatric Disorders Increases Accuracy of Risk Prediction for
Schizophrenia, Bipolar Disorder, and Major Depressive Disorder. American Journal of Human
Genetics, 96(2), 283–294. http://doi.org/10.1016/j.ajhg.2014.12.006
McNally, K. (2011). Definitions of schizophrenia, 1908-1987: The failed essentialism. Theory &
Psychology, 22(1), 91-113. Retrieved March 10, 2016.
Mohn, A.R., Gainetdinov, R. R., Caron, M. G., & Koller, B. H. (1999, August 20). Mice with
Reduced NMDA Receptor Expression Display Behaviors Related to Schizophrenia. Retrieved
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Steele, D., Moore, R. L., Swan, N. A., Grant, J. S., & Keltner, N.L. (2012). Biological
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