Anorexia Nervosa: finding the genetic background of rigid behavior an endophenotype of
Anorexia Nervosa
H.C. den Boer, 1 July 2011
Abstract
Anorexia Nervosa is from all the psychiatric diseases the disease with the highest mortality rate.
Anorexia Nervosa is currently under intense investigation, however the underlining cause of Anorexia
Nervosa is still not known primarily because its complexity. AN consist of several distinct behavioral
component that contribute to the disease in distinct manners. Human genetic studies have tried to
find the genes responsible for AN, however because of heterogeneity due to a lack of statistical
power only several studies developed significant results. Several studies that did develop significant
result pointed towards DRD4, DRD2, 5-HT2A, BDNF, HRTD1 and ORPD1 as susceptible genes that
contribute to the disease. The technique to perform Genome Wide Association studies might help to
solve this problem. Additionally the use of endophenotyping will help to solve heterogeneity in the
patient groups by focusing on behavioral domains such as activity, rigidity or anxiety. Besides the
human genetic studies mice studies have contributed to our knowledge of AN. Distinct mice models
and KO mice have resulted in the association of multiple genes to AN such as leptin, POMC, NPY,
AGrP, dopamine, M3, CRCH2 and the anx gene. Mice models combined with endophenotyping might
result in the discovery of the genetic background of specific behaviors. The specific behaviors that
contribute to AN consist of anxiety, rigidity, depression, perfectionism and activity. Therefore I
proposed a set shifting test in mice to discover the genes responsible for rigid behavior. Rigid
behavior was selected because it is an endophenotype that makes the disease more sever and the
duration of the disease longer. Rigid behavior is tested with a set shifting task in mice in which BL6 is
compared to AJ. Further QTL analysis with CSS (Chromosomal Substitution Strains) should result in
susceptible regions on the chromosomes of the mice. First Data of the comparison between the BL6
and AJ for the set shifting task already shown
Author: H.C den Boer
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Contents
page, 2
Abstract
page ,1
Introduction
page , 3-29
1.
2.
3.
4.
5.
6.
Anorexia Nervosa criteria
Psychiatric complexity of Anorexia Nervosa
The genetic research in Anorexia Nervosa
Animal models
Endophenotypes
Rigidity
page, 3,4
page, 4,5
page,6-10
page, 10-16
page, 16-23
page,24-29
Materials and methods
page, 30
Results
page, 30-32
Discussion
page, 32
References
page, 33-37
Author: H.C den Boer
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1.0 Anorexia Nervosa criteria
Without food we humans cannot survive. So food gets an important place in our lives because we
need to eat a broad spectrum off food to keep our body running normally. We use this food to
retract energy from and to build up our cells. During evolution we received multiple mechanisms to
cope with our need for food. Such mechanisms make sure we eat enough food, but also make sure
we do not eat too much. However deficits in these coping mechanisms result in abnormal eating
behavior or an eating disorder such as Anorexia Nervosa. Anorexia Nervosa is besides a physical
pathology also a mental disorder. The disorder Anorexia Nervosa is investigated over the years from
a psychiatric point of view but also from a biological perspective. However most of the genetic
background of Anorexia Nervosa stays unidentified. The goal of this paper is to provide insight in the
disease Anorexia Nervosa and the different ways to investigate Anorexia Nervosa. What do we know
about the disease and the mechanisms that contribute to the disease? What is the genetic
background of Anorexia Nervosa? The second goal of this paper is to propose an experiment to
investigate the genetic background of rigid behavior. Rigid behavior contributes to Anorexia
Nervosa, however the genes and neuronal systems responsible for this behavior are not fully known.
1.1 Criteria of eating disorders
To investigate Anorexia Nervosa we need to know what Anorexia Nervosa is. What are its criteria?
What makes it such a large problem throughout the world? And what makes it different from other
eating disorders? There is a large spectrum of eating disorders. These eating disorders comprise
Anorexia Nervosa (AN), Bulimia Nervosa (BN), purging disorder, Binge eating disorder or other eating
disorders. The most used definition for these eating disorders is provided by the American Psychiatric
Association’s Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV)( 1 ).
The criteria that are provided by the DSM-IV for Anorexia Nervosa are the refusal to maintain a body
weight at or above a minimal normal weight for age of height. This will lead to the maintenance of a
body weight less than 85% of what is normal. Someone with Anorexia Nervosa has an intense fear of
gaining weight or becoming fat even when they are underweight. They have a disturbance in the
experience of there body shape and weight. This influences there self evaluation or brings them in
denial of the seriousness of there current low body weight, furthermore females with AN have
amenorrhea. Amenorrhea is the absence of at least 3 consecutive menstrual cycles. The disease
manifestates itself in two distinct forms, a restricted type or a binge eating/purging type (2, 3).
The criteria for Bulimia Nervosa according to DSM-IV are recurrent binge eating characterized by:
Eating a lot more than normal people will do in a discrete period of time and the sense of losing
control over eating during this time. Patients with BN have compensatory mechanisms for the binge
eating behavior like: misuse of laxatives or other medication, self vomiting, excessive exercise and
fasting. This binge eating takes place at least twice a week over a period of 3 months. And the self
evaluation of BN patients is influenced by there body and weight. For BN there are two types, the
purging type and the non purging type (2, 3). Both AN and BN are distinct eating disorders besides
there resemblances. This is important to know because different biological processes or genetic
deficits can be responsible for the distinct diseases.
Anorexia Nervosa patient will suffer physically from there mental illness. Their abnormalities in food
intake will lead to signs of malnutrition. This results in pubertal delay or poor growth. Significant
weight loss will result in pale look, sunken eyes, thin limbs and protruding ribs. Heart rate,
temperature and blood pressure will decrease with insufficient food intake. Subcutaneous tissue and
musculature will disappear. The skin may become cold and dry. Patients with AN will show a lot of
Author: H.C den Boer
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starvation symptoms like anemia, leukopenia, thrombocytopenia, hypoglycemia. Also AN patient can
evolve heart failure and failure of other organs (1).
1.2 Epidemiology
To further understand Anorexia Nervosa we need to know which people are vulnerable to get the
disease. AN is a large problem throughout the world. In the United States alone there are around 24
million people who have an eating disorder. From the AN patients 85-90 % is women. The onset of
AN is typically during adolescents, though certain studies reported cases of AN onset before the age
of 13 (6). Anorexia Nervosa exhibits the highest mortality rate of all mental illnesses, however the
exact mortality rate for AN is unknown. Multiple studies and sources show different rates. This is
because AN patients ultimately die off heart failure, organ failure, malnutrition or suicide. These
cases generally display the medical complications as the cause of death and not the mental health of
the patient. According to the Journal of Psychiatry the mortality rates of AN are around 4 % and for
BN the mortality rates are around 3, 9 % (4, 5).
2.0 Psychiatric complexity of Anorexia Nervosa
Investigating Anorexia Nervosa cannot solely be done with the previous stated criteria. The
underlying causes of AN patients are more multifaceted than these criteria state. AN patients display
an extensive variety abnormal behaviors that can contribute to the onset, severity and outcome of
the disease. Such abnormal behaviors are hyperactivity, anxiety, depression, inflexibility or rigidity,
obsessiveness and compulsivity. In this section these abnormal behaviors and there contribution to
the disease will be described.
2.1 Hyperactivity
When someone is fasting during a longer period the body will adopt a state in which it can survive
during this period. That means a switch in its energy sources. The body will use its adipose tissue to
keep its plasma glucose levels within an acceptable range for our brain and neurons. And the body
will break down muscles or even other tissue to support the energy need of the body. Besides the
change to different energy sources the body will try to limit its energy expenditure to the minimum.
Someone who is fasting will put less energy in keeping up its temperature and will stop synthesis for
growth and will even break down tissue also effecting the reproductive functioning. At the same time
someone would lower the expenditure of the body by moving less. But in AN patients this is not
always the case. In AN patients 80 % will even show hyperactivity. These AN patients show a
constant, agitated restlessness when they are emaciated. This hyperactivity is associated with the
acute stage off the illness (7). The hyperactivity makes the disease more severe given that in a semistarved state all the energy is needed for survival and not for movement. The hyperactivity seen in
AN patients is unique. Someone in a state of semi-starvation is not able to be active. AN patients
even seem to be more alert than someone who is on a normal diet. The exact nature for this
hyperactivity is still to be clarified but appears to be that of neurobiological factors and a conscious
attempt to loose more weight (8).
2.2 Anxiety, Obsessiveness and Compulsivity.
Research indicates that AN frequently co-occurs with anxiety disorders. Multiple studies show that a
significant part of AN patients experience one or more anxiety disorders. The lifetime prevalence for
anxiety disorders varies from 25% to 75 % of AN patients. In most cases the onset of the anxiety
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disorder precedes the onset of the eating disorder. This leaded to the suggestion that early onset
anxiety disorder predispose individuals to the development of an eating disorder later in life (9).
Clinical observations and investigation showed co-occurrence between AN and Obsessive Compulsive
disorder (OCD). These studies showed that the OCD onset mostly predated the onset of AN,
Therefore it could be a risk factor for AN. Individuals with OCD demonstrate a longer history of an
eating disorder as well as the development of an eating disorder at an early age (10). The symptoms
of OCD seen in AN patients are regularly aimed at the regulation of food restriction. Though
symptoms related to checking and cleaning compulsions can, symmetry and order and aggressive
obsessions can also be present (9). In addition perfectionism and Obsessive Compulsive Personality
Disorder (OCPD) are as well connected to eating disorders; however this association will be discussed
in the section inflexibility.
AN patients can show symptoms of social anxiety as well. Some studies show that individuals with AN
have significantly higher levels of social phobia than is seen in control women. These social phobias
include Agoraphobia, panic disorder and specific phobia. Furthermore Post traumatic stress disorder
is seen in AN patients, although this is considered to be a non-specific risk factor (9).
2.3 Inflexibility
Multiple obsessions and compulsion are seen in AN patients. These include cleanliness, orderliness,
perfectionism, rigidity and miserliness. The presence of the obsessions and compulsions gives the
suggestion that these are traits important in the development and maintenance of the eating
disorders. These traits are seen in people with OCPD which shows co-occurrence with AN. OPCD is
characterized by inflexibility and the strictness in procedures, perfectionism and orderliness.
AN patients are thought to show super perfectionism related to their body. This perfectionism is a
negative predictor for the outcome for eating disorders.
Furthermore AN patients can be rigid in their eating patterns. Often this rigidity exhibits a ritualistic
element, for example eating slowly, eating foods in a particular order, or eating ‘save’ foods. The
thought behind this behavior can be that a change in eating pattern will result in catastrophic
outcomes, for instance uncontrolled rapid gaining of weight (11).
2.4 Depression
Both clinical and associational studies have consistently revealed an association between major
depression and AN. In population studies lifetime major depression was reported in 50 % of all the
women with AN (12). Depression can negatively affect self-esteem in patients and as a result this can
increase severity and chronicity of the illness. Additionally AN patients show co-morbidities with
intellectual disability and dissociative disorders. Moreover AN patients demonstrate elevated levels
of suicide and suicide attempts making AN a very deadly mental illness.
2.5 Summary Psychiatric complexity of Anorexia Nervosa
The co-occurring symptoms described above all contribute to the disease AN. Some characteristics
contribute to the onset of the disease while others contribute to the severity of the disease.
Furthermore not all the characteristics are present in each AN patient. This makes the study of
Anorexia Nervosa very complicated and hard to study. To study AN these components and there
influence on the disease should be taken into account. In addition to the presence of these
symptoms there is also the question what the contribution of the environment and the genetics is in
AN. However there are some ways to investigate the role of these symptoms, the genetics and
environment on AN. With new techniques and animal models the genetic background of AN begins
to produce answers to these questions.
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3.0 The Genetic Research in Anorexia Nervosa
Anorexia Nervosa is a mental illness that has a sociocultural component (the environment) and a
biological component (the genetics). The evidence for this biological component came from studies
with twins and families. These studies made way for other genetic studies to investigate psychiatric
diseases like AN. Because of the Twin and Families studies and genetic sequencing, linkage studies
could be performed. These linkage studies are a way to investigate genes of known mechanisms and
their associations with AN. Genome Wide Association Studies (GWAS) are the future for this kind of
genetic research, since they make it possible to compare the whole genome of patients with each
other. The sight of these genetic research methods looked promising; however the results that were
found raised skepticism. This is for the reason that some studies showed significant results while
other studies could not. The reason for this was the heterogeneity in the subject groups. Reducing
the heterogeneity should be a large priority with these methods. To reduce heterogeneity
subphenotyping and endophenotyping could be used. The insights that came from these genetic
research methods will be described in this section.
3.1 Twin and Family studies
The first evidence that showed that there is a substantial genetic contribution to the etiology of AN
came from Twin and Family studies. Twin studies try to differentiate genetic and environmental
effects by comparing similarity for a disorder between identical twins and fraternal twins. Identical
twins are monozygotic and fraternal twins are dizygotic. The comparison made in these studies is
based on the principal that if there is a genetic component for a disorder the identical twins should
correlate more than fraternal twins. Several studies indicate that the genetic contribution to AN is
roughly 58 % to 76 % (13, 14, 15). The findings of family studies likewise demonstrate that eating
disorders can be tracked back trough family history. First-degree relatives of individuals with AN have
approximately three-fold greater lifetime risk of developing AN than unaffected individuals. Likewise
research suggests an increased risk for any eating disorder in relatives. These findings opened the
door to more genetic research on this subject (13).
3.2 Linkage analysis
Linkage analysis is a tool to find the chromosomal regions that harbor genes that contribute to the
selected trait of interest. Linkage analysis might be performed with the use of anonymous genetic
markers that are identified while the genomes of individuals were mapped. The markers are named
single nucleotide Polymorphisms (SNP) and are DNA sequence variations that occur when a single
nucleotide is altered in the genome sequence of an individual. Each individual possesses 10 million of
these SNPs and that creates a unique sequence for every person. In linkage analysis cases with the
same disorder are mapped for these SNPs and the candidate genes are then located under linkage
peaks. The linkage peaks are areas where the cases are genetically the same or almost the same.
After this the linkage peaks might be explored by case control association studies (13). Linkage
studies can be performed by several distinct methods. One method is to search for linkages in
different regions or genes that are thought to be susceptible. For example in several genes that
contribute to the serotonin pathway in the brain. Another method to search for linkages within the
genome is with the use of GWAS. With the technique and means we have currently the mapping of
the whole genome can be performed relatively cheap and fast. For both these linkage studies the
phenotyping for the disorder should be done very accurately to reduce the phenotypic and genetic
heterogeneity, because else the results will not be statistically significant (13).
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3.3 The finding of a susceptible gene on chromosome 1
To find genes that are susceptible to contribute to AN GWAS might be performed. The finding of a
susceptible gen on chromosome 1 was due to the GWAS method. Grice D.E et al performed a GWAS
within a group of 192 families with at least one affected relative pair with AN or BN. And to reduce
heterogeneity and increase power they also did a linkage study in 37 families. There first results
presented modest evidence for any linkage on all the chromosomes. The single multipoint NPL
(nonparametric multipoint linkage) above the 1.5 was at marker D4S2367 on Chromosome 4. The
marker showed a NPL of 1.80. The modest results could reflect either to the fact that there are
several genes that all contribute to the found effect, however weakly, and/or it could reflect to large
sample heterogeneity (16). That is why they reduced the amount of families that were under
investigation by defining a subgroup. The subgroup consisted of 37 families with relatives that have
restricted Anorexia Nervosa to get a group that should show more genetic homology. As a result 9
peaks were discovered with the highest peak discovered at marker D1S3721 on chromosome 1. The
marker D1S3721 showed an NPL score of 3.03. (Fig 1) The discoveries suggest a linkage between
chromosome 1 and AN nonetheless it showed that there is a lot of heterogeneity within AN and the
possibility of multiple genes that all contribute to the disease (16).
Figure 1. The figure shows the linkage peaks of the markers on chromosome 1 with the NPL score of 3.03 at
marker D4S2367. This region at marker D4S2367 can hence susceptible genes that might contribute to AN (16)
3.4 Case control association studies
Case control association studies can be used solely as a method for a research; additionally it might
be used as a follow-up study (16). Case control association studies (CCAS) rely on genetic,
physiological, biochemical or pharmalogical evidence to determine the involvement of a specific gene
to the analyzed phenotype. CCAS have to consider some clinical observation to get good results with
low heterogeneity. They have to consider that AN patients and BN patients prevalence is higher in
females than in men. They have to consider that the manifestation periods of these illnesses are
predominantly expressed during puberty and adolescence and that 30 % of AN patient later on
develop BN. And at last they have to consider that Anorexia Nervosa has high comorbidity rates with
several mental illnesses (17). The complexity of an eating disorder and the results of other complex
disorders suggests that the genetic basis for AN is likely to be polygenic and that the effect size of a
predisposition of an allele is likely to be small. That is why the focus in case control association
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studies has been on neurobiological disturbances that still persist after recovery. CCAS investigated
the serotonergic, dopaminergic systems and weight regulation systems (17).
3.4.1 Serotonergic system
Serotonin (5-hydroxytryptamine; 5HT) is involved in a broad range of function. It is implicated that
serotonin is important in the regulation of appetite and eating behavior. Serotonin reuptake
inhibitors have been used as a treatment component for AN (13). Serotonin is a neurotransmitter
and in that system tryptophan hydroxylase, the 5-HT transporter and 5-HT receptors are included.
The suggestion that serotonin was important in eating disorders came from the establishment that
after long-term weight restored patient of AN the levels of cerebrospinal 5-hydrroxyindoleacetic
were still elevated. The focus on the serotonergic system resulted in some statistical significant
results nonetheless multiple studies failed to provide significant results. This is manly due to a small
sample size that might not achieve enough statistical power. The few acceptations that did get
statistical significant results will be discussed.
The study from Brown K.M et al focused on the susceptible region found on chromosome 1 and tried
to further establish its relationship with AN. The research groups they used consisted of 226 AN
patients and a control population of 687 age-matched healthy volunteers. The group of AN patient
consisted of 122 of the restricted AN type and 104 of the binge-purging AN type. In total the study
tested 176 SNPs for their association with AN. They discovered 4 polymorphisms in the HTR1D gene
and 6 polymorphisms in the OPRD1 gene. From the HTR1D gene 2 SNPs showed significant statistical
evidence of an association with AN. And in the OPRD1 gene 3 SNPs showed some degree of
association with AN. The marker rs569356 showed a significant association with both the restricting
and binge-purging AN type. And at this marker the G, T genotype seems to be more common in the
disease population compared to the control population (figure 2) (18). The exact mechanisms in
which these findings impact the biological basis of AN is unclear. Since there is no evidence that
suggests that these polymorphisms have biological consequents for the activity of the opoid delta
receptors or the serotonin 1D receptor (18).
Figure 2. The figure shows the genotyping that is done for marker rs5699356 on the ORPD1 gene. A significant
difference is seen between the frequencies of cases and control (p=0.0011). The frequencies of the C.T was
increased in AN patients compared to controls. This finding is complemented by a decreased frequency of T.T
genotype in An patients compared to controls.
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An alternative study focused on the 5-HT2A receptor as a strong candidate for an association with
AN. The A allele of the -1438 G/A polymorphism in HTR2A has been investigated in some case control
association studies. But these studies do not present the same results, so it merely gives us an
indication that 5-HT2A is a candidate gene (17).
3.4.2 Dopaminergic system
The dopaminergic system has been associated to be implicated in multiple symptoms of AN. For
example symptoms like: repulsion to food, hyperactivity, weight loss, OCD and the distortion of body
image. Reduced metabolites were found in the cerebrospinal fluid in AN patient during AN periods
and after recovery. This implicates that dopamine dysfunction can account for some symptoms.
Most CCAS in the dopaminergic system is performed on the D2 and D4 receptor genes (13, 17.)
Multiple studies focused on the D2 receptor. The DRD2 is a trans membrane G protein Linked
receptor that is most prominently found in the striatum, nucleus accumbens and the olfactory bulb.
The DRD2 is previously investigated in numerous other psychiatric diseases. These studies showed
that there are certain functional polymorphisms of the DRD2 gene that affect its function, for
example the transcriptional efficiency. Bergen et al executed a CCAS with the AN-ARP and data set
consistent of 196 eating disorder families. The -141 C/- insertion/deletion was statistical significant
association with AN. This Polymorphism has shown to affect transcriptional efficiency. Similarly the
SNPs 939Y and 957Y showed statistical significance and has been shown to exhibit decreased stability
and reduced translation of the DRD2 transcript in vitro (19, 20).
The study of Bachner-Melman R. et al focused on the D4 receptor. The DRD4 receptor is thought to
play an essential roll in satiety. Of this receptor several functional polymorphisms are known. These
are an exon 3 repeat, a promoter region tandem duplication and a promoter region SNP. The study
examined a group of 202 trios (AN daughters and parents) and tested these Polymorphisms. They
showed significant association between the C-521T SNP. This means that the C-allele that is less
transcriptionally active is transmitted to AN subjects (21).
3.4.3 Weight regulation system
The first research on this subject was on Leptin. Leptin is a key hormone in the regulation of the
energy balance and is involved in behavioral alterations associated with changes in energy storage.
Low leptin levels are associated with a low fat mass or BMI. The hypoleptinemia discovered after the
recovery of an eating disorder suggests that reduced leptin might be a trait for eating disorders.
Nevertheless CCAS have not confirmed this association.
Other CCAS did confirm associations between the weight regulation system and AN. One such
association is between BDNF (Brain-derived neurotropic factor) and AN. BDNF is a key modulator
that regulates synaptic efficiency in neurotransmitter pathways associated with AN. Changes due to
the modulation of these neurotransmitters might be involved in the pathology of AN. BDNF is
expressed in the hypothalamic nuclei that is associated with weight regulation and feeding control. In
acute AN patients they discovered reduced peripheral BDNF concentrations suggesting that it is
involved in the severity of the disease. Ribasés M. et al showed in a group of 143 eating disorder (ED)
patients that there is in fact an association between the Val66Met SNP and ANR (Anorexia nervosa
restricted type)(13, 17,22).
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3.4.4. 182 candidate genes
Pinheiro A.P. et al performed a slightly different case control study. 182 candidate genes were
selected from 3 linkage studies. The 182 were susceptible for AN based on the location under
reported linkage peaks. Pinheiro A.P. et al tested each SNP individually as well as the haplotypes of
the genes. They tested a group of 1.085 AN patients, 687 patients were the no binge eating type and
421 were the ANR type, the control group consisted of 677 individuals. However the study did not
show any significant results, underscoring the importance of large samples to detect genotype
differences (66).
3.5 Summary human genetic studies.
Human genetic studies discovered associations between Anorexia Nervosa and the genes DRD4,
DRD2, 5-HT2A, BDNF, HRTD1 and ORPD1. Nonetheless most of the case control association studies
failed to present significant result. This is largely due to a lack in statistical power but also because of
a lot of heterogeneity in the case groups. That is why within the last 3 years the GWAS studies are
the future for this kind of research. In GWAS a large number of genetic markers might be used.
GWAS rely on the assumption that linkage disequilibrium (LD) enables one SNP to act as a surrogate
marker for association sequence variations in the same region (23). The genotyping of a large
amount of SNPs gives a GWAS a good chance to find at least one SNP that will be in a LD where there
are also common functional variants present. The GWAS can lead to more associations found for AN,
however subphenotyping and endophenotyping will still be very important to reduce heterogeneity.
Besides that within human genetics there is no control over the environment of the subjects. This
control is evident in animal models. Because of the control over the environment and the fact that
genetic modification can be made in animals there is likewise genetic research in animals for AN.
4.0 Animal models
Rodents and humans share a lot of genetic information, this is named homology. Because of this
homology we can investigate the genetic background in rodents and compare it with humans. Animal
models are very convenient because we cannot perform all research in human because of ethics. In
rodents, especially in mice we can perform a lot of research with the benefit that mice can be
genetically modified. That is why there are numerous mice models in which AN is investigated;
however the complexity of Anorexia Nervosa makes it hard to investigate AN in mice. There is no
Anorectic mouse available that is why distinct characteristics and mechanisms of Anorexia Nervosa
have to be investigated in distinct setting with genetically distinct mice. The distinct mice models that
leaded us to a larger understanding of AN will be discussed in the next section. The results that came
from these models will be discussed just as the benefits and failings of the animal models. The
knowledge we gained from these models is important for the investigation of rigid behavior as well,
since rigid behavior is present in animals as well.
4.1 Activity based AN (ABA) mouse model
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An model that is a suitable option to investigate anorexia behavior in rodents is the Activity based
anorexia model. The model reproduces AN behavior through restricted food intake. The restricted
food intake leads to characteristics seen in AN patients such as: restricted food intake in the presence
of hunger, weight loss, the drive for activity, and the physiologic consequences of malnutrition (24).
Mice that are in a restricted feeding schedule and have access to a running wheel will show excessive
wheel running. This results in weight loss, amenorrhea and even death. Normally when mice are in a
limited food access model they will consume more food during the limited time they can eat,
however with increased activity (due to the running wheel access) the animals will no longer
consume more food. Epling and Pierce et al suggested a role in the evolution for this behavior. In a
time of food scarcity, animals will either hibernate and conserve there energy or the animal will
become mobile and migrate to search for food (25). Certain studies on this subject pointed to leptin
as an important regulator for this activity based AN model (26, 27). Current studies used this ABA
model to investigate hyperactivity in distinct mouse strains.
4.1.1 Association of leptin and Activity based AN mouse model
Leptin was one off the first genes associated with the hyperactivity during the activity based AN
mouse model. Leptin is thought to be a parameter in AN patients although there is no evidence for a
disturbance in the leptin gene in AN patients, however low leptin levels might still interfere with the
disease. In the ABA model the rodents will demonstrate semi-starvation hyperactivity (SIH). The
hyperactivity demonstrated during the ABA model can be reduced in rodents with peripheral leptin
treatment. Rodents were even rescued from ISH with peripheral leptin treatment (26). This gave rise
to some controversy because leptin is thought to limit food intake through the stimulation of proopiomelanocortin (POMC)/cocaine-amphetamine-regulated transcript neurons and inhibition of
neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons in the arcuate nucleus of the
hypothalamus. The study of Hillebrand et al showed that leptin reduces wheel running, however
leptin decreased food intake and increased thermogenesis as well (Figure 3). These observations are
confirmed in human AN patients. The AN patients demonstrated the most hyperactivity when leptin
levels were on the lowest. The variances between human individuals even correlate with the
variances in there hyperactivity. There might be role for leptin in the treatment for AN by reducing
hyperactivity, however leptin cannot be used for that purpose because of its role in food intake and
thermogenesis (27). The leptin pathway with POMC and AgRP is still thought to be important in AN
and multiple studies suggested a role for these genes in the disease.
4.2 Food restriction models
Just as the Activity based Anorexia model in the food restricted model the food will be available for
rodents during limited time. The food limitation mimic certain neuroendocrine changes observed in
AN, though the large limitation of this model is that the restriction to food is not voluntary. Voluntary
food restriction is present in AN patients and cannot be exactly mimicked in an animal model. The
food restriction may benefit or impair cognition and motor performances depending on the degree
of restriction. Diet restriction of 60 % will result in improved eight-maze performance (model to
investigate cognition in rodents). When rodents are restricted to 40 % of normal food intake it will
result in a very high mortality rate, meaning that distinct diet restrictions give opposing outcomes
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(28). Due to food restriction models changes in brain catecholamine’s are linked with semi-starvation
and malnutrion is linked with cholinergic changes. The involvement of the opiate system and the
food restriction provides insights in the role of the opiate system in AN and might help us in the
evaluation of weight loss on the serotonin, cholinergic, adrenergic and opiate system. The food
restriction is no more than a model for AN but not a true replica of the human disease and therefor
the results cannot be directly linked with AN (28).
Figure 3. The figure illustrates the results of rats during the ABA model. The rats were exposed to the ABA
model and were given leptin treatment or a vehicle. Leptin treatment reduced the Run Wheeling activity
significantly compared to the vehicle treatment. (26)
4.3 Genetic animal models.
The food restriction model and the activity based model are helpful in exploring which way different
neurotransmitter pathways are involved in weight loss. But they can not copy the symptoms of the
disease voluntarily. That means that there is no Anorexia Nervosa mouse in the world. However
currently there are certain genetic models in which certain symptoms of anorexia Nervosa are
present and in these models the genetic background of AN can be investigated. The search for genes
that are important in regulating food intake and satiety resulted regularly in the obesity phenotype
and not in the AN phenotype. There are several possibility’s why it was so hard to find genes that
could lead towards the AN phenotype. First of all it might be because an Anorexia Nervosa
phenotype animal will die by starvation and malnutrition. Secondly the AN phenotype might lead to
infertile animals, so the genetic mutation leading to the AN phenotype will not be passed on to the
next generation. And finally from an evolutionary prospective, there might be a lot of genes involved
in the anorectic pathway to prevent starvation when sources are scarce, in that view the defense
against weight loss is larger than the defense against weight gain. Accordingly no individual lesion
will lead to the AN phenotype (28). In recent years certain models are created to investigate the AN
phenotype and these will be discussed.
4.3.1 ANX/ANX
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The anx/anx mouse is the only spontaneous mouse mutation that leads to the anorexic phenotype.
The anx/anx mouse is characterized by a recessive anx mutation resulting in poor appetite. The mice
fail to regulate their food consummation and this results in reduced body weight, emaciated
appearance, body tremors, head weaving, hyperactivity, and uncoordinated gait. The phenotype
becomes apparent at day 5 to 8 after birth and the mice have reduced serum leptin levels from
postnatal day 8 due to a lack of adipose tissue. The mice die when they are around 3 to 5 weeks old
because of there genetic background (28, 29).The anx gene is mapped and is discovered on
chromosome 2 (32). In this anx/anx mouse the role of the hypothalamus and the hypothalamic
arcuate nucleus is investigated in numerous studies, due to the fact that the hypothalamus is thought
to regulate food intake.
In the hypothalamic arcuate nucleus two messenger molecules neuropeptide Y (NPY) and proopiomelanocortin (POMC) appear to play an antagonistic role in the regulation of food intake.
Injections of NPY are thought to increase food intake and deficiencies in the melanocortin-4 receptor
results in obesity. Anatomic and pharmaceutical evidence suggests that the balance of those
messenger molecules regulates food intake (30). The abnormalities that are caused by the anx
mutation could be due to an imbalance in those messengers. The anx/anx mice display decreased
levels of POMC mRNA and decreased levels of Y1R and Y5R mRNA that inhibit POMC (Figure 4). The
POMC neurons showed an affected morphology suggesting atrophy and degeneration of this
population of cells (30).The imbalance might be explained by a lack in the development of the AgRP
neurons as well. With immunohistochemistry they discovered that in anx/anx mice the AgRP system
does not develop to the same extend as in normal mice. This might be a result of the low leptin
levels. Leptin is suspected to play an additional role in the development of other hormonal systems
and neuronal development. The low levels of leptin might possibly affect the development of the
AgRP neurons (31). Altered NPY and AgRP peptides distribution in neurons is discovered as well.
Accumulation of NPY and AgRP in anx/anx mice at the cell bodies and decreased levels of those
peptides at the terminals were seen. This suggests improper release of these peptides (32).
Figure 4. The figure illustrates the altered mRNA levels of the anx mice compared to control mice (colored
bars). POMC, the Y1R and Y5R showed significantly less mRNA levels in the anx mice compared to the control
mice (30).
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The anx mutation is associated within a locus on chromosome two, the exact mechanism by which
manner this results in the AN phenotype is not yet known. That question might be answered after
sequencing the anx gene and studying its functions. The studying of the anx gene will additionally
provide more answers of exactly how the hypothalamus regulates food intake and which systems are
important for the development of these systems or eating disorders in general. The anx/anx mouse
has a single large shortcoming; the mouse dies around week 4. This large shortcoming makes the
investigation of the anx/anx mouse extremely limitated. Therefor it can not be used in any behavioral
testing method. Gene Knock out mice models might life prolonged compared to the anx/anx mouse
and do provide the option to use them for behavioral testing methods.
4.3.2 Gene knock-out models
Distinct gene-knock out models exist that are intended for the Anorectic phenotype. Several of those
are the result for a search in genes that influence food consumption and the energy pathway. A few
were coincidently found and resulted in the anorectic phenotype. In that kind of a gene- knock-out
(KO) model one gene is infected and gives rise to the phenotype. The influence of one gene can be
investigated. Nonetheless the negative side is that when one gene is Knocked-out and it does not
provide an obvious phenotype the model is not inconclusive (28). Several KO exit and it is important
to know what they contributed to our understanding of AN.
Dopamine deficient mice
The dopamine deficient mice do not have a dopamine gene in his genome. Dopamine is important in
natural reward functions and in the learning of goal directed behavior. Food is included in the natural
reward functions and dopamine antagonists have shown to impair the food reward of Dopamine.
Dopamine deficient mice are born normal but will rapidly become hypoactive and hypophagic. They
will die due to lack of nutrients after 3 to 4 weeks. The lack of nutrients is not caused by motor
deficits in the mice, since the mice appeared to move as much as normal mice and their ingestive
behaviors in response to novel food were initially the same as those of normal mice. However, they
only consume small amounts of food even when it is very palatable and accessible. Restoration of DA
production within the caudate putamen restores feeding in DD mice (28, 33). The Dopamine
deficient mice provides insight in the role of dopamine on feeding behavior and the motivational role
of food in AN.
M3 receptor KO
Mice deficient in the M3 muscarinic receptor (M3R−/−) display a significant decrease in food intake,
reduced body weight and peripheral fat deposits and very low levels of serum leptin and insulin (28,
34). M3 is a muscarenic receptor; it is a G-coupled acetylcholine receptor important in several
neuronal systems. The M3 receptor subtype is extensively expressed throughout the central nervous
system including the diencephalons. During the first postnatal week the mice look normal, however
at the end of week two or three they start to show a decrease in body weight and the mice
consumed 30 % calories a day less than the control mice. This implied a role in food regulation for
the M3 receptor. Food regulation is strictly regulated in the hypothalamus. In wild-type mice the
muscarinic receptors were located in high density in the hypothalamus. In the M3 receptor KO mice
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the density of muscarenic receptors in the hypothalamus was 50 % lower, indicating that the M3
receptor is expressed in high levels in this brain area (34). In the Hypothalamus several critical
neuropeptides are present and they were altered in there expression (discussed in section in 4.1.1
and 4.4.1). Hypothalamic AGRP mRNA levels were increased, whereas hypothalamic POMC mRNA
expression was reduced in M3R−/− mice as compared to wild-type mice. These observations are in
agreement with the hypophagic characteristics seen in the M3 deficient mice. Melanin concentrating
hormone (MCH) (discussed in the section MCH KO) was also affected in these mice. (Figure 5) The
study of Yamada M. et al proposes that the cholinergic pathway acts downstream of the
hypothalamic leptin pathway and upstream of the MCH system (Figure 6) (28, 34).
Figure 5. The figure illustrates the expression of hypothalamic neuropeptide mRNAs in wild-type and M3R-/mice. Hypothalamic AGRP mRNA levels were increased, whereas hypothalamic POMC mRNA expression was
reduced in M3R−/− mice as compared to wild-type mice. MCH levels were reduced in M3R -/- mice compared
to wild type mice as well (34).
Figure 6. The expression of M3 is located primarily in the lateral hypothalamus (LHA), suggesting a role for M3
in the responsiveness of the MCH pathway that lies downstream of the hypothalamic leptin pathway.
Cholinergic input is coming to LHA from the laterodorsal tegmental and the tegmental nuclei. MCH neurons
receive synaptic input from both the AGRP/POMC system of the arcuate nucleus (34).
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MCH KO
MCH has a central role in feeding behaviour and promotes feeding behaviour. The levels of MCH
mRNA rise when the body is in starvation or when the mice are leptin deficient. MCH -/- mice have
reduced body weight and are hypophagic. The animals NPY and AgRP expressions were unchanged;
however POMC mRNA levels were suppressed. The investigations indicated that MCH is a critical
regulator downstream of leptin and the POMC system (Figure 6) (28, 35).
Corticotropin releasing hormone 2 (CHRC2) KO
The CHRH2 is important in the Hypothalamic–pituitary–adrenal (HPA) axis. This HPA axis is activated
by stress. The response for stress is the releasing of the Corticotropin hormone that interacts with its
receptors. This interaction is responsible for the release of adrenocorticotropic hormone (ACTH) into
the bloodstream. The HPA axis is involved in numerous mood disorders like anxiety disorders, bipolar
disorders and AN. Mice deficient for the CHRH2 have anxiety like behaviours and are hypersensitive
to stress. In addition the CHRH KO mice consume 75 % less food after a 24 hour period of food
deprivation compared to control mice (28, 36).
Contactin-1 gene KO
A mutation in the contactin-1 gene resulted in an ataxic and anorectic phenotype in mice. The
phenotype initiates at postnatal day 10. The animals die due to starvation at day 19. The mutation
displays some resemblance to the anx/anx mouse and that is why some investigators made a
Contactin-1 KO to investigate its role in feeding behaviour. Contactin-1 is an immunoglobulin like
adhesion/recognition protein. Contactin-1 regulates molecular interaction in the developing nervous
system and regulates synaptic plasticity in the hippocampus. The KO showed the same results for
NPY and AgRP neuropeptides as in the anx/anx mouse. The neuropeptides were accumulated in the
cell bodies of the arcuate nucleus and there was a drastic reduction of those neuropeptides in the
nerve terminals. POMC was decreased in these mice as well (37).
The mouse models provide a useful way to investigation of Anorexia. It already thought us a lot
about feeding behavior and which genes are important in this behavior. The combination of those
models and the correct experiments might lead to answers about the cause of AN, however the
models are not ideal for Anorexia. The mouse models cannot grab hold of the complexity in a whole
of AN. Especially the complex behavioral traits are difficult to examine in the mouse models. That is
why several researchers suggested a role for subphenotypes and endophenotypes to further
investigation the disease within humans and in mice.
5.0 Endophenotypes
Due to the lack of animal models that cover the whole disease, especially the psychiatric complexity
of the disease (section 2.0) several investigators have turned their focus on endophenotyping. They
make the switch from complex syndrome genetics to interspecies trait genetics to solve the genetic
substrates important for several behaviors. In complex diseases multiple genes make up the genetic
component of the disease. Those genes are hard to find in human studies due to heterogeneity.
Furthermore the environmental factors important for the development of the disease are not
controllable in humans. It is important to get a grasp on both of these factors. Linkage studies in
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human have not succeeded to get a grasp on both factors, mainly due to the lack of homology in
there patients group. In animals this control over genetics and environment is indeed possible,
however to investigate complex psychiatric disease a new approach is needed. The anx/anx mouse
and the gene KO mice will not be sufficient. An approach is the behavioral domain concept. This
concept focuses on naturally occurring behavioral domains and the variation between animals in
those behavioral domains. The endophenotype and the behavioral domain concept are relatively
new; however they look very promising to solve the distinct behavioral traits in AN. Both the
concepts provide a theoretical basis for an experiment on rigid behavior and its association with AN.
The behavioral domain concept and the endophenontype approach will be discussed in this section.
Besides that the validity of animal models of human behavior will be addressed as well (38).
5.1 Behavioral domain concept
In AN there is overlap with other mental diseases (section 2.0). The overlap results in overlap in the
underlining neurobiology. Because of this overlap the boundaries between different diseases are not
as sharp as we thought, resulting behavioral domains. There is a lot of overlap in AN compared with
BN, however there is also overlap between distinct diseases like schizophrenia, bipolar disorders and
psychoses. The overlap is for example present in a gene like BDNF. Polymorpisms in this gene are
associated with several diseases like schizophrenia, bipolar disorder, major depression, anorexia
nervosa and bulimia nervosa (Figure 7). Consequently the etiology of the disease is not directly linked
to our psychiatric diagnosis. That implies that the genes important for the disease are rather linked
to the behavioral domains underlining the disease than the psychiatric diagnosis of the disease (38).
Figure 7. The figure illustrates that the biological border of distinct psychiatric diseases like schizophrenia,
unipolar depression and bipolar disorder are not the same as the diagnostic borders. BPD stands for bipolar
disorder (38).
The behavioural domain concept is based on the idea of the endophenotype. The endophenotype is
the internal phenotype discoverable by a ‘‘biochemical test or microscopic analysis’’. A phenotype is
often an imperfect indicator of the genotype and for a specific phenotype there are genetic and
nongenetic factors that contribute to the phenotype. The complexity of the brain and the complexity
of psychiatric diseases makes AN a difficult to study disease, Therefore endophenotypes might be the
outcome. Those endophenotype is not specific for one disorder; it overlaps and affects multiple
disorders. Thus the endophenotype concept might simplify the phenotypes what might result in less
heterogeneity in the genetic analysis (Figure 8).To successfully use the endophenotype concept, a
phenotype should have certain defined characteristics. There are multiple characteristics for the
endophenotype. First of all an endophenotype is associated with the illness in the population.
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Secondly the endophenotype is heritable. Thirdly the endophenotype is present whether the
individual’s illness is active or not active. Fourthly within families the endophenotype and illness cosegregate (39).
Figure 8. A decrease in the complexity of the phenotype and the genetic analysis will lower the amount of
genes involved and will provide more homogenous results than the investigation of complex phenotypes (39)
In complex disease like AN the environmental interactions with the genotype is off eminent
importance. To successfully study these interaction animals can be used, however animals do not
develop paranoid ideas or suicidal thoughts. To investigate the right behavioural traits they have to
be complementary between the animal and the human. Such behavioural traits are conserved during
the evolution and should be based on common survival mechanisms. Adaptations during the
evolution originated in proper behavioural responses to the external environment. In rodents the
basic behavioural traits are found in humans as well. These behavioural traits include anxiety,
activity, cognition and social interaction. Moreover these behavioural traits are distorted in complex
psychiatric disease and are therefore important traits to investigate (Figure 9).
Figure 9. The behavioural domains contribute to multiple complex diseases. The behavioural domains are
linked to susceptible genes and this relationship will be more direct than the link between the genes and the
complex psychiatric disorder. The environmental factors that might contribute to the development of
psychiatric disease or specific behaviours can be controlled and manipulated in rodents so the genetics of
behavioural traits are more easily discovered than in humans (38).
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Although the concept of the behavioral domains and the endophenotype look promising there is still
skepticism towards this approach. This skepticism is based on the validity of animal models of human
behavior. To make sure the results are valid to the investigated genotype-phenotype relation in
animals and humans the gene should affect the phenotype in both species. Thus the gene should be
conserved over the course of the evolution (Figure 10).
Figure 10. The figure illustrates the theory to investigate behavioral traits of complex psychiatric disease. The
small circles in the left part of the figure are the behavioral domains that are currently of interest for the
investigation of psychiatric disorders. The right part of the figure shows where there has to be homology
between human and animal to get valid results.
Besides the concept of endophenotyping the concept of domain interplay has also been suggested
for the research of psychiatric diseases. The domain interplay is based on the fact that multiple
psychiatric diseases show co-morbidity. For example the co-morbidity between anxiety and autism,
these disorders overlap. When you use several behavioral domains at the same time you can make
your research even more specific (Figure 11). For example a genetic model that focuses on social
interaction deficit and anxiety. Consequently this generates a focus specifically on the overlapping
domains of psychiatric disorders, which could lead to the pathogenetic process or system responsible
for those overlapping domains (ref 40). The domain interplay concept is not yet used for actual
research; however it might provide highly specific results.
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Figure 11. The figure illustrates the theory that when two ore more deficits or behavioral traits (interplay
domain) are investigated it could lead to a pathogenetic process underling that specific interplay domain. In
this figure OCD, social anxiety and autism are used as an example (40).
5.2 Forward genetics and QTL mapping
The use of forward genetics, Quantitative trait loci mapping and chromosome substitution strains are
tools to investigate the proposed behavioral domains. When these tools are combined with the
correct behavioral investigating methods they provide a sufficient way to investigate behavioral
domains of psychiatric diseases. The use of forward genetics and QTL mapping has resulted in
chromosomal loci associated with the behavioral domains of anxiety, activity and depression.
However it has not yet leaded to the discovery of chromosomal loci associated with rigid behavior.
This is partly due to the lack of an appropriate investigating method in mice for this behavioral trait.
In this section the tools to investigate the behavioral traits will be described. And the examples of
activity, anxiety and depression will be used to show the progress due to these tools. In section 6 the
implications for these tools and the investigation of rigid behavior will be discussed.
With forward genetics we look at a specific phenotype and try to solve which genes are important for
the phenotypical behavior.
QTL mapping is a genetic mapping approach which tries to find the DNA segment responsible for the
phenotype; this approach uses inbred mouse strains. Distinct mouse strains have different physical
and behavioral phenotypes. The strains differ from each other in characteristics like anxiety, activity
and cognition. In a strain the whole population has identical genetics in comparison to each other. In
that way you can easily compare two distinct strains and discover which genes are responsible for
the phenotype. From the both inbred strains that the data of there DNA should be present as a result
the distinct DNA loci can be determined. SNPs are used to determine which locus belongs to which
strain. The two inbred strains are crossed resulting in F1 animals (44). Those will be crossed as well
resulting in F2 animals. The F2 animals will be genetically mapped and phenotypically mapped to
determine which segments are important for the behavior.
For QTL mapping the use of chromosome substitution strains (CSS) will be of great importance. QTL
mapping uses two inbred strains with distinct phenotypes for a specific behavior. The chromosome
substitution strains are created of these strains containing all the chromosomes of one strain except
for two homologous chromosomes of the other strain. For example chromosome 1 is of strain 1 and
the other chromosomes are from strain 2. With the CSS a link between a specific behavior and a
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specific chromosome can be found. The chromosomes of strain 1 that give the best link with the
phenotype might be further investigated. This might be done by crossing the CS strain of the
chromosome of interest with strain 2 to generate F1 and F2 progeny. The F2 progeny are tested for
there association with the phenotype. The progeny will be mapped to find the homologues
sequences that belong to strain 1. At these sequences lie the susceptible genes important for the
investigated phenotype (42, 43). In the next section some studies important for AN that are
performed with QTL and CSS will be described.
5.2.1 Activity
Activity is an important behavioral domain that is apparent in AN patients. The generation of motor
activity is under tight neuronal control. Which Neurobiological mechanisms contribute to motor
activity is under the investigation. For AN patients it might be of great importance to find which
genes contribute to hyperactivity. The study of the behavioral domain hyperactivity is executed in
several ways, for example with a home cage model (45, 46) or with the Activity based Anorexia
model. Both models and outcomes of studies with these models will be discussed.
Multiple studies tried to discover association between chromosomal loci and activity, indicating for
potential QTLs on multiple chromosomes, however most of these studies addressed chromosome 1
for potential QTLs. To identify where on the chromosome the QTL for this trait lays Kas M.J.H et al
proposed an experiment with CSS derived from the C57BL/6J (host) strain and the A/J (donor) strain.
The animals were tested in a home cage environment to asses motor activity levels. The home cage
was chosen to get minimal human interference enhancing the behavioral resolution. Besides that
motor activity can be assessed independent of sheltering preference (45, 46). When the C57BL/6J
mouse was compared to the CS strain1 mouse they discovered lower motor activity levels in the CS
strain1 mouse. Motor activity was monitored with a video tracking system above the home cages. To
map the genetic QTL responsible for motor activity the C57BL/6J mice were crossed with the CS
strain1 mice resulting in the F1 progeny. The F1 progeny were crossed with each other resulting in
the F2 progeny. The resultants were 82 mice that were monitored in the home cage for 3
consecutive days. The mice showed lower basic activity compared to the C57BL/6J mouse. To map
the QTLs 15 dispersed SNPs were used. One LOD score showed a peak at 79 970 253 bp (Figure 13)
(46).
Figure 13. The figure illustrates the LOD scores for the F2 progeny of the CS strain1 animals. The Dots represent
the 1-LOD support interval in which further genetically fine mapping was performed (46).
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Data of 2445 genetically heterogeneous mice were used to further investigate the QTL. The resultant
was a QTL region of 312 kb containing the A830043J08Rik gene and a U6 small nuclear element. In
the A830043J08Rik gene they found a nucleotide deletion in its 3’0 UTR compared to the genome
sequence of the C57BL/6J mouse strain. This suggests that the A830043J08Rik gene could be
important for motor activity (46).
Disturbed motor activity can be addressed within an ABA model as well. The study of Kas M.J.H et al
used an ABA model. They used C57BL/6J mouse as the host strain and used the A/J mouse as the
donor strain. After CSS analysis they discovered disturbed running wheel activity in CS strains 4, 12
and 13 when the mice were exposed to a restricted feeding paradigm (Figure 14).
Body weights were assessed by Kas M.J.H et al as well. They obtained the lowest body weight in CS
strain 12 and 13 after a three days restricted feeding paradigm. The result suggests that there are
important QTLs for this behaviour on chromosomes 4, 12 and 13 what is consistent with several Loci
obtained from studies in humans (16, 47).
Figure 14. The figure illustrates the disturbed Wheel running. The disturbed wheel running was assessed in the
CS strains compared to the C57BL/6J. Chromosomes 4, 12 and 13 showed the most significant association with
the disturbed wheel running behaviour.
5.2.2 Anxiety
Anxiety related behavior is an important domain that is apparent in AN. Anxiety and anxiety like
behavior is easily tested in an open field assay. In the assay the mouse will be set into a big round box
that is open at the top. The amount of time the mouse will spend at the sites or in the middle of the
box are used as parameters for anxiety behavior. This is based on the fact that a mouse spending
more time in the middle of the box is less afraid than a mouse that spends all of his time at the sides
of the box. The middle of the box is naturally scarier because the mice can be seen by predators,
however interpreting the results is hard since mice are very curious animals as well.
For the anxiety like behavior different QTLs are obtained on Chromosome 1, 12 and 15. The studies
on anxiety like behavior used divergent mouse strains like the C57BL/6J and the A/J that present
divergence in their phenotypes as well. However the study of Bailey et al found a QTL for this
behavior using two strains (C57BL/6J and C58/J) that are relatively similar genetically and
phenotypically. They made this choice based on the expectation that the closely related inbred strain
would decrease the potential interaction with the mutation and the background of the C58/J (48).
Secondly, it is not necessary for two parental strains to differ in phenotypic values associated with
complex traits to be able to identify QTL in their intercross progeny (49). Since novel combinations of
two strains might even lead to extreme phenotypes (48). The study of bailey et al tested the
C57BL/6J, the C58/J and their F2 progeny. QTLs associated with time spend in the centre of the box
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were obtained on chromosome 1, 8 and 13 (Figure 15). The genetic background of the F2 progeny
was tested for homozygosis at these QTL. Mice that were homozygote for C57BL/6J at Chromosome
8 spend less time in the middle than the other F2 progeny mice, however at chromosome 1 and 13
homozygote mice for C58/J spend less time in the middle than the other F2 progeny. Association
between the QTL and other obtained QTLs were also present in certain other studies. However the
obtained QTLs are large and still need elucidation (48, 49). In the regions of the QTL on chromosome
1 the RGS2 gene is included. The RGS2 gene is thought to affect anxiety like behavior. RGS2 is widely
expressed in the brain. Homozygote mutations in the RGS2 gene compared to a C57BL/6 mouse
showed that the mutation makes mice more anxious. This might be one of the components in the
QTL important for the phenotypical behavior (50, 51).
Figure 15. The figure illustrates the LOD scores of the percent time spend in the centre are shown, with high
LOD scores for Chromosomes 1, 8 and 13 (48).
5.2.3 Depression
Depression related behaviour is apparent in AN patients. Depression is mainly manifested in the
inability to cope with stress. The inability to cope with stress is tested in several distinct tests, like the
tail suspension test or the forced swimming test. These tests are mainly used to test anti-depressants
because they are rapidly executed. Besides that the tests are sensitive to various factors that are
influenced by depression or influence depression, like changes in food intake (52).
The research with the tail suspension test or the forced swimming test made a big contribution on
the development of anti-depressants. Several studies have been performed for anti-depressant.
Additionally the genetic background for these tests has been investigated. QTL mapping of the F2
progeny derived from the C57BL/6J and the C3H/He were used. QTL mapping showed loci for the Tail
suspension test (TST) and the forced swimming test on chromosome 8 and 11. Genome wide
interaction linkage revealed GABA(A) receptor subunits as possible candidates for genes linked to the
tests (53). A similar study conducted with only the TST revealed the Gabra3 gene, a subunit of the
GABA(A), as a possible candidate. Further investigation with an agonist for this subunit resulted in
less immobility during the TST and raised the suggestion that Gabra3 could be a good target for antidepressant drugs (54).
QTL mapping of behavioural traits has in the current years leaded to associations with genes and
chromosomal loci. However these studies are not yet conducted for rigid behaviour, while rigid
behaviour is very important in the outcome of AN. Further investigation into the genetic background
of rigid behaviour is needed and forward genetics; QTL mapping and CSS are tools that will help to
discover important loci and genes.
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6.0 Rigidity
As discussed in section 2 Anorexia Nervosa co-morbid with different diseases. Approximately 50 % of
AN patients suffer from Obsessive Compulsive disorders. Even the AN patients that do not suffer
from OCDs demonstrate elevated levels for these diseases and other anxiety related diseases. AN
patients have higher levels of Childhood rigidity and perfectionism than BN and than normal people.
Childhood obsessive-compulsive personal traits demonstrated a high predictive value for the
development of an eating disorder. Subjects of eating disorders that reported perfectionism and
rigidity in childhood demonstrated co-morbid with OCD later in life (55). Rigidity and perfectionism
increase by onset of Anorexia Nervosa. The obsessional behaviors seen in AN patients will increase
with weight loss and is seen to decrease with weight gain. The rigidity to be rule bound and the
striving for perfection can facilitate the restriction in food intake and the control of appetite. This
behavior could make the Anorexia patient feel like they are in control (56).
As seen above rigidity is an important part in the maintenance and the outcome of AN (see also
section 2). However what is rigidity. Rigidity means stiffness or inflexibility, the inability to change or
a person’s resistance to change as a personal trait. AN patients show elevated levels of rigidity and
rule bound behavior what makes rigidity an important endophenotype to study. Certain neuronal
brain areas and neurotransmitter systems are associated with rigidity. The exact mechanism of rigid
behavior is not yet known. Set shifting tasks in humans and animals might lead to answers, however
the test methods are not yet perfected. In the next section what is known of the biological
components of rigid behavior will be discussed just as the methods that leaded to this knowledge. As
a result of this knowledge I want to propose an experiment to investigate rigid behavior in mice in
section 7.
6.1 Neuronal regions
For rigid behavior there are certain brain regions involved. The brain regions need to integrate a lot
of information to plan the correct behavior for the situation a person is in. The brain regions and
circuitries likely to be involved in rigid behavior will be described in this section.
6.1.1 Frontal association cortex.
Functional deficits due to frontal lobe lesions are diverse and devastating. This part of the brain is
important in the maintenance of a person’s personality. It does so by integrating complex sensory
and motor cortex information and information from the association cortexes. The resultant is a selfimage in reflection to the world around the person and the ability to plan complex behaviors and
execute them. Lesions in this area might even result in changes in behavior or character of the
person involved. Multiple cases of frontal lobe lesions have been reported and these cases show
deficits in a wide range of cognitive disabilities, such as preservation, disordered thought, impaired
restraint, inability to plan appropriate action. Deficits of these abilities are also apparent in people
with Anorexia, such as planning of the appropriate action. The appropriate action for AN patients is
to start eating differently, but even when they know that they have to eat differently they still
cannot. An easy way to test the cognitive function of the frontal lobe is with the Wisconsin Card Test.
This test is a widely used to test rigidity in mice and human.
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6.1.2 Limbic system
The limbic system is important for the rigid behavior seen in AN patients as well. The limbic system
controls the emotional behavior. The experience of emotion has a powerful influence on other
complex brain functions, including the neural circuitry responsible for rational decisions and
interpersonal judgment that guides social behavior. Clinical observation indicated that the amygdala,
prefrontal cortexes and the striatal and thalamic regions are involved in such processes. The limbic
system is important in the guidance of goal-directed behavior as well. They regulate the rewarding
effects of natural agents such as food. Deficits in the rewarding capacity of food might lead to dietary
restriction seen in AN. Besides that the limbic system is important in associative learning in which the
hippocampus is important too. Impairment in associative learning can result in inflexibility or rigid
behavior.
In the study of Zastrow A. et al they illustrated that multiple regions are associated with cognitive set
shifting abilities and behavioral set shifting abilities. The associations were obtained with eventrelated functional MRI. The AN patients showed higher error rates than the healthy control group.
And they showed reduced activity in multiple brain regions such as, the left and right thalamus,
ventral striatum, anterior cingulate cortex, sensorimotor brain regions, and cerebellum. The results
imply a hypo activation in the ventral anterior cingulate-striato-thalamic loop that is involved in
motivation-related behavior. In contrast, anorexia nervosa patients demonstrated predominant
activation of frontoparietal networks that is indicative for effortful and supervisory cognitive control
during task performance (57). This implies a role for the prefrontal loop and the limbic loop of the
basal ganglia circuitry for the obtained rigidity in AN patients.
6.2 Set shifting
To test inflexibility and rigidity there are several tests that might be executed. The tests are mainly
set shifting tests in which the brain is tested in its ability to cope with changes. Several of these tests
will be discussed.
6.2.1 Wisconsin card sorting task
The Wisconsin car sorting task (wcst) is a test in which extradimensional (EDS) and intradimensional
(IDS) shifts can be measured. In the test the examiner will put 4 cards before the test person. The
test person has to put the appropriate card in front of the stimulus card based on the sorting rule, for
example the color of the card, the shape of the card or the number on the card. After 10 consecutive
correct responses the rule will be changed and the person has to shift to another sorting rule. The
rule can be changed extradimensional, for example a change from shape to color. The previously
irrelevant dimension is than the stimulus. Intradimensional shifting is possible too, two novel stimuli
will be presented nonetheless the sorting rule will stay similar. Mastering the EDS is more difficult
than mastering the IDS. Mastering the EDS requires cognitive flexibility, associative ability and
selective attention (61). The WCST is used to test cognitive flexibility, but is also widely used to test
frontal lobe dysfunction and its severity. The amount of errors made in the test and the number of
categories completed in the test is the degree of set shifting.
Author: H.C den Boer
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6.2.2 Brixton task
In the Brixton task the participant must predict the movement of a bleu circle across ten circles. The
movement pattern of the bleu circle will change throughout the test and the number of incorrect
errors will be used as a measure of set shifting ability (58, 59).
6.2.3 The Trail making test
In the trail making tests the person will be presented with an 18-item alphabetic sequence and then
an 18-item alphanumeric sequence is presented and these are connected in order. The letters and
numbers will then be mixed up on a computer screen so the participant will have to alternative
between these in sequence (58, 59).
6.2.4 Picture set test
During the picture set test the subject is presented with a series of 18 trials. The subject is shown
four objects on a computer screen and has to determine which object is the odd one. To apply the
odd one the subject has to discover what the sorting rule is. The set shifting performance will be
measured by the amount of errors made (59).
6.2.5 Human set shifting studies
The implication that set shifting is a measurement of rigidity and that this is an endophenotype for
AN has to be proven by experiments. Certain studies investigated this implication with the above
discussed set shifting tests. Roberts M.E et al tested the implication with AN and BN patients. In his
study 270 women participated, of these women 98 had an eating disorder, of which 35 were AN
restricted type. Another 30 women were fully recovered AN patients, and the control groups
consisted of 50 sisters of the ED women and 88 healthy control. The WCST demonstrated that ED
patients made more errors and completed fewer categories compared to the healthy compare
group. Similar results were present by the brixton task and the tail making task. The recovered AN
group demonstrated poor WCST ability compared to the Healthy control group, however they did not
differ in the other tests. The unaffected sisters demonstrated poor set shifting results compared to
the healthy group too (Figure 16) (58). Individuals with poor set shifting were further compared with
individuals with intact set shifting abilities to obtain an association between set shifting ability and
the duration of the illness, self-reported anxiety and depression. The results demonstrated that
individuals with poor set shifting had a significantly longer duration of the illness and scored
somewhat higher on the self-reported anxiety and depression. This suggests that poor set shifting is
an important factor in AN and that it is apparent (in minor degrees then during the illness) in
recovered AN patients and unaffected sisters suggesting that it is not merely due to the nutritional
state (58)(59). Similar results were obtained in the study of Tchanturia K. et al. They tested AN
patients, recovered AN patients and a healthy control group. The AN patients performed significantly
less compared to the other subjects groups in the trail making test, the brixton test, and the picture
making test. The recovered AN group also demonstrated significantly more errors in the picture set
test and a perceptual task compared to the healthy control group. The recovered group was the
intermediate of the AN group and the healthy control groups. Accordingly it can be stated poor set
shifting is partially independent of current disease or nutritional state, even after weight gain the
Author: H.C den Boer
Page 26
subjects did not improve in there set shifting abilities (59).additionally the results suggest that set
shifting is an endophenotype for AN. Moreover it is not merely an endophenotype but a behavioral
domain too that is seen in other diseases like bipolar disorder, schizophrenia and other psychiatric
diseases (60).
Figure 16. The HC group showed significant less persons with poor set shifting compared to the other groups.
Recovered AN patients were intermediate compared to the Healthy control and the ED groups (58).
6.3 Rodent models
Further investigation to the genetic background of rigidity mice models might be used; however the
set shifting tasks for humans do not directly apply for mice. Therefor multiple models have to be
made to asses different types of set shifting in rodents. The mice models have to asses the correct
set shifting ability so the results might ultimately be linked to set shifting in humans. Set shifting
models will be discussed just as several results due to set shifting tests in mice.
6.3.1 Rodent models for set shifting
The rodent models mainly used looks like the WCST. The sand digging paradigm is such a model. The
sand digging method consists of a series of seven tasks. The tasks include a simple discrimination, a
compound discrimination, a reversal of the compound discrimination, IDS, EDS and a reversal of the
EDS. The test starts with a training session in which the rodents are trained to dig for bits of food in a
bowl with digging medium. In the training session the rodents have to habituate to the setting and
learn to dig for bait. They learn to form an attentional set. Following the training session the mice are
given three simple discrimination tasks. The bait will be placed in one of two bowls which differ in
texture, odor or medium. The rodents are trained to the criterion of six consecutive correct choices.
A choice is defined by the first bowl the rodent digs in. After the training the test will start. At the
start of every task the rodents has four discovery trials and after that the digging will be scored for
correct or incorrect. (61)
The first task the mice perform is a simple discrimination. In the task there is a pair of stimuli
presented to the rodent from which it has to choose the right stimulus. The stimuli are within one
Author: H.C den Boer
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dimension. After the six consecutive correct choices the compound discrimination begins. In the
second task a second dimension will be introduced. So in the first task there was only one dimension,
for example texture. However in the second task there are two dimensions, for example texture and
odor. The second dimension is an irrelevant dimension, so the rodent still has to discriminate
between two different textures. The third task is the compound discrimination reversal. Here the
texture is still the right dimension; however the correct texture is changed into the incorrect texture.
The IDS follows. Texture stays the right dimension only there are now two different textures to
choose from. Following is the intradimensional reversal. The correct texture is changed into the
incorrect texture. The EDS follows. New textures are introduced, however texture is not the relevant
dimension anymore, the relevant dimension will change for example in odor. The last of the series is
the extradimensional reversal in which the correct odor is changed into the incorrect odor. During
the within set reversal animals are learning an attentional set and will improve with consecutive
within set reversals. However the more the rodents are trained the worse they will perform in a set
shift. Accordingly the formation of an attentional set improves the performances from the compound
discrimination to the IDS. Hence EDS as a measure of set shifting is only valid when it is a decrease in
performance compared to the IDS (61, 62).
The san digging task described is in rats and cannot be exactly replicated in mice as in rats. The EDS
results compared to the IDS results did not show a decrease of performance in the EDS task. This
suggests that perhaps the sand digging paradigm is not valid to asses set shifting in mice. For that
reason Garner J.P et al proposed some modifications. Firstly they subjected mice to overtraining and
demonstrated that overtraining approved the performance on the CD, CDR and IDS and IDR
confirming the formation of an internal construct. Mice that are not able to make an internal
construct will show similar performances in IDS as in EDS. Overtraining thus confirmed set formation
making the EDS task a good measure for set shifting. Bisonette et al confirmed demonstrating that a
single IDS discrimination is insufficient to form an attentional set in mice. Thus Overtraining is an
approach to improve the test (Figure 19) (62, 64). Secondly they made several other modifications to
improve the test. Those were 8/10 training criterion instead of 6 consecutive correct choices. Testing
was done late during the light phase consequently the mice would be more alert than tested during
their night phase. Lastly they used the animals as there own control so they could control between
individual variations (62).
Figure 19. The figure illustrates that over trained mice perform worse in the EDS. In the graph you will see that
over trained mice need more trials to learn the EDS and make more errors learning the EDS (62).
Author: H.C den Boer
Page 28
Besides the sand digging paradigm there is a two dimensional computer graphic stimuli as well. The
choice making is performed by a nose-poke on a touch screen. Furthermore the seven series of tasks
will be performed on visual discriminations. The difference between this test and the digging test is
that the IDS and EDS are made within the same sensory system. While this is not the case for the
digging test in which the dimensions switches from texture to odor. The two dimensional computer
graphic stimuli could provide a more valid test for cognitive set shifting since it resembles the tests
for humans better than does the digging test (63). However as seen in Brigmann et al the test did not
lead to the decreased performance on the EDS task compared to the IDS. This might be due to the
visual stimuli in general and might imply that the visual stimuli are not able to generate the
formation of an attentional set. Though it might likewise be due to the lack of reversal tasks in
Brigmanns test as a result the animals were not able to learn the set formation (62, 63).
6.3.2 Prefrontal cortex and mice set shifting
In rats it is demonstrated that frontal lobe lesions led to behavioral deficits comparable of those
observed in humans. Lesions of the medial prefrontal cortex (MFC) areas reduced the formation of
an attentional set. Lesions of the orbital frontal cortex (OFC) impaired goal-directed behavior and
reversal learning. The lesion demonstrated correlation between the prefrontal cortex and impaired
cognitive abilities in rats. These studies are likewise reproduced in mice. In the study of Bissonette et
al they made lesions in the OFC and the MFC and the Murine brain. The mice were tested for reversal
learning and set shifting through the proposed set shifting experiment of Garner et al. Lesions in the
OFC-region caused selective deficits in reversal learning. In contrast mice with lesions in the MFCregion demonstrated deficits in set shifting ability. Similar results were obtained in rats and primates.
6.3.3 NMDA antagonist
In humans it is established that ketamine (NMDA receptor antagonist) impairs attentional set shifting
measured with the WCST. Ketamine actions were likewise tested in mice with an attentional set
shifting task like Garner et al. The test demonstrated that ketamine produces specific deficits in
cognitive flexibility. The deficits might be reversed by the NR2B subunit selective antagonist Ro 256981, suggesting pro-cognition action for this antagonist. This was likewise demonstrated in other
studies where Ro 25-6981 appeared to promote impulsive-type responding resulting in an
improvement in the task performances. The results propose an important role for NMDA receptors in
cognitive inflexibility (65).
6.3.4 Summary rigidity
Rigidity is an important part of Anorexia Nervosa. Set shifting is a way to measure this cognitive
rigidity. In humans with Anorexia Nervosa set shifting was impaired not only during the illness but
likewise after recovery suggesting that set shifting is an endophenotype for AN. This resulted in the
formation of several tests for set shifting in rodents to effectively asses set shifting and to solve the
genetic background for impaired set shifting. Garner et al, Bissonette et al and Kos et al have
effectively modified the mice model to investigate attentional set shifting. With these models the
Author: H.C den Boer
Page 29
genetic background of set shifting can be investigated with QTL mapping and forward genetics as
described in section 5.2.
Materials and Methods
Animals, The animals that will be used are of the C57BL/6J and the A/J strain. CSS with the C57BL/6J
are the host strain and the A/J mice are the donor strain (46) Lod scores will be obtained for the
individual CSS, so further QTL mapping might be performed.
Reversal/set shifting task, the set shifting task is the same task performed by Bisonette et al (64).
Testing is performed over 4 days. Mice are tested through a series of discriminations where the
exemplar pair was changed, but the dimension (odor or medium) of the correct choice remains the
same. The dimension is relevant if its attributes predict outcome. For example, if odor is the relevant
dimension the mouse requires to choose the correct odor from each pair and ignore the attributes of
the digging medium. In this example, the digging medium is considered the irrelevant dimension. The
discriminations are as follows: (1) a single series of SDs in which the mouse is presented with two
choices of the relevant dimension and one choice of the irrelevant dimension (i.e., two odors within
the same medium); (2) a single series of CDs in which the mouse is presented with the same choices
of relevant dimension as in the SD and two choices of irrelevant dimensions (the exemplar used in
the SD and a new exemplar); (3) a series of four IDS in which the mouse is presented with compound
discriminations using two novel exemplars from the relevant and irrelevant dimensions for each IDS
(the relevant dimension of the correct choice (i.e., odor) is maintained throughout the
discriminations); (4) a IDR in which the mouse is presented with the same set of exemplars as in IDS,
but the stimulus–reward pairing was reversed within the relevant dimension; and (5) an EDS in which
the mouse is presented with a novel compound discrimination, except for the first time the correct
choice was an exemplar that was previously from the irrelevant dimension (the previously relevant
dimension has become irrelevant) (64).
The baited bowl was randomly presented on either side of the testing cage, and the relevant
exemplar was randomly presented with the irrelevant exemplars. The trial will be stopped if the
mouse does not dig within 3 min in the testing cage. The order of discriminations and exemplars are
the same for all mice, but the direction of the EDS (odor to medium or medium to odor) is
counterbalanced within each experimental group. A criterion of eight consecutive correct trials was
required to complete each task. Data are reported as the number of trials to criterion and the
number of errors required for each discrimination. (64)
Results
Results for the set shifting test were obtained with the C57BL/6J mice and the A/J mice. They were
tested for the SD, CD, IDSI till IDSIV, IDSIVRev, EDS, EDSRev. This resulted in several distinct data,
trials to criterion, number of error trials. Significant difference between the two strains was obtained
at three discriminations of the set shifting test, the IDSIII, the IDSIVRev and the EDSRev. A/J mouse
needed more trials compared to the C57BL/6J to reach to the criterion of eight consecutive correct
trials at the IDSIII and the EDSRev (Figure 20). The A/J mouse made more errors per trial for the
IDSIII, IDSIVRev and the EDSRev (Figure 21).
Author: H.C den Boer
Page 30
Figure 20, the illustrations shows the differences between the AJ and BL6 for the amount of trials to reach the
criterion. In the IDSIII and the EDSRev the AJ mouse needed significantly more trials to reach the criterion.
Figure 21, the illustration shows the difference between the AJ and BL6 for the amount of error trials. AJ
demonstrated a significant difference in the IDSIII, the IDSIVRev and the EDSRev for errors per trial
Author: H.C den Boer
Page 31
Mean latency measured in the set shifting test did not show any significant difference, implicating
that they do not differ in the time they need to make a decision. In the number of correct trials there
was one significant result. In the EDSRev the AJ mouse demonstrated significantly more correct trials
compared to the BL6 mouse (Figure 22).
Figure 22, the illustration demonstrates the number of correct trials in each distinct discrimination. The AJ
mouse demonstrates a significant difference in the correct number of trials compared to the BL6 in the EDSRev.
__________________________________________________________________________________
Discussion
In mice several distinct mice strains have been breaded. The distinct strains are genetically different
and exhibit distinct phenotypical behavior. The phenotypical difference for rigid behavior can be
measured through the sand digging paradigm. The sand digging paradigm has been perfected for its
function to test mice by Bissonette et al (64).The sand digging paradigm has been used to measure
the phenotypical difference in the AJ and BL6 mouse. The data of the test suggest a difference in the
AJ and the BL6 strain. The AJ strain demonstrates a difference between the IDS and the EDS
demonstrating that the mice create an attentional set. The BL6 appears to construct an attentional
set as well. Furthermore the AJ mouse demonstrates differences compared to the BL6 at the IDSIII,
IDSIVRev and EDSRev. Demonstrating poorer set shifting ability compared to the BL6 mice. The
significant differences at these three discriminations are important for the QTL analysis. The IDSIII,
IDSIVRev and EDSRev can be used as a reverence for the QTL analysis. Further analysis might be
executed with the CSS described in the materials and methods. The use of CSS will show which
chromosomes are important for the phenotypical difference between the AJ and BL6.
Author: H.C den Boer
Page 32
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