genetic analysis of stress responsiveness in a mouse model
TRANSCRIPT
Genetic analysis of stress responsiveness in a mouse model
MARK MURPHY1, REBECCA E. NEWMAN1, MAGDALENA KITA1, YVETTE M. WILSON1,
SASH LOPATICKI2, & GRANT MORAHAN2
1Department of Anatomy and Cell Biology, University of Melbourne, Melbourne, Victoria, Australia and 2Walter and Eliza
Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, Victoria, Australia
AbstractThe purpose of the present paper was to look for genes that might be involved in anxiety-related behaviours by undertaking agenetic analysis of a simple mouse model of stress responsiveness. Two inbred mouse strains have been identified that showeither high or low stress responsiveness. These strains were crossed to generate F1 progeny, which were then crossed togenerate F2 progeny, and in which there is segregation of genotype within individual animals. DNA was isolated from theseanimals and a genome scan was conducted in order to find regions on the genome that correlate with the stressresponsiveness. Several regions on the mouse genome show significant linkage with the stress phenotype. One region inparticular, on chromosome 12, was further characterised and the most significant linkage was found between 32.8 and44.8 cM. These chromosomal regions may contain genes encoding proteins that are involved in the underlying neuralcircuitry involved in stress responsiveness.
The stress response is a very important and natural
aspect of human behaviour and, in a healthy
individual, is a natural and necessary response to
external stimuli. However, chronic or extreme
reactions to stress are directly related to anxiety
and can lead to pathological conditions such as long-
term anxiety states, depression and panic disorders
(Gold & Chrousos, 2002; Jetty, Charney, & God-
dard, 2001; Kendler, Kessler et al., 1995; Lopez,
Akil, & Watson, 1999). Stress-related disease is not
limited to psychopathologies, but also contributes to
other major health problems such as hypertension,
atherosclerosis, and disorders of the immune system
(Vanitallie, 2002). These disease states include some
of the major medical problems of our times and
stress-related illnesses affect at least 25% of the
population. In the next 20 years just one of these
diseases, generalised depression, is predicted to
become the second greatest cause of death and
disability in the world, whereas ischaemic heart
disease, which is directly associated with hyperten-
sion and atherosclerosis, will become the greatest
cause of death and disability (Murray & Lopez,
1996).
It is now becoming increasingly clear that a
significant part of the individual variation in the
stress response has a genetic component (Bouchard,
1994; Eley & Plomin, 1997). In twin studies, the
genetic component for the stress-related personality
trait, neuroticism, is estimated to be 40 – 50%
(Bouchard, 1994; Pedersen, Plomin, McClearn, &
Friberg, 1988). Furthermore, there is significant
comorbidity for the anxiety-related traits and
depression, suggesting direct causal links between
these disorders (Kaufman & Charney, 2000).
However, there is no clear genetic picture emerging
to show that one set of genes may underpin all of
these pathologies (Kendler, Walters et al., 1995).
Analysis of genetics of these disorders in humans is
complicated by many factors, including accuracy of
diagnosis, the likely multifactorial inheritance, and
influence of environment. This may make it
particularly difficult to identify genes that are
associated with these disorders using conventional
genetic linkage analysis. Attempts to undertake such
analyses for other complex psychiatric disorders,
such as bipolar disorder and schizophrenia, have so
far had limited success (Baron, 2002; Moller, 2003;
Correspondence: M. Murphy, Department of Anatomy and Cell Biology, University of Melbourne, Melbourne, Vic. 3010, Australia. Tel.: + 61 3 8344 5785.
Fax: + 61 3 9347 5219. E-mail: [email protected]
Australian Journal of Psychology, Vol. 56, No. 2, September 2004, pp. 108 – 114.
ISSN 0004-9530 print/ISSN 1742-9536 online # The Australian Psychological Society Ltd
Published by Taylor & Francis Ltd
DOI: 10.1080/00049530410001734883
Pulver, 2000), even given that there is evidence for
a strong genetic contribution to both of these
disorders.
Because of the difficulties associated with human
analysis, an attractive alternative is to use animal
models of reactivity to stress, often termed emotion-
ality (Eley & Plomin, 1997). The studies of genetics
of emotionality often involve the use of inbred
strains of mice, where each animal within an inbred
strain is genetically identical, but different strains
are genetically different. If there are significant
differences in behaviour between inbred strains
raised in the same environment, then these have a
genetic basis. A number of studies have used inbred
strains for the genetic analysis of emotionality (Flint
et al., 1995; Talbot et al., 1999; Talbot et al., 2003;
Tarricone, Hingtgen, Belknap, Mitchell, & Nurn-
berger, 1995). These studies have often used the
Open Field Activity (OFA) test as a measure of
fearfulness or emotionality and have established
genetic linkage between this phenotype and a
number of regions on different mouse chromo-
somes.
In this study we have used a similar approach to
analyse the genetics of emotionality. We have used a
modification of the OFA test, an elevated OFA
(eOFA), to look for differences in emotionality
between two different mouse strains, C57/Bl6 (B6)
and DBA2/J (D2). We find very significant differ-
ences in locomotion between these strains. We have
undertaken a genetic analysis in an F2 cross of these
two strains, and find several regions of the mouse
genome that show significant association with this
phenotype.
Methods
Animals and breeding.
The inbred mouse strains, B6 and D2, were obtained
from the Walter and Eliza Hall Institute Animal
Production Facility (Kew, Victoria, Australia). Upon
weaning, all animals were housed in same-sex groups
at the Departments of Anatomy and Cell Biology/
Pathology Animal Facility at the University of
Melbourne. The mouse room was maintained at a
temperature of 22+ 18C with a 12:12 light – dark
cycle. The mice had feed (standard rodent feed
GR2+ , Barastoc, Vic., Australia) and water available
at all times. All animal care was conducted according
to the Australian Code of Practice for the Care and
the Use of Animals for Scientific Purposes, 1997.
An outcross between B6 female mice and D2 male
mice was conducted to generate first filial (denoted
B6D2F1) progeny, and these animals were subse-
quently crossed to generate second filial progeny
(B6D2F2) for mapping studies.
Elevated Open Field Activity test
All behavioural testing was conducted between 10:00
and 17:00 hours. The animals to be tested were taken
to the Behavioural Laboratory, within the Depart-
ment of Anatomy and Cell Biology, at least 1 hr prior
to testing. We used a modification of the OFA test,
the eOFA. In this test, OFA was measured under
intense white light (2 6 250 W) for 3 min upon a
rectangular arena (766 102 cm), 74.0 cm above the
floor and with grid lines drawn in 20.0 6 20.0-cm
sections. The 80 – 85-day-old mice were individually
placed into a cylindrical chamber (11.3 cm diameter,
11.4 cm height) in the center of the arena. The 3-min
trial commenced once the chamber was lifted via a
pulley system by the experimenter and its shadow was
free from the arena. The parameters measured were
total time each mouse was exploring the arena, the
total number of section boundaries crossed and the
number of faecal boli deposited by the mouse during
the 3-min trial. After each trial the arena was wiped
clean with a damp cloth to eliminate any olfactory
cues affecting the behaviour of subsequently tested
mice.
The animals were given an overall stress score,
which combined three measures of OFA: time
moving, grid crossings and rate (grid crossings/time
moved). For each of these values, the means for B6
mice were assigned a value of 0, and for D2 mice, a
value of 1. There was a negative correlation for time
moved and grid crossings, and a positive correlation
with rate. Defecation rate was excluded from the
overall score because it showed no correlation with
the other measures of stress responsiveness in the F2
cross. Initially 460 F2 animals were generated and
phenotyped using the OFA. These animals were
divided into two groups of 230 and DNA from 30 of
the most D2-like and 30 of the most B6-like animals
from one of the cohorts of 230 animals were subject
to a genome scan.
DNA preparation and genome scan
DNA was prepared from animals’ tails by incubation
in 100 ml 25 mM NaOH/0.2 mM ethylenediamine
tetraacetic acid (EDTA), pH 12.0 at 958C for
60 min. After incubation the tails were left to cool
at room temperature for approximately 2 min and
then shaken vigorously to release the DNA into the
solution. Finally, 100 ml 40 mM Tris-HCl, pH 5.0
was aliquoted into each eppendorf tube. For
genotyping, initially 140 MapPrimerTM pairs (Re-
search Genetics, Huntsville, Alabama, USA) were
selected from the Whitehead/Mit mouse genome
databases to scan the whole genome at approximately
10 – 15-cM intervals. All primer pairs generated
products of different sizes in B6 or D2 mice, allowing
Genetics of stress-responsiveness 109
the assignment of a particular region of the mouse
genome to either of these parental strains. Different-
sized microsatellite products were generated by
polymerase chain reaction (PCR) in a volume of
10 ml where 5 ml was diluted DNA solution. The
remaining 5 ml consisted of 66-nM primer; 16 PCR
Buffer (minus Mg2+ , GibcoBRL); 2 mM MgCl;
0.1 mM deoxynucleotide triphosphates (dNTPs);
0.015 MBq [a32P] deoxyadenosine triphosphate
(dATP) and 0.2 U Taq polymerase (GibcoBRL).
After an initial denaturation at 948C for 1 min, the
reaction conditions were 35 cycles at 958C for 20 s,
538C for 20 s, 728C for 20 s and a final cycle of 728Cfor 2 min and 258C for 2 min. The PCR products
were resolved on 5% polyacrylamide gels and
products detected by autoradiography. The data
generated from this genotyping were analysed for
linkage using the nonparametric MAPMAKER/QTL
program (Whitehead/MIT Center for Genome Re-
search, Cambridge, MA).
Further genotyping at a subregion of chromosome
12 of the B6D2F2 animals was undertaken in order
to do further linkage analysis with the stress
phenotype (as well as a nonstress phenotype). This
was done by selecting both samples that had either
high or low stress scores from both cohorts of 230
B6D2F2 animals and genotyping them as above for
markers lying only in chromosome 12.
Linkage analysis
A chi-square contingency table was utilised to
observe if a specified genotype on chromosome 12
correlated to either high stress or low stress (stress
nonresponsive).
Results
Activity of mouse strains on elevated open field activity
test
In initial observations of B6 and D2 mice, clear
qualitative differences in behaviour were observed.
The B6 mice were relatively easy to pick up and
did not show any signs of internal shaking of
shivering. In comparison, D2 were more difficult
to pick up in their cages, running quickly around
the cage and flattening their bodies to the walls of
the cage. They also appeared to have a shiver as
well as constant head movements and appeared
hyperactive. We thus believed that they could have
been stress prone or highly stress responsive, and
tested them in an eOFA. The OFA is an example
of measuring rodent behaviour in a novel environ-
ment. The open field arena, being white and well
lit, produces an unwelcoming and potentially
frightening environment for rodents (Archer,
1973; Flint et al., 1995). Therefore exploratory
activity upon the open field is a useful measure for
stress responsiveness.
We tested both strains on our eOFA, which is
somewhat different to the OFA test used by others in
that the arena is raised above the floor and has no
sides. It may be more threatening than a sided open
field test because there is nowhere to hide. To
determine the behaviour of different strains of mice
on this arena, we placed them in the centre of the
open field, in an enclosed cylinder, and then
removed the cylinder and observed their behaviour.
The B6 mice moved freely on the open field and
constantly investigated the entire arena, including
the edges. In comparison, most D2 mice moved very
reluctantly from the centre of the arena, as indicated
by repeated back and forward movements of their
upper bodies and heads, and investigated much less.
When they did move, they were inclined to move in
rapid bursts. A comparison of time moved and grids
crossed showed that B6 mice moved 4 – 5 times more
than D2. In addition, the rate of movement of the D2
mice was significantly higher than that of B6 (Table
I), which is consistent with the D2 animals moving in
quick bursts. The number of defecations was also
significantly greater in the D2 animals, which is
another indicator of stress in the animals.
Generation of F1 and F2 mice
D2 and B6 mice were mated to generate F1 mice and
subsequently these F1 mice were mated to generate
460 F2 mice. The eOFA tests were undertaken on
both sets of animals. Analysis of the F1 mice showed
an intermediate distribution of movement, between
that observed for B6 and D2 animals (Figure 1).
However, the mean activity was closer to that
observed for D2 animals than B6, suggesting that
the low movement, or stress phenotype was semi-
dominant. An analysis of the distribution of OFA in
the F2 mice revealed an extensive variability in
movement, from no movement at all through to
movement above that of parental B6 animals.
However, the frequency distribution was very skewed
towards low movement times (Figure 1), similar to
the D2 animals, which further indicated that the low
Table I Quantification of activity of mouse strains B6 and D2 on
the elevated open field
B6 D2
Time moved (s/3 min) 110+ 14 22+ 25
Grid crossings 50+ 9 17+ 12
Rate (no. grid crossings/
time moved)
0.46+ 0.09 0.87+ 0.32
Defecations 0.1+ 0.32 2.4+ 1.78
110 M. Murphy et al.
OFA-stress phenotype was dominant. There were
few F2 mice with high OFA scores.
Genome scan
A genome scan was conducted on DNA from a
group of 30 F2 mice that had the lowest OFA scores
and 30 F2 mice that had the highest OFA scores
from a cohort of 230 of the F2 animals, to look for
quantitative trait loci (QTL) on the mouse genome
associated with stress-like behaviour on eOFA. A
number of QTLs were found that showed either
suggestive or significant linkage to stress-related
behavior (Table II). In particular, two loci, on
chromosomes 5 and 12 showed significant linkage
(LOD score 4 4.3). Of these, the locus on chromo-
some 12 gave the highest LOD score.
Analysis of quantitative trait loci on chromosome 12
The significant QTL seen for a locus on chromo-
some 12 and the stress phenotype was further
analysed with a greater number of mice and more
markers covering chromosome 12. High-stress F2
animals were selected from the entire 460 F2 animals
for further analysis. Table III shows the chi-square
contingency table, which compares the observed
genotypes with the expected genotypes of the
B6D2F2 animals that exhibited high stress respon-
siveness. It can be seen that there is significant
linkage with several D2 alleles across chromosome
12; in particular at the proximal end at 17.5 cM, and
at the distal end, from 41.5 cM to 51.4 cM.
In addition, we separately analysed mice that were
low stress responsive in the F2 population. We
reasoned that because these mice represented such a
low proportion of the F2 population, then a more
limited number of possible genotypes would be
responsible for low stress responsiveness than for
high stress responsiveness. Thus, if a particular B6
allele(s) on chromosome 12 were involved in low
stress responsiveness, it would be more likely to be
present in low-stress F2 animals compared to D2
alleles at the same locus in the high-stress popula-
tion. Table IV shows the results of this analysis. It
can be seen that there is significant linkage across a
contiguous region of chromosome 12 from 17.5 cM
to 44.8 cM. Furthermore, the p values are generally
more significant compared to those seen in the high-
stress analysis (cf. Table III with Table IV). In
particular, there are highly significant loci at 32.8 –
44.8 cM (Table IV). These data provide strong
support that a region on chromosome 12 is involved
with the stress phenotype in these strains of mice.
Discussion
In our initial studies on the characterisation of
different strains of mice, we looked for mice that
may have had multiple behavioural characteristics.
One of the tests we undertook was the Barnes
circular maze (Barnes, 1979), which involves mice
learning the location of an escape box under a hole
in a spatially defined location. Whereas B6 mice
learned this test relatively easily and were able to
solve the test using spatial cues, the D2 mice were
unable to solve this test. The D2 mice, when
released on the arena of the Barnes maze, moved
very quickly or not at all (Murphy, 1997). They
developed a stereotypic behaviour on the arena
over successive trials, which involved running
Figure 1. Frequency distribution of open field activity (OFA) in
B6D2F2 mice. Shown are the distribution of animals versus time
moved on the elevated OFA test (eOFA) for the first cohort of 230
B6D2F2 animals generated. Also shown are M+SD for both
parental strains, B6 and D2, as well as M+SD for a group of 30
B6D2F1 animals.
Table II Quantitative trait loci for different measures of activity
associated with open field activity
Phenotypes showing
linkage Chromosome
LOD score for
Stress phenotype
Stress and time moved 1 3.0
Stress and grids crossed 2 2.9
Stress and time moved 3 3.3
Stress and rate 5 4.39
Stress, rate and time moved 12 4.86
Note. Qote: QTL=quantitative trait loci; LOD score= logarithm
of the likelihood for the presence of a QTL.
LOD scores shown are those associated with the composite stress
score for each animal. Stress was a composite comprising open
field time moving, grid crossings, and rate of grid crossings, as
described in Methods. In addition, a number of these loci also
showed linkage to the individual components of the stress score, as
indicated.
Genetics of stress-responsiveness 111
around the edge of the arena very quickly, so
quickly that in most cases they were unable to
determine which hole in the arena led to an escape
box. In some cases, the mice become so agitated
that they fell off the arena. Further, a significant
proportion of the mice underwent a mild seizure
on the Barnes maze arena, which lasted for approx
1 min, and was followed by recovery. Other data
indicate that the D2 mice are anxiogenic. Thus in
both light – dark exploration and in elevated plus
maze, this strain shows moderate to high levels of
anxiogenic behavior (Crawley et al., 1997).
We then developed a modified form of the OFA
test that was similar to a conventional open field
test, but was elevated and unenclosed, similar to the
Barnes maze. Using this test, we found significant
differences in activity between B6 and D2 mice. In
particular, the D2 mice moved very little on the
open field, in contrast to their behavior both on the
Barnes maze and in their home cages. When they
did move, they did so in quick bursts. We suggest
that these behaviours reflect a stress-proneness in
the D2 mice, which, under the conditions of the
Barnes maze, results in hyperactivity and sometimes
seizure, and in the eOFA results in freezing or little
movement. Although our eOFA is highly reflective
of conventional OFA, it is also possible that it is
detecting other behaviours in addition to stress-
proneness, such as stress-independent ambulatory
activity. It is interesting to note that the D2 mice
are also susceptible to audiogenic seizure, although
this has been observed only between 3 and 5 weeks
of age (Seyfried, 1979). It is possible that the
seizure induced by the conditions experienced on
the Barnes maze, and that induced by audiogenic
stress, have similar or related underlying genetic
mechanisms.
There is evidence that low levels of exploratory
activity on the open field arena correlate with high
blood concentrations of corticotropin-releasing hor-
mone (CRH), adrenocorticotropic hormone
(ACTH) and corticosterone (Sternberg et al.,
1992). In addition, high levels of these hormones
have been correlated to high levels of stress respon-
siveness in humans. The OFA has been used for
many years in behavioural research to look for stress-
related behaviours (Sutanto & de Kloet, 1994) and
has been utilised as the animal model to test drugs
for anxiolytic effects, because it would be expected
that these drugs would induce an increase in
exploratory activity in fear-prone animals (Radcliffe
& Erwin, 1998).
Stress-responsiveness shares phenotypic and genetic
similarity with emotionality as open field activity is the
common parameter measured
The results of this study suggest that high stress
responsiveness is a dominant trait in the B6 6 D2
cross, with the exception of the defecation rate
component. The F1 generation displayed total
exploratory time, sections crossed and rate means
similar to the D2 parental strain, supporting the
notion that stress responsiveness is a dominant trait.
With defecation rate the F1 generation exhibited an
intermediate mean to the B6 and D2 parental strains,
suggesting that the presence of D2 alleles additively
influence defecation rate.
Previous studies of emotionality have linked OFA
with defecation rate (Archer, 1973). Emotionality is
a term used to describe rodent behavioural responses
in novel environments, such as the open field arena
(Archer, 1973), and this trait is similar to the stress
responsiveness trait investigated in this study. High
emotionality has been proposed to be characterised
as low open field exploratory activity coupled with a
high defecation rate and therefore a negative
correlation would be expected between these two
measures (Flint et al., 1995). Our study did not find
this for the B6 6 D2 F2 cross, and there was no
Table III Chi-square analysis for high stress responsiveness in F2 mice of B66D2 cross and in loci on chromosome 12
Marker 182 172 34 158 239 121 99 17
Chromosomal
location
2.2 17.5 23 32.8 39.3 41.5 44.8 51.4
Observed Het 28 31 13 28 25 21 30 39
DD 22 26 8 14 15 18 15 25
BB 13 10 4 9 10 5 5 9
Total 63 67 25 51 50 44 50 73
Expected Het 31.5 33.5 12.5 25.5 25 22 25 36.5
DD 15.75 16.75 6.25 12.75 12.5 11 12.5 18.25
BB 15.75 16.75 6.25 12.75 12.5 11 12.5 18.25
w2 p 0.187 0.018 0.51 0.47 0.6 0.02 0.049 0.025
Note. Het=heterozygous; DD=homozygous for D2; BB=homozygous for B6.
112 M. Murphy et al.
correlation between defecation and other aspects of
the OFA (data not shown).
Flint et al. (1995) employed a B6 6 BALB/c (an
albino mouse strain) cross to identify loci associated
with the emotionality trait, where BALB/c mice are
classified as displaying high emotionality. In that
study, three loci were found that showed significant
linkage with high emotionality, on chromosomes 1,
12 and 15. To date, the genes at these loci have not
been identified. For the chromosome 12 locus the
linked markers span a region 13.1 – 31.7 cM from
the centromere. This coincides with the region
identified in our studies. It is thus quite possible
that these two studies have identified the same locus
for stress responsiveness/emotionality. Likewise, our
initial genome scan identified a region on chromo-
some 1, which could be analogous to that found by
the Flint group as well as to that of other studies
(Caldarone et al., 1997; Gershenfeld et al., 1997;
Tarricone et al., 1995)
A subsequent study from the Flint group (Talbot
et al., 1999) further supported, from an eight-way
cross with different inbred mouse strains (three of
these being B6, D2 and BALB/c), that there was
significant linkage between chromosome 12 and high
emotionality. The most highly linked marker,
D12Mit190, in that study corresponds to approxi-
mately 22 cM in chromosome 12. The Flint group
concluded that this locus was different to the one
found in the initial B6 6 BALB/c genome scan
because the linked marker does not distinguish
between BALB/c and B6 alleles (Talbot et al.,
1999). The implication is that there must be two
loci influencing emotionality with this region of
chromosome 12 (13.1 – 31.7 cM). However, this
marker actually can distinguish between B6 and D2
alleles (128 bp and 114 bp, respectively; Whitehead
Institute/MIT Center for Genome Research, Genet-
ics and Physical Maps for the Mouse Genome: http://
carbon.wi.mit.edu:8000/cgi-bin/mouse/index).
Thus, the detected linkage by (Talbot et al., 1999)
on chromosome 12 may be due to the detection of
D2 alleles in their eight-way cross and is a similar
region to that detected in our initial B6 6 D2
genome scan. It follows that the region responsible
for this linkage may be a single locus. Overall this
evidence suggests that a region on chromosome 12 is
associated with OFA, because this is the common
parameter in stress responsiveness and emotionality.
In humans, there have been a limited number of
studies that have addressed the genetic basis of
stress. The most significant finding is from a
candidate gene approach (Lesch et al., 1996), which
found that polymorphisms in the regulatory region of
the serotonin transporter gene, and which result in
decreased expression of this gene, can account for up
to 9% of the genetic component of the anxiety-
related trait of neuroticism in that group of subjects.
This is a very exciting finding, but it leaves open the
way for the discovery of genes that underlie the great
majority of the trait for neuroticism and related
anxiety-associated traits.
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Marker 182 172 34 158 239 99 17
Chromosomal
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Observed Het 20 18 11 22 24 25 18
BB 10 14 13 22 22 22 12
DD 5 3 4 9 7 5 5
Total 35 35 28 53 53 52 35
Expected Het 17.5 17.5 14 21.5 21.5 21 17.5
BB 8.75 8.75 7 10.75 10.75 10.5 8.75
DD 8.75 8.75 7 10.75 10.75 10.5 8.75
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