JAD-04549; No of Pages 9
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Journal of Affective Disorders xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Journal of Affective Disorders
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j a d
Research report
Selective breeding for dominant and submissive behavior in Sabra mice
Yuval Feder a,b, Elimelech Nesher a, Ariel Ogran a, Anatoly Kreinin c, Ewa Malatynska d,
Gal Yadid b, Albert Pinhasov a,⁎
a
b
c
d
Department of Molecular Biology, Ariel University Center of Samaria, Ariel, 40700, Israel
Leslie Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan 52900, Israel
Tirat HaCarmel Mental Health Center, Tirat HaCarmel, Israel
Eli Lilly and Company, LRL/LCC, Indianapolis, IN 46285, United States
a r t i c l e
i n f o
Article history:
Received 25 August 2009
Received in revised form 31 January 2010
Accepted 23 March 2010
Available online xxxx
Keywords:
Animal models
Dominance
Submissiveness
Selective breeding
Antidepressants
Depression
Mania
a b s t r a c t
Background: The Dominant–Submissive Relationship (DSR) model used here was developed
for mood stabilizing and antidepressant drug testing. Treatment of submissive animals with
known antidepressants significantly reduced submissive behavior in a dose-dependent
manner. We hypothesized that if submissive behavior in DSR is a valid model of depression,
it should be possible to show a genetic predisposition for this trait, since clinical studies support
a genetic component for depression.
Methods: To test this hypothesis, we applied selective breeding on outbred Sabra mice based on
DSR paradigm.
Results: Here we have demonstrated that the frequency of DSR formation gradually increased
across four generations of outbred Sabra mice, when animals inbred for the dominant trait
were paired with those inbred for the submissive trait. Chronic imipramine administration
(10 mg/kg) significantly reduced submissive behavior in the F2 generation consistent with the
effect seen in unselected C57BL/6 J mice.
Conclusions: We conclude that increased frequency of DSR formation suggest a genetic
component of these two phenotypes, and strengthens the predictive and face validity of the
DSR test. Selective breeding may aid in a better understanding of the genetic basis of dominant
and submissive behavior, important elements in the etiology of affective disorders.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Dominant–Submissive Relationship (DSR)-based tests,
the reduction of dominant behavior model (RDBM) and the
reduction of submissive behavior model (RSBM), were developed to study the effects of chronic anti-manic or antidepressant drug treatments to facilitate the development of
new medications for mania and depression (Malatynska et al.,
2002; Malatynska and Knapp, 2005; Malatynska and Kostowski, 1984; Malatynska et al., 2007a, b; Malatynska et al.,
2005; Pinhasov et al., 2005). In this test, drugs used in the
⁎ Corresponding author. Tel.: + 972 3 937 1480; cell: + 972 54 774 0271;
fax: + 972 3 937 1422.
E-mail address: [email protected] (A. Pinhasov).
clinic to treat mania reduced dominant behavior (Malatynska
and Knapp, 2005; Malatynska and Kostowski, 1984), while
drugs used to treat depression reduced animals' submissive
behavior (Malatynska et al., 2002; Malatynska and Knapp,
2005; Malatynska et al., 2005; Pinhasov et al., 2005).
Depending on the selection criteria, from 25% to 40% of
animals (rats or mice) form DSR (Malatynska et al., 2002;
Malatynska and Kostowski, 1984; Malatynska et al., 2007b;
Pinhasov et al., 2005). The rest of the animals do not form
statistically distinguishable dominant–submissive relationships (for review see (Malatynska and Knapp, 2005)). Thus,
DSR pair selection, which is the first and most important step
in these paradigms, gives a consistent measure of the fraction
of the population showing susceptibility to the expression of
DSR behavior.
0165-0327/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jad.2010.03.018
Please cite this article as: Feder, Y., et al., Selective breeding for dominant and submissive behavior in Sabra mice, J. Affect.
Disord. (2010), doi:10.1016/j.jad.2010.03.018
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2. Materials and methods
tunnel, allowing a constant supply of sweetened milk (3% fat,
10% sugar). The tunnel had narrow slits cut on both sides of the
feeder for easy gate insertion and removal (Fig. 1A). In this way,
the paired mice had an equal time starting position at the
beginning of the session.
DSR tests were carried out on five consecutive days per
week. During each 16 h period preceding testing, the mice
were deprived of food, but water was provided ad libitum. The
animals had free access to food for two days between testing
periods until the night before the next five day testing period.
The animals were housed in groups of five per home cage. The
pairs of mice with relatively similar weight (average weight
42 ± 3 g) and from different home cages were tested daily.
DSR testing of each pair was conducted for a single 5 min
session on each day of the consecutive five day testing period.
During this 5 min session milk drinking scores were recorded
manually by a human observer.
DSR testing was conducted over a two week period to
select mice for breeding. The first week was used to adapt the
mice to the testing conditions, while the selection of pairs
showing a DSR was based upon scores measured during the
second week. Dominant or submissive status for mice in a
pair was defined on the basis of three criteria (Malatynska
and Knapp, 2005; Malatynska et al., 2007b; Pinhasov et al.,
2005). First, there had to be a significant difference between
the average daily drinking scores of both animals in a pair.
Second, the dominant animal's drinking time had to be at
least 40% greater than the submissive animal's drinking time.
Third, there must be no “turnaround” during second week of
DSR testing, where the submissive animal's drinking time is
higher than its dominant partner on isolated occasions. The
member of a pair that had a significantly lower drinking time
(p b 0.05) in the selection week and fit the remaining selection
criteria was defined as submissive and its counterpart was
defined as dominant.
2.1. Animals
2.3. Selective breeding
Two-month-old outbred Sabra mice (Harlan Laboratory,
Jerusalem, Israel) were subjected to DSR test-based selective
breeding in-house. Sabra mice were originally developed at
the Hebrew University, Jerusalem, Israel, and Harlan Laboratories, Ltd. has been maintaining this strain by contract with
the Hebrew University since 1993. Animals were given
standard laboratory chow and water available ad libitum.
The colony room was maintained on a 12 h L:12 h D cycle
(lights on 01.00–13.00 h). The experiments were conducted
in compliance with the NIH/USDA guidelines, under the
approved Institutional Animal Care and Use Committee
(Protocol number IL-13-10-06).
For breeding, a single dominant or submissive male was
housed together with two or three female mice selected for
the same trait. Females were removed and relocated into
individual cages two weeks later. At 21–25 days from birth,
offspring were weaned from the mother, weighed, ear
clipped for identification, and housed in groups of five by
sex throughout all experimental phases of the project.
Selective breeding experiments were started in August
2007. The founder dominant or submissive animals were
designated generation P; offspring generations were designated F1, F2, F3, and F4. Males of the P generation that formed
DSR during two week test period were bred with females
with the same traits: submissive males with submissive
females and dominant males with dominant females. In each
subsequent generation, the number of animal pairs subjected
to the DSR test was dependent on the number of offspring
(Table 1). In consecutive (F1–F4) generations, offspring of the
dominant mice were paired and subjected to the two-week
DSR test with offspring of the submissive animals. Since a
relatively small number of animals of P and F1 generations
developed DSR, animals which met partially the defined
criteria were also used for selective breeding. Thus, P and F1
animals with drinking time difference of less than 40% were
Developing lines of animals based on behavioral features
is an important approach to study the biochemical and
genetic basis of a trait of interest. Several lines of animals
were developed to study the genetic and biochemical origins
underlining mood and anxiety disorders (Brunelli and Hofer,
2007; File et al., 1999; Gonzalez et al., 1998; Henn and
Vollmayr, 2005; Touma et al., 2008).
Here we demonstrate the inheritance of dominance and
submissiveness as determined in the DSR-based tests,
providing new support for the face validity of the RSBM as a
model of depression. We used selective breeding for
dominant and submissive traits of fixed-pair outbred Sabra
mice that form DSR in a food competition test. This is an
established outbred line (Chia et al., 2005) that is used in
neuroscience and pharmacological research (Avraham et al.,
2005; Gobshtis et al., 2007; Kogan et al., 2007; Pick and Yanai,
1989; Sumariwalla et al., 2004; Yanai et al., 1981). The
presence of a genetic component of dominant and submissive
traits, as expressed in the formation of DSR, was evaluated
using two measures: frequency of the trait in the population,
and latency of DSR development. We hypothesized that the
number of pairs that form DSR would increase in consecutive
generations of Sabra mice selected for dominant and
submissive behavior, and the time necessary to develop DSR
would decrease. Three control groups were used to test the
validity of the selective breeding study. A parallel group of
mice were randomly bred without behavior testing during
the period of selective breeding to show that the historical
ratio of mouse pairs developing DSR remained constant over
the course of this study. We also examined if changes in
locomotor activity or activity in the elevated-plus maze were
associated with selection for susceptibility to formation of
DSR.
2.2. Dominant–Submissive Relationship test
The DSR paradigm was performed as described in rats
(Malatynska and Kostowski, 1984; Pinhasov et al., 2005) and
mice (Malatynska et al., 2005), with minor modifications. The
apparatus (Fig. 1A) was constructed of red Plexiglas, chosen to
reduce outside disturbances. It consists of two identical chambers (12 × 8.5 × 7 cm) joined by a tunnel (2.5 × 2.5 × 27 cm). A
0.5 cm diameter hole was cut in the bottom center of the
tunnel. A self-refilling feeder (Fig. 1B) is connected to the
Please cite this article as: Feder, Y., et al., Selective breeding for dominant and submissive behavior in Sabra mice, J. Affect.
Disord. (2010), doi:10.1016/j.jad.2010.03.018
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3
Fig. 1. Apparatus for testing of Dominant–Submissive Relationship (DSR). The apparatus consists of two chambers connected with a tunnel only large enough to
allow one mouse to pass through at a time. In the floor, at the mid-point of the tunnel is a container of sweetened milk (A). Schematic depiction of self-refilling
feeder (B).
Table 1
Number of Sabra mice pairs forming Dominant–Submissive Relationships
during four consecutive generations of selective breeding.
Gender
Generation
Number of
tested animal
pairs
Number of
pairs that
formed DSR
Percent (%) of
pairs that
formed DSR
Males
P
F1
F2
F3
F4
P
F1
F2
F3
F4
12
10
10
20
20
8
10
10
15
20
4
5
6
13
15
2
4
6
10
16
33
50
60
65
75
25
40
60
66
80
Females
also used for selective breeding. This strategy was applied
only for P and F1 animals. Inbreeding was minimized by
breeding animals that were least related to one another.
Concurrently, we maintained a line of Sabra mice in which
breeders were randomly assigned without any behavioral
selections. This control group of animals was used to assess
heritability of dominant/submissive traits.
2.4. Elevated plus-maze (EPM)
The elevated plus-maze was constructed with two open
(10 × 45 cm) and two enclosed arms (10 × 45 × 40 cm) that
extended from a common central platform (10 × 10 cm). The
apparatus was made from black wood and was elevated on a
wooden box to a height of 60 cm above floor level.
Experiments were performed under dim illumination between 10:00 AM and 12:00 AM. One hour prior to the test the
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Fig. 2. Percent of pairs forming Dominant–Submissive Relationship (DSR)
across generations (P, F1–F4). Data were fitted by linear regression using
GraphPad Prism, r was 0.95 and 0.977 for males and females, respectively,
and slope was significantly different from unity at p b 0.01 for males (■) and
at p b 0.01 for females (●). Number of DSR formed pairs for each generation
that fit selection criteria described in the Materials and methods section is
depicted in Table 1.
animals were placed in the experimental room for habituation.
Baseline testing was followed by one 5 min test for each mouse.
The number of entries onto open and closed arms and the times
spent in open and closed arms were manually scored. The maze
was cleaned with 75% ethanol solution and rinsed with water
after each test (Gobshtis et al., 2007).
2.5. Open field activity
Animals were placed in a transparent “open field” glass box
(30 × 40 cm, divided into 20 squares of equal size). Recording
of horizontal (the number of squares crossed) and vertical (the
number of rears) activities have been done manually for 9 min
(Gobshtis et al., 2007). The glass box was cleaned with water
after each test.
2.6. Drugs and treatment
Submissive mice from the F2 generation were treated with
imipramine hydrochloride (Sigma-Aldrich, Cat no. I0899) at
10 mg/kg, while dominant mice received injections of the
vehicle alone (sterile water) for four consecutive weeks,
starting at the beginning of the third week after selection and
ending after the sixth week of the experiment. The solutions
were administered by the intraperitoneal (i.p.) route.
To test the stability of the DSR reaction, dominant and
submissive F4 generation animals were treated (i.p.) with the
vehicle for three consecutive weeks starting at the beginning
of the third week and ending after the fifth week of the DSR
test.
2.7. Data analysis
Animals from parental and offspring generations were
tested for DSR formation for two weeks. For each pair of mice,
data were tested for statistical significance using the two-tail
t-test. All pairs that met the criteria described above were
selected. Two weeks' data for these selected dominant and
submissive mice were plotted daily and were fitted using a
non-linear regression analysis.
Dominance level values were calculated to measure the
social relationship between paired subjects. Dominance level
(DL) is calculated as the difference between the total daily
feeding time of the dominant member of a pair and that of its
submissive partner. Data were fitted by non-linear regression
analysis to measure relationship onset time (ROT). ROT was
estimated from the curve as the time to reach 50% of the
maximum DL response.
Data from the imipramine and vehicle treatment study
were averaged for each week for dominant and submissive
mice and were fitted by non-linear regression analysis.
Non-linear regression analysis was done using GraphPad
Prism software. Statistical significance of difference between
dominant and submissive mice was evaluated using Two-Way
ANOVA with Bonferroni-corrected post-hoc t-test analysis.
3. Results
In the parental (P) generation, 33% of Sabra males and 25%
of the females formed DSRs (Table 1, Fig. 3A and F). The first
generation of offspring (F1) produced 50% of pairs with DSR
among males and 40% among females (Table 1, Fig. 3B and G).
Progressively greater numbers of pairs developing DSR were
observed in F2, F3, and F4 generations: 60%, 65%, and 75%
among males and 60%, 66%, and 80% among females, respectively (Table 1, Fig. 2). There was significant difference in
number of pair formations between generations (p b 0.05).
Bonferroni-corrected post-hoc analysis showed significant
difference only in number of pairs formed between parental
and F3 and F4 generations. There was no statistically significant
difference between the number of DSR pairs formed by males
and by females.
In the parental generation, the differences in time spent
on the feeder by the paired mice was significantly different
for males (p b 0.01) and females (p b 0.05) in the second week
evaluation period. This difference was not significant in the
first week acclimation period (Fig. 3A and F). For all filial
generations, the time spent on the feeder by paired animals
started to be significantly different during the first week acclimation period. The significant difference in time spent on
feeders by dominant versus submissive males in the F1
generation begins on day four for both sexes (Fig. 3B and G;
p b 0.001) and in the F2 generation it appears for the first time
on day five for males and on day four for females (Fig. 3C and H;
p b 0.0001). In the F3 generation this difference is noticeable as
early as day one for females (Fig. 3I), but not until the fourth day
for males (Fig. 3D; p b 0.0001). Similar pattern of DSR formation
onset was observed in the F4 generation. This is shown for
males on Fig. 3E and for females on Fig. 3 J.
For better comparison, the relation onset time (ROT) was
calculated from non-linear regression analysis performed
Fig. 3. The formation of Dominant–Submissive Relationship (DSR) in pairs of Sabra mice across generations tested daily for two weeks. The formation of DSR pairs
is shown on panels A–E for males (dominant (■) , submissive (□)), and panels F–J for females (dominant (●), submissive (○)). DSR pair formation in the parental
generation is shown on panels A and F and filial generations F1 (B and G,), F2 (C and H), F3 (D and I), and F4 (E and J) males and females, respectively. The statistical
significance between groups of dominant (Dom) and submissive (Sub) animals was assessed using two-way ANOVA with Bonferroni post-hoc corrected analysis.
Data points marked (*) are significant at p b 0.05, (**) at p b 0.01, and (***) at p b 0.001.
Please cite this article as: Feder, Y., et al., Selective breeding for dominant and submissive behavior in Sabra mice, J. Affect.
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using the dominance level of pairs from different generations
(Fig. 4A and B). The ROT for F1 (3.1 ± 0.4 days), F2 (4.9 ±
0.6 days), F3 (3.6 ± 0.4 days), and F4 (3.6 ± 0.3 days) generations in males and for F1 (3.1 ± 0.3 days), F3 (3.8 ± 0.8 days),
and F4 (1.7 ± 0.3 days) generations in females were significantly shorter than the ROT for parental animals (P1 males,
8.0 ± 0.6 days; P1 females 8.5 ± 0.4 days; Fig. 4C). In the F2
females, the ROT of 8.0 ± 0.7 days was not significantly
different from the parental females (Fig. 4C). The ROTs for
males and females were not significantly different with the
exception of the F2 groups.
The effect of imipramine on submissive behavior was
examined on F2 Sabra males. Daily imipramine injections
(i.p.) at a dose of 10 mg/kg for four weeks gradually reduced
their submissiveness (Fig. 5A). In this experiment, a significant
difference in drinking time between groups of dominant and
submissive animals was already present after the first week
of testing (p b 0.001) and increased during the second week
(p b 0.0001, Fig. 5A). Submissive animals were injected daily
with imipramine, and their dominant counterparts were injected with sterile water from the start of the third week of the
study. A reduction of submissive behavior was observed after
the second week of imipramine treatment, and was eliminated
after the third and fourth weeks of treatment (Fig. 5A).
Fig. 4. Dominance levels in pairs of male (A) or female (B) Sabra mice that
formed Dominant–Submissive Relationship (DSR) across the P, F1–F4 generations. The onset of DSR of the P animals was compared to the filial generations (C). Data points marked (*) are significant at p b 0.05, (**) at p b 0.01,
and (***) at p b 0.001. Number of pairs in each generation was as follow for
males: P (n = 4), F1 (n = 5), F2 (n = 5), F3 (n = 13) and F4 (n = 15); for females:
P (n = 4), F1 (n = 5), F2 (n = 5), F3 (n = 13) and F4 (n = 16).
Fig. 5. Effect of chronic 10 mg/kg i.p. imipramine (IMI) administration on
behavior in submissive Sabra mice of the F2 generation (A). Effect of chronic
i.p. injection of vehicle (sterile water) on dominant–submissive behavior (B).
Changes in dominance level of imipramine-treated compared to vehicletreated animals (C). Data points significantly different between dominant
(Dom, n = 5) and submissive (Sub, n = 5) groups of animals are marked (*)
at p b 0.05, (**) at p b 0.01, and (***) at p b 0.001.
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The stability of the Dominant–Submissive Relationships
was tested on males of the F4 generation. After selection, both
dominant and submissive mice were treated (i.p.) with sterile
water for three consecutive weeks. Chronic vehicle administration did not change previously established DSRs among
these animals (Fig. 5B). A comparison of dominance levels
between animals treated with imipramine and those who
received vehicle is depicted on Fig. 5C.
Sabra male that were subjected to selective breeding were
evaluated with the Elevated plus-maze (EPM) and Open Field
tests. No significant differences in behavior between dominant, submissive and control (randomly bred) groups were
found in the EPM test (Table 2). The Open Field test did not
reveal any changes in the horizontal activity between
experimental groups (Fig. 6A). However, a significant
difference in the vertical activity (rearing) was found
between F4 submissive and control Sabra males (Fig. 6B).
4. Discussion
This work's major findings are twofold: that breeding
Sabra mice for dominant and submissive traits resulted in
progressively larger numbers of pairs in each consecutive
generation of animals meeting the DSR criteria, and that
statistically significant DSRs were formed more rapidly in
filial than in the parental generations. These findings suggest
that Dominant–Submissive Relationships defined in the DSR
test have an inheritable genetic component. This finding
supports the face validity of the RSBM described here as a
disease model of depression since clinical studies suggest that
major depression has a genetic component. This conclusion is
strengthened by the use of outbred animals that are more
genetically diverse than the inbred strains used in previous
studies. Like the other mouse strains studied, the outbred
Sabra mice form discrete numbers of DSR pairs, and the
submissive members of these pairs respond to imipramine
treatment with reduced submissive behavior. While encouraging, this is a limited result and further studies need to be
Fig. 6. Horizontal (A) and vertical (B) activity of F4 generation dominant
(Dom, n = 10), submissive (Sub, n = 10) and randomly bred (control, n = 10)
Sabra mice in the Open Field test. Data points significantly different between
submissive and control group of animals are marked (**) at p b 0.01.
done to support the use of these mice in this behavioral
model. Previously we have shown that mice can form DSR
using the inbred C57BL/6J strain (Malatynska et al., 2005). We
show here that Sabra animals are also capable of developing
stable Dominant–Submissive Relationships (Fig. 3A and F).
Several researchers have used increased frequency of a trait
in populations subjected to selective breeding as a measure
of the genetic component of the trait under investigation
Table 2
Behavior data of four generations (P, F1,3,4) of Sabra dominant (Dom), submissive (Sub) and control male mice in the EPM test.
P
F1
Dom (n = 4)
OE
CE
TE
OT
CT
Sub (n = 4)
Cont (n = 8)
Dom (n = 5)
Mean
SEM
Mean
SEM
Mean
SEM
12.50
21.75
34.25
66.00
117.00
1.76
1.11
1.03
6.10
10.62
16.00
17.75
33.75
91.25
107.80
2.68
0.85
3.47
9.63
10.35
11.38
17.25
28.63
73.38
118.10
2.28
1.40
2.42
12.53
8.48
F3
OE
CE
TE
OT
CT
8.80
13.80
22.60
61.60
99.60
Cont (n = 10)
SEM
Mean
SEM
Mean
SEM
2.31
0.80
2.77
14.12
11.48
13.40
12.20
25.60
109.20
87.80
0.98
1.32
1.33
12.15
10.20
10.20
14.20
24.40
81.10
102.80
1.24
0.79
1.47
10.52
5.73
F4
Dom (n = 10)
Sub (n = 10)
Mean
Mean
12.40
16.30
28.70
104.60
106.00
Mean
Sub (n = 5)
SEM
1.08
1.43
1.84
8.49
6.70
9.80
15.80
25.60
80.10
124.30
SEM
0.83
0.99
1.45
6.91
6.29
Cont (n = 12)
Dom (n = 16)
Sub (n = 16)
Mean
Mean
Mean
10.83
13.75
24.58
97.67
112.90
SEM
0.98
1.07
1.55
7.48
5.83
12.75
15.81
28.51
74.56
139.60
SEM
1.54
1.07
2.27
7.73
7.04
12.13
18.31
30.44
52.63
118.90
Cont (n = 16)
SEM
0.99
0.77
1.48
6.02
5.01
Mean
SEM
11.06
15.94
27.00
54.81
136.6
1.17
0.93
1.67
6.09
10.96
The values are means ± SEM with the number of animals for each generation.
OE — open arms entries; CE — closed arms entries; TE — total entries; OT — open arms duration (s); CT — closed arms duration (s).
Please cite this article as: Feder, Y., et al., Selective breeding for dominant and submissive behavior in Sabra mice, J. Affect.
Disord. (2010), doi:10.1016/j.jad.2010.03.018
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(Hitzemann et al., 2008; Pulido et al., 1996; Verle et al., 2000).
For example, genetic involvement in catalepsy was shown by
the increasing percent of animals with catalepsy across
generations of animals selected for this trait (Kondaurova
et al., 2006). Breeding submissive and dominant animals separately strengthened the presence of these two behavioral
traits in the population, as reflected in the greater number of
pairs forming DSR, and by the shorter time needed to develop
DSR between paired animals in later generations. There was a
progressive increase in the number of pairs forming DSR across
generations (parental and four generations of offspring) as
shown in Fig. 2A. Statistically significant differences in post hoc
analysis were achieved between parental and F3 and F4 generations. Two-Way ANOVA showed no significant difference
between number of pairs formed by males and females across
generations. This finding is consistent with our previous results
conducted on Sprague Dawley rats showing no difference
between sexes in number of pairs developing DSR (Malatynska
et al., 2002).
The second measure of the genetic component of the traits
under investigation was latency of DSR formation. In randomly
bred Sabra as well as C57BL/6J mice and Sprague Dawley rats,
significant DSR developed in the second week of interaction
(Fig. 3A and F (Malatynska et al., 2002; Malatynska and Knapp,
2005; Malatynska et al., 2007b)). To compare the DSR latency
between generations in our work, we defined a relationship
onset time (ROT) value as the time necessary to reach 50% of
the maximal dominance level of the DSR pairs of a given
generation. Maximal dominance level value was derived from
non-linear regression analysis (Fig. 4A and B). Generally, the
latency of DSR development was significantly shorter for selectively bred male and female Sabra mice than for randomly bred
Sabra mice (Fig. 4C). This may indicate a genetic component of
dominant and submissive traits. Although ROT values remained
constant across offspring generations, some non-linearity occurred in the F2 generation (Fig. 4C). This ceiling effect, which
appeared exclusively in generation F1 may be attributed to
several factors, including relatively small sample size, analysis
of two traits simultaneously, and the complexity of inheritance.
It is well established that valid animal models of diseases
such as depression or mania should be characterized by three
measures: predictive, face, and construct validity (Willner,
1984, 1995). Genetic control would support the face validity
of our work, which is based on the presence of neurochemical
changes observed in the model and during the illness and
genetic control is expressed by a neurochemical features observed in psychiatric disorders. There is common agreement
that the spectrum of bipolar disorders is under the control of
several genes (Carter, 2007; Elashoff et al., 2007). There are
several reports related to genetic aspects of dominance and
subordination traits (Chapais and Shulman, 1980; Jarrell et
al., 2008; Jozifkova and Flegr, 2006; Kaesermann and Baettig,
1977; McCobb et al., 2003; Vera Cruz and Brown, 2007).
However, the detailed nature of the genetic control of dominant and submissive traits remains to be elucidated. In this
work, we produced data supporting that genetic mechanisms
hold at least partial control over dominance and submissiveness in Sabra mice. Our findings are consistent with the study
by Masur and Benedito (1974) which found that the frequency of dominant or submissive behavior increases among
rats bred for these traits. The graded increase in pair forma-
tion frequency and latency for DSR onset are suggestive of a
multi-gene rather than a single-gene trait. This is consistent
with existing evidence for a genetic predisposition to mania
and depression.
It has been previously shown that submissive behavior of
mice and rats measured in pair food competition tests was
sensitive to antidepressants desipramine, imipramine, amoxapine, and fluoxetine (Malatynska et al., 2002; Malatynska et
al., 2005; Pinhasov et al., 2005). In our study, we have shown
that submissiveness of male Sabra mice is reduced by treatment with antidepressant imipramine (10 mg/kg) for three
weeks and remains significantly reduced after a fourth week
of treatment, the longest time studied (Fig. 5A). At the same
time, chronic treatment of F4 animals with vehicle (sterile
water) for three consecutive weeks did not affect animals'
dominant–submissive behavior (Fig. 5B). This is similar to the
stability of controls in rat DSR (Malatynska et al., 2007b;
Pinhasov et al., 2005). Analyses of animals' behavior in the
EPM and Open Field tests did not reveal any significant differences between dominant, submissive and control species
(Table 2, Fig. 6A). The only significant difference between F4
submissive and control males has been observed in vertical
activity (Fig. 6B). This phenomenon should be further evaluated. The results of our experiments strengthen the potential of
submissive Sabra mice to serve as a model of depression.
However, a full characterization of its predictive validity should
be conducted.
In summary, we have demonstrated genetic control of dominant and submissive behavioral traits as expressed in fixedpair competition tests in Sabra mice. These results strengthen
the face validity of DSR based models for depressive and manic
disorders. Use of these selectively bred animals with prominent
dominant and submissive features in drug testing could reduce
the number of animals entering experiments, while increasing
throughput for testing putative antidepressant or antimanic
drugs. Clearer genotypes can aid in better understanding of
submissiveness and dominance traits, which are important
elements in the etiology of depressive and manic disorders,
respectively.
Role of funding source
Nothing declared.
Conflict of Interest
No conflict declared.
Acknowledgement
We thank Dr. Richard Knapp for reading the manuscript
and for helpful discussions and suggestions.
References
Avraham, Y., Menachem, A.B., Okun, A., Zlotarav, O., Abel, N., Mechoulam, R.,
Berry, E.M., 2005. Effects of the endocannabinoid noladin ether on body
weight, food consumption, locomotor activity, and cognitive index in
mice. Brain Res. Bull. 65, 117–123.
Brunelli, S.A., Hofer, M.A., 2007. Selective breeding for infant rat separationinduced ultrasonic vocalizations: developmental precursors of passive
and active coping styles. Behav. Brain Res. 182, 193–207.
Carter, C.J., 2007. Multiple genes and factors associated with bipolar disorder
converge on growth factor and stress activated kinase pathways
controlling translation initiation: implications for oligodendrocyte
viability. Neurochem. Int. 50, 461–490.
Please cite this article as: Feder, Y., et al., Selective breeding for dominant and submissive behavior in Sabra mice, J. Affect.
Disord. (2010), doi:10.1016/j.jad.2010.03.018
ARTICLE IN PRESS
Y. Feder et al. / Journal of Affective Disorders xxx (2010) xxx–xxx
Chapais, B., Shulman, S.R., 1980. An evolutionary model of female dominance
relations in primates. J. Theor. Biol. 82, 47–89.
Chia, R., Achilli, F., Festing, M.F., Fisher, E.M., 2005. The origins and uses of
mouse outbred stocks. Nat. Genet. 37, 1181–1186.
Elashoff, M., Higgs, B.W., Yolken, R.H., Knable, M.B., Weis, S., Webster, M.J.,
Barci, B.M., Torrey, E.F., 2007. Meta-analysis of 12 genomic studies in
bipolar disorder. J. Mol. Neurosci. 31, 221–243.
File, S.E., Ouagazzal, A.M., Gonzalez, L.E., Overstreet, D.H., 1999. Chronic
fluoxetine in tests of anxiety in rat lines selectively bred for differential
5-HT1A receptor function. Pharmacol. Biochem. Behav. 62, 695–701.
Gobshtis, N., Ben-Shabat, S., Fride, E., 2007. Antidepressant-induced undesirable weight gain: prevention with rimonabant without interference
with behavioral effectiveness. Eur. J. Pharmacol. 554, 155–163.
Gonzalez, L.E., File, S.E., Overstreet, D.H., 1998. Selectively bred lines of rats
differ in social interaction and hippocampal 5-HT1A receptor function: a
link between anxiety and depression? Pharmacol. Biochem. Behav. 59,
787–792.
Henn, F.A., Vollmayr, B., 2005. Stress models of depression: forming
genetically vulnerable strains. Neurosci. Biobehav. Rev. 29, 799–804.
Hitzemann, R., Malmanger, B., Belknap, J., Darakjian, P., McWeeney, S., 2008.
Short-term selective breeding for high and low prepulse inhibition of the
acoustic startle response; pharmacological characterization and QTL
mapping in the selected lines. Pharmacol. Biochem. Behav. 90, 525–533.
Jarrell, H., Hoffman, J.B., Kaplan, J.R., Berga, S., Kinkead, B., Wilson, M.E., 2008.
Polymorphisms in the serotonin reuptake transporter gene modify the
consequences of social status on metabolic health in female rhesus
monkeys. Physiol. Behav. 93, 807–819.
Jozifkova, E., Flegr, J., 2006. Dominance, submissivity (and homosexuality) in
general population: testing of evolutionary hypothesis of sadomasochism by Internet-trap-method. Neuro. Endocrinol. Lett. 27, 711–718.
Kaesermann, H.P., Baettig, K., 1977. Genetic and environmental effects on
food-competition in paired rats [proceedings]. Act. Nerv. Super. (Praha)
19, 257–258.
Kogan, N.M., Schlesinger, M., Peters, M., Marincheva, G., Beeri, R., Mechoulam, R., 2007. A cannabinoid anticancer quinone, HU-331, is more potent
and less cardiotoxic than doxorubicin: a comparative in vivo study.
J. Pharmacol. Exp. Ther. 322, 646–653.
Kondaurova, E.M., Bazovkina, D.V., Kulikov, A.V., Popova, N.K., 2006. Selective
breeding for catalepsy changes the distribution of microsatellite D13Mit76
alleles linked to the 5-HT serotonin receptor gene in mice. Genes Brain
Behav. 5, 596–601.
Malatynska, E., Knapp, R.J., 2005. Dominant–submissive behavior as models
of mania and depression. Neurosci. Biobehav. Rev. 29, 715–737.
Malatynska, E., Kostowski, W., 1984. The effect of antidepressant drugs on
dominance behavior in rats competing for food. Pol. J. Pharmacol. Pharm.
36, 531–540.
Malatynska, E., Goldenberg, R., Shuck, L., Haque, A., Zamecki, P., Crites, G.,
Schindler, N., Knapp, R.J., 2002. Reduction of submissive behavior in rats:
a test for antidepressant drug activity. Pharmacology 64, 8–17.
9
Malatynska, E., Rapp, R., Harrawood, D., Tunnicliff, G., 2005. Submissive
behavior in mice as a test for antidepressant drug activity. Pharmacol.
Biochem. Behav. 82, 306–313.
Malatynska, E., Pinhasov, A., Creighton, C.J., Crooke, J.J., Reitz, A.B., Brenneman,
D.E., Lubomirski, M.S., 2007a. Assessing activity onset time and efficacy for
clinically effective antidepressant and antimanic drugs in animal models
based on dominant–submissive relationships. Neurosci. Biobehav. Rev. 31,
904–919.
Malatynska, E., Pinhasov, A., Crooke, J.J., Smith-Swintosky, V.L., Brenneman,
D.E., 2007b. Reduction of dominant or submissive behaviors as models
for antimanic or antidepressant drug testing: technical considerations.
J. Neurosci. Methods 165, 175–182.
Masur, J., Benedito, M.A., 1974. Genetic selection of winner and loser rats in a
competitive situation. Nature 249, 284.
McCobb, D.P., Hara, Y., Lai, G.J., Mahmoud, S.F., Flugge, G., 2003. Subordination
stress alters alternative splicing of the Slo gene in tree shrew adrenals.
Horm. Behav. 43, 180–186.
Pick, C.G., Yanai, J., 1989. Studies into the mechanisms of strain differences in
hippocampus-related behaviors. Behav. Genet. 19, 315–325.
Pinhasov, A., Crooke, J., Rosenthal, D., Brenneman, D., Malatynska, E., 2005.
Reduction of Submissive Behavior Model for antidepressant drug activity
testing: study using a video-tracking system. Behav. Pharmacol. 16, 657–664.
Pulido, F., Berthold, P., van Noordwijk, A.J., 1996. Frequency of migrants and
migratory activity are genetically correlated in a bird population: evolutionary implications. Proc. Natl. Acad. Sci. U. S. A. 93, 14642–14647.
Sumariwalla, P.F., Gallily, R., Tchilibon, S., Fride, E., Mechoulam, R., Feldmann,
M., 2004. A novel synthetic, nonpsychoactive cannabinoid acid (HU-320)
with antiinflammatory properties in murine collagen-induced arthritis.
Arthritis Rheum. 50, 985–998.
Touma, C., Bunck, M., Glasl, L., Nussbaumer, M., Palme, R., Stein, H., Wolferstatter,
M., Zeh, R., Zimbelmann, M., Holsboer, F., Landgraf, R., 2008. Mice selected
for high versus low stress reactivity: a new animal model for affective
disorders. Psychoneuroendocrinology 33, 839–862.
Vera Cruz, E.M., Brown, C.L., 2007. The influence of social status on the rate of
growth, eye color pattern and insulin-like growth factor-I gene expression
in Nile tilapia, Oreochromis niloticus. Horm. Behav. 51, 611–619.
Verle, P., Nhan, D.H., Tinh, T.T., Uyen, T.T., Thuong, N.D., Kongs, A., Stuyft, P.,
Coosemans, M., 2000. Glucose-6-phosphate dehydrogenase deficiency in
northern Vietnam. Trop. Med. Int. Health 5, 203–206.
Willner, P., 1984. The validity of animal models of depression. Psychopharmacology (Berl) 83, 1–16.
Willner, P., 1995. Animal models of depression: validity and applications.
Adv. Biochem. Psychopharmacol. 49, 19–41.
Yanai, J., Bergman, A., Shafer, R., Yedwab, G., Tabakoff, B., 1981. Audiogenic
seizures and neuronal deficits following early exposure to barbiturate.
Dev. Neurosci. 4, 345–350.
Please cite this article as: Feder, Y., et al., Selective breeding for dominant and submissive behavior in Sabra mice, J. Affect.
Disord. (2010), doi:10.1016/j.jad.2010.03.018