Genetic independence of mouse measures
of some aspects of novelty seeking
Christopher L. Kliethermes* and John C. Crabbe
Department of Behavioral Neuroscience, Oregon Health & Science University and Portland Alcohol Research Center, Veterans Affairs Medical Center,
Portland, OR 97239-3098
Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved February 5, 2006 (received for review
November 8, 2005)
High novelty seeking is a complex personality attribute correlated
with risk for substance abuse. There are many putative mouse
models of some aspects of novelty seeking, but little is known of
genetic similarities among these models. To assess the genetic
coherence of ‘‘novelty seeking,’’ we compared the performance of
14 inbred strains of mice in five tests: activity in a novel environ-
ment, novel environment preference, head dipping on a hole-
board, object preference, and a two-trial version of the spontane-
ous alternation task. Differences among strains were observed for
all tasks, but performance in any given task was generally not
predictive of performance in any other. To evaluate similarities
among these tasks further, we selectively bred lines of mice for
high or low head dipping on the hole-board. Similar to results from
the inbred strain experiments, head dipping was not correlated
with performance in the other measures but was genetically
correlated with differences in locomotor activity. Using two ap-
have found little evidence that these partial models of novelty
unified construct called novelty seeking. Based on the substantial
influence of genetic factors, ease of implementation, and relative
independence from general locomotion, head dipping on a hole-
board is a good task to use in the domain of novelty seeking, but
multiple tasks, including others not tested here, would be needed
to capture the full genetic range of the behavioral domain.
drug abuse ? inbred strains
theme in many personality scales. Novelty seeking (1), sensation
seeking (2, 3), and impulsivity?behavioral approach have been
described as extensions of Eysenck’s personality model (4). They
that are associated with an underlying biological substrate such as
monoamine level or turnover, which provides a plausible basis for
natural selection of the trait. Polymorphisms in human genes
involved in monoaminergic, and particularly dopaminergic, signal-
ing are predictive of the degree of novelty seeking (5, 6), although
some associations are controversial (7). Whichever personality
construct is considered, a well replicated finding is that individuals
characterized as high novelty seekers are more likely to abuse or be
dependent on drugs of abuse than are low novelty seekers (see ref.
8 for a review).
Animal models of novelty seeking rely on the overt expression of
a behavior and are often described by the degree to which the
animal either explores a novel stimulus (such as an open field, e.g.,
ref. 9) or demonstrates a preference for novel as compared with
Individual differences in some aspects of novelty seeking are also
inferred from tasks assessing neophobia (or preference) for novel
interaction with conspecifics, among many others. All of the above
tasks involve competing tendencies of the animal to explore and
avoid. A human temperament and personality inventory (1, 11, 12)
he idea that an individual’s preference for new and exciting
stimuli describes some core aspect of personality is a central
distinguishes subscales, including novelty seeking, harm avoidance,
avoidance and reward dependence in this scale. Other domains of
human novelty seeking such as impulsivity have traditionally been
measured in rodents by using operant procedures (e.g., ref. 13). It
is unreasonable to expect an animal model for a complex human
trait to capture all aspects of that trait (14, 15). Rather, certain key
features can be targeted by multiple, partial animal models.
Many rodent tasks conflate a reaction to a novel situation with
a tendency to seek novelty. Thus, these simple models of novel
environment reactivity do not allow a dissociation of general
activity that exists under all situations, including the home cage,
from activity that is specific to a task. Furthermore, they are likely
seeking tendencies. For example, Piazza et al. (16) demonstrated
that rats that show higher activity in a novel environment also have
higher levels of corticosterone during the behavioral assessment,
but whether corticosterone is a cause or an effect of locomotion
over familiar stimuli could be considered a more direct reflection
of novelty seeking, because a comparison can be made to chance
levels of preference for novel relative to familiar stimuli within an
animal. These tasks, though, are hindered by more complicated
and, consequently, long-term memory-dependent experimental
procedures and often reintroduce the stress?novelty seeking con-
found during the preference assessment. Interpretation of partial
rodent models of ‘‘novelty seeking,’’ therefore, must include con-
sideration of memory (in novelty preference tasks) and stress. In
addition, some of the same tasks (e.g., exposure to an open field)
are used to model anxiety-like states (17), and induction of an
anxious state may underlie neophobia in some circumstances (18).
measures of novelty seeking as well as many other tests of emo-
tional-like behaviors. The existence of many inbred mouse strains,
each composed of genetically identical individuals, allows compar-
ison of the influence of genes on multiple behavioral endpoints,
because naı ¨ve mice can be used for each behavioral test. Because
all same-sex members of any inbred strain are genetically identical,
when animals from multiple strains are tested under controlled
environmental conditions any differences among inbred strains
reflect those allelic differences, because they are modulated by
prenatal and postnatal environmental factors, including maternal
behavior. Individual differences within a strain are caused by the
(19). Furthermore, knowing how inbred strains perform relative to
each other in one task can sometimes predict their relative perfor-
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Freely available online through the PNAS open access option.
Abbreviations: HEB, high exploratory behavior; LEB, low exploratory behavior.
*To whom correspondence should be sent at the present address: Ernest Gallo Clinic &
Research Center, Department of Neurology, University of California at San Francisco,
5858 Horton Street, Suite 200, Emeryville, CA 94608. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
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mance in other tasks. For example, inbred strain rank order in
reactivity to a modified mirrored chamber is correlated with their
rank order reactivity in an elevated plus maze (20). Because of this,
the general interpretation of these strain mean correlations is that
common genes are influencing performance in the correlated tests
(21); hence, the tests are measuring a similar underlying biological
We sought to determine whether common genes influence
performance in several mouse tests putatively modeling some
aspects of novelty seeking. We examined activity in a novel envi-
and object preference on a hole-board (e.g., ref. 22), and sponta-
neous alternation (23). These choices were based on both their
frequency of use in the literature and, in the case of novel
environment preference, the degree to which the task could indi-
cate a relative preference for novelty relative to familiarity. Other
studies have addressed similar issues targeting the construct ‘‘anx-
iety,’’ in some cases using measures similar to those we report.
Beuzen and Belzung (17) studied eight inbred mouse strains using
a light?dark box, a ‘‘free exploration’’ test (similar to our novel
responses in the light?dark and learning tests (factor 1) from those
in the exploration test (factor 2). Although most strains did not
differ in the exploration task, BALB?c female mice were highly
exploratory whereas CBA mice scored lowest (17). A similarly
conceived effort compared individual F1and F2hybrid mice for
responses in tests where a novel object was introduced to a familiar
light?dark, elevated plus maze, and hole-board tests (24). Among
plus maze responses (factor 2) from novel object and environment
preference (factor 1), but it was not designed to detect genetic
The approach we took was to compare the patterns of difference
in performance of inbred strains of mice on the five putative
measures of novelty seeking. Genetic correlations among the tasks
were estimated from the means of 14 inbred strains measured in all
tasks. We identified exploratory head dipping on a hole-board as a
highly heritable trait and subsequently selectively bred mice from a
B6D2F3 starting population for divergent expression of head
mice in three of the other novelty seeking tasks at each generation
of selection as a second technique to identify genetic correlations
among the tasks.
For more information about results, see Tables 1–7 and Figs. 4–6,
which are published as supporting information on the PNAS
seeking are described in Supporting Text, which is published as
supporting information on the PNAS web site, and shown in Fig. 4
A–F. The strain-effect sizes for all dependent variables are shown
in Table 1, and strain-effect sizes within each of the two passes of
the experiment are shown in Table 2. Strain-effect sizes were
and head dipping (R2? 0.55).
Genetic Correlations. To simplify the analysis, only the principal
dependent variable and a measure of activity from each test were
included in the correlational analysis; the variables discussed are
indicated in Table 3, which shows all pairwise genetic correlations.
of novelty seeking were moderately to strongly correlated: degree
of preference for a novel environment, head dipping, and rearing
(all r ? ?0.46?). However, the correlations with novel environment
preference seemed to be driven by strain A?J, which showed a
the inbred strain means of the novelty seeking mea-
sures. Each point represents an inbred strain mean,
and the lines represent the least squares regression
of rears during the initial 30-min activity assessment,
and ‘‘Activity’’ refers to the distance traveled (in cen-
timeters) during this same test. ‘‘Novel Environment
Preference’’ refers to the amount of time spent in the
head dips on the hole-board. ‘‘Object Preference’’ in-
dicates the percentage of time spent head dipping for
holes relative to the total time spent head dipping,
strain that chose the novel arm. NOD refers to strain
NOD?LTJ and is labeled to indicate this strain’s influ-
ence on the correlations with spontaneous alterna-
tion, and A?J is similarly shown as an outlier for the
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March 28, 2006 ?
vol. 103 ?
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strong aversion to the novel compartment. Removing this strain
from the analysis reduced these values (r ??0.39?; data not shown).
On the other hand, spontaneous alternation and object preference
were generally uncorrelated with the other novelty seeking mea-
sures (r ? ?0.27?), except that the correlation of spontaneous
alternation and rears tended to be higher (r ? ?0.46).
A single strain, NOD?LTJ, appeared strongly to influence the
magnitude (and perhaps direction) of the correlations with spon-
taneous alternation. Removing this strain resulted in lower values
for these correlations (all r ? 0.19; P ? 0.54). The degree of
preference for objects was not significantly correlated with any
initial test of activity in a novel environment was relatively strongly
0.77), and both measures were highly genetically correlated with
Locomotor activity in the initial test in a novel environment was
highly correlated with activity recorded concurrently in all other
novelty seeking tests (all r ? 0.8 and P ? 0.01; see Fig. 5) as well
as with the number of head dips and preference for a novel
environment. Given this strong relationship between locomotion
and most dependent measures, we examined the pattern of corre-
of locomotion by regressing the main dependent measure from
each task on locomotion as measured in that task. The result was
a residual score for each mouse that reflected the degree to which
this individual deviated from the best-fit regression line, and this
residual was explicitly uncorrelated with locomotion (r ? 0). Strain
means were then calculated from individual residual scores, and
genetic correlations among the mean residual scores were calcu-
lated. Residuals had a heritable component equal to or slightly less
than that of the main dependent measure for each task (see Table
1), and the overall pattern of correlations among the remaining
dependent measures was unchanged, with only the correlation of
the residual score of head dipping and rearing approaching signif-
icance (r ? 0.53, P ? 0.06; data not shown). Thus, a genetically
similar, heritable component exists between these two behaviors
that cannot be completely explained by locomotor activity.
The pattern of genetic correlations across the inbred strains
suggests that object preference and spontaneous alternation are
genetically distinct from each other and largely unrelated to any of
the other tasks, whereas activity in a novel environment is corre-
lated with most other measures. This pattern is similar to that
obtained from correlating the dependent measures of each mouse
from all tests; i.e., the phenotypic correlations among dependent
measures are similar to the genotypic correlations (see Table 4).
To characterize the relationships among the variables further we
also performed a principal-components analysis of the individual
phenotypic data from all of the novelty seeking measures. This
analysis is described in Supporting Text, and the results are shown
in Table 5. Unlike the genetic correlations, the principal-
components analysis indicated that, at the phenotypic level, novel
each other than they are with any other trait. Therefore, we wished
to test the relationships among the tasks further by using a second
genetic approach: selective breeding for high and low head dipping
as measured in the hole-board apparatus.
Selected Lines: Response to Selection. The head dipping scores for
the B6D2F3 founding population were normally distributed with a
range of 21–116 head dips (data not shown), a range similar to that
deal of variation in the starting population from which to select for
high and low head dipping. Small differences between high explor-
atory behavior (HEB) and low exploratory behavior (LEB) mice
were apparent after one generation of selection (see Fig. 2 A and
B; generation S1, combined R2? 0.03) and were larger after two
generations (R2? 0.11). By the fifth selected generation, selected
line accounted for 15% of the total variation. The selection
parameters at each generation of selection are shown in Table 7.
The realized heritability estimate was slightly larger in both upward
replicates (h2? 0.14 for HEB-1 and 0.20 for HEB-2) than in the
downward direction (h2? 0.11 for LEB-1 and 0.04 for LEB-2).
Heritability for the divergence between HEB and LEB mice was
estimated as 0.13 for both replicates.
Selected Lines: Correlated Responses to Selection. LEB mice of both
of selection (main effect of line at S5, F1,128? 39.48; P ? 0.0001;
see Fig. 2 C and D) as well as during all other correlated response
testing (data not shown). The mice selected as breeders for both
LEB lines moved substantially less than the population mean and
particularly less than the HEB breeding pairs. The HEB mice
selected as breeders, however, did not differ substantially from the
population mean for locomotion at any generation of selection,
indicating that the selection response for head dips in the upward
The overall dissociability of locomotion from head dipping is
supported by an analysis of covariance, in which the proportion of
variation in head dipping that can be accounted for by locomotion
is removed before any potential difference in head-dipping due to
the selected lines is considered. At generation 5, a significant main
effect of selected line on the number of head dips was still present
by analysis of covariance (F1,127? 8.03; P ? 0.01).
At each generation of selection, all mice were tested first on the
hole-board and, the next day, in the object preference task. One to
and 1 day later they were tested for spontaneous alternation in a
generations of selection, only the results from selection generation
novel environment preference were observed, there were no dif-
ferences between the selected lines in the degree of preference
(Fs1,132 ? 1.04) and no effect of replicate or line by replicate
interaction (Fs1,132 ? 2.70; P ? 0.1). There were likewise no
differences in the numbers of HEB and LEB mice that chose the
of the tests considered, and, in the case of preference for objects,
and LEB mice. (A and B) The head dipping response across five generations of
selection. Connected points indicate the line means, and the unconnected
points represent the mice selected at each generation as breeders that pro-
duced the offspring of the following generation. (C and D) Activity (beam
breaks) in HEB and LEB mice across the entire experiment (HEB and LEB mice
were not selected for this trait). Means ? SEM are depicted.
www.pnas.org?cgi?doi?10.1073?pnas.0509724103Kliethermes and Crabbe
from the inbred strain experiments suggests that, whereas most of
the behaviors putatively indexing aspects of novelty seeking cor-
related relatively highly with locomotion, the novelty-related be-
haviors are at best modestly genetically correlated with each other.
This pattern was also seen in the selectively bred HEB and LEB
lines, which showed large, correlated differences in locomotor
activity in all apparatus but did not consistently differ in perfor-
mance in other tasks that putatively measured aspects of novelty
seeking other than head dipping. Given the very different modal-
ities of responses recorded in each task (locomotion, time spent in
an area, choice behavior, etc.), this finding of few correlations may
not be unexpected. However, it implies that the trait being mea-
sured by the various tasks either is not isomorphic across the
behavioral assays or is phenotypically complex, and each task is
that most clearly reflected the actual preference for novelty, was
moderately influenced by genotype (R2? 0.33). In a similar
apparatus, but using a different procedure, Belzung and her col-
exploratory procedure, which generally indicated few strain differ-
ences, although more differences among other inbred strains were
found in response to predator odor exposure. These results imply
that genotype might play a relatively small role in the expression of
to be sensitive to different factors, such as age at weaning or other
experiences (27–30). Data in Fig. 1 and Table 4 show that sponta-
neous alternation is largely unrelated to all of the other tasks.
Head dipping on a hole-board is a commonly used task in many
types of pharmacological, genetic, or neural lesion experiments as
a measure of exploratory and?or anxiety-like behavior (31, 32). In
the inbred strain experiment, a strong genotypic correlation be-
tween head dipping and locomotion was observed (r ? 0.77). If
genotype is ignored and individual values for head dipping and
locomotion correlated, a similar strong correlation was found (r ?
0.63, see Table 4). However, within a given inbred strain the
magnitude of the phenotypic correlation varied greatly: some
strains showed strong positive correlations, whereas others showed
correlations between head dipping and locomotion (see Fig. 6).
HEB and LEB mice at the fifth generation of selection also
exhibited a genetically correlated difference in locomotor activity,
and within each line and replicate the phenotypic association was
variable (r ? 0.22–0.67; data not shown). Collectively, these results
indicate a somewhat unpredictable relationship between head
suggest that the choice of mouse strain used for an experiment with
the hole-board can have a large influence on this correlation.
Despite a high heritability estimate from the inbred strain
experiment, the response to selection for head dipping proceeded
LEB mice of both replicates, with a realized heritability estimate of
0.13 for each replicate after five generations of selection. In
retrospect, the modest response is most likely due to the choice of
differences were observed between these two strains in our first
inbred strain comparison, this difference was not found in the
second, which indicated in the aggregate that only modest additive
genetic variance relating to head dipping existed in the starting
population. Given the slow response to selection, it is probable that
trait. It does not appear that there are a few genes with large effect
on head dipping present in the cross, or else a much more rapid
divergence would have been expected.
The presence of a genetic correlation between two tasks is
typically taken to indicate the action of common genes in influ-
encing performance in the tasks, a situation called pleiotropy. The
implication is that a survey of genotypes in two similar tasks should
different or the strain means do not correlate, the tasks could be
measuring independent processes or genetically unrelated aspects
of the same process (33). The reliability of each task must also be
influence between the measures, but it could indicate that one or
both of the tasks are simply not reliable enough to detect a
correlation that might actually exist. Furthermore, detection of a
genetic correlation depends on a measurable genetic contribution
to each task, and the larger the genetic effect size for each trait, the
more likely a genetic association is to be detected.
With these limitations in mind, locomotion and the measures
most closely associated with it, rearing and head dipping, are the
most heritable and interrelated measures in the current experi-
ments. Selective breeding of HEB and LEB mice resulted in
populations of mice that displayed correlated differences in loco-
motion during the selection test. Object and novel environment
preference, which demonstrated much smaller between-strain dif-
ferences, tended to resemble each other phenotypically more than
they did any other measure. Spontaneous alternation likewise
tended to be unrelated to the other measures. Similar to the results
obtained with the inbred strains, HEB and LEB mice did not show
consistent differences in any of these measures, including the
phenotypically similar object preference task, further indicating
that these measures are largely dissimilar.
Task-specific stress or anxiety-like states could also contribute to
the current pattern of results, but the relationship to novelty
depends on the circumstances of testing. Handling mice activates
the hypothalamic–pituitary–adrenal axis, leading to elevations in
to the novel environment preference test used in the current
experiments; mice that were habituated to one compartment for
dashed line indicates chance performance in all measures. Above-chance levels were observed in all tests, but no line differences were apparent. (n ? 24–44 per
genotype.) Means ? SEM are depicted.
Object preference (A), novel environment preference (B), and spontaneous alternation (C) in HEB and LEB mice after five generations of selection. The
Kliethermes and CrabbePNAS ?
March 28, 2006 ?
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24 h showed no elevations of corticosterone when allowed to
explore a novel compartment. However, mice forced to remain in
levels, as did mice placed into a different, novel environment (34,
35). In our studies, mice were habituated to the familiar compart-
because they are stressful, seeking to reduce stress through famil-
iarity. Alternatively, strains could select a novel environment be-
cause the familiar one has become stressful (36).
To explore the role of stress in the HEB?LEB differences, we
performed two additional experiments (see Supporting Text). HEB
and LEB mice were tested in the hole-board, and corticosterone
levels were determined immediately after the test or from untested
animals in the room. Untested HEB and LEB mice did not differ
in corticosterone levels (HEB, 4.4 ? 1.4 ?g?dl; LEB, 4.4 ? 1.4
?g?dl; not significant). Both genotypes showed significant eleva-
tions in corticosterone after exposure to the task, but the increases
were similar (HEB, 16.6 ? 1.7 ?g?dl; LEB, 15.5 ? 2.1 ?g?dl; not
significant). Thus, although stress clearly accompanies the hole-
board exploration test, it cannot explain the genotypic differences
in this behavior. We also tested separate groups of HEB and LEB
mice in the novel environment exploration task after habituating
them for 23–24 h in the familiar compartment, rather than the 15
min we used in the other experiments. In this task there was again
no significant genotypic difference in corticosterone levels that
resulted from free exploration of the novel compartment (HEB,
8.6 ? 1.6 ??dl; LEB, 6.4 ? 1.0 ?g?dl; not significant). After longer
familiarization, these intermediate corticosterone values appeared
to reflect the expected reduction in stress (36). Both genotypes
showed an aversion to the novel compartment: HEB mice spent
405 ? 116 sec of the possible 1,200 sec in the novel compartment,
and LEB spent a comparable 490 ? 75 sec there. These responses
are similar to some inbred strains tested in the free exploratory
procedure previously reported (17, 25, 26), suggesting that the
relative degree of familiarity with an apparatus can have a large
effect on the degree of novelty preference observed. Clearly, the
behavioral differences between HEB and LEB mice cannot easily
be explained on the basis of differences in stress responses to the
testing situation and furthermore demonstrate that stress and
exposure to novelty are related in most test situations.
examined aspects of human novelty seeking that reflect self-
the extent that they reflect responses to novelty given the complex
interactions of novelty, neophobia, anxiety, and activity, these tasks
may most closely correspond to one of four subdimensions named
by the Tridimensional Personality Questionnaire (1, 11) and elab-
orated in the Temperament and Character Inventory (12), called
exploratory excitability. However, the traditional indices of rodent
exploration, activity in a novel environment and head dipping on a
the novel environment preference test, which relies on the overt
expression of preference for the novel compartment. This last test
could reflect some aspects of the impulsivity component of novelty
seeking, because the expression of preference will be influenced by
latency to enter the novel compartment.
Overall, these data suggest that, although there may be an
construct is genotypically complex and confounded with other
factors (notably general activity, anxiety, and stress), and no single
task can claim to measure it. Earlier studies have also tended to
exploratory, anxiety-like, and novelty seeking tendencies (17, 24),
and our data suggest that this is also true at the level of genetic
influences. The current findings highlight the need for a better
models of personality traits, particularly in studies attempting to
identify genetic contributions to individual differences.
Materials and Methods
Subjects and Husbandry. Inbred strain experiments. Male and female
mice of the following inbred strains from The Jackson Laboratory
were used: 129S1?SvImJ, A?J, AKR?J, BALB?cByJ, BTBR?J,
C3H?HeJ, C57BL?6J, C57L?J, DBA?2J, FVB?NJ, NOD?LTJ,
NZB?B1NJ, PERA?EiJ, and PL?J. All mice were received at ?45
days of age and housed in the animal facilities of the Portland
Department of Veterans Affairs Medical Center for a minimum of
2 weeks before beginning the experiments. The experiments were
conducted in two passes that consisted of different strains (n ? 6
per sex per strain) within each pass, but 12 C57BL?6J and 12
DBA?2J mice were included in each pass to determine any effects
Selected line experiments. B6D2F2?J mice were purchased from
The Jackson Laboratory. Random mating pairs were established
to produce an F3hybrid cross, which was subsequently used as
the founding population. Each F3 individual was genetically
unique, possessing either C57BL?6J or DBA?2J alleles at each
gene locus, and had experienced multiple recombinations of the
progenitor strains’ chromosomes. One hundred eleven 55- to
65-day-old naı ¨ve B6D2F3 mice (53 male and 58 female) were
tested on the hole-board as described for the inbred strain
experiments. The 12 male and 12 female mice that showed the
most head dipping behavior in the 10-min task were randomly
divided into two replicate lines of six breeder pairs each to form
the HEB-1 and HEB-2 lines, whereas the lowest-scoring 12 male
and 12 female mice were paired to form the LEB-1 and LEB-2
lines. A mass selection procedure was used; mice were randomly
assigned mates so long as the selected mate was not a sibling.
This procedure was chosen to produce the fastest rate of
response to selection at the expense of somewhat higher rates of
inbreeding. After being selected from the initial, common F3
parental population, each line and replicate was a closed breed-
ing population in which all offspring were tested at each gener-
LEB mice selected as breeders for each subsequent generation
of selection. Six breeder pairs were maintained in each line and
replicate throughout the experiment.
Procedure. All inbred mice were tested serially through the follow-
ing five tests in this order: activity in a novel environment; novel
environment preference; head dipping on a hole-board; object
preference on the hole-board; and spontaneous alternation in a
Y-maze. In the first pass of these experiments, novel object recog-
nition was also evaluated, but, because of problems with the
procedure, this task was not used in the second pass, and the data
are not shown. At each generation of selection of HEB and LEB
mice, the testing procedure was similar to that used for the inbred
on the hole-board, followed by object preference, novel environ-
ment preference, and spontaneous alternation. The testing order
the trait for which the mice would be selectively bred, in experi-
mentally naı ¨ve mice. Activity in a novel environment was not
included in the report of the selection because of high correlations
of behavioral testing are given in Supporting Text.
Activity in a Novel Environment. Mice were placed into one of 12
identical Accuscan automated activity monitors, and locomotion
(distance traveled in centimeters) and the number of rears were
recorded for 30 min by infrared beam disruptions.
Novel Environment Preference. Mice were placed into one side of
four identical two-compartment boxes that were constructed of
acrylic (40 ? 40 cm total area) and consisted of one black-walled
side with a white floor and a white-walled side with a black floor,
www.pnas.org?cgi?doi?10.1073?pnas.0509724103Kliethermes and Crabbe
both separated by a vertically sliding door. Initial placement into
the black or white compartments was counterbalanced within all
genotypes. After a 15-min exposure to one of the compartments,
the door was opened, and the time spent and activity (beam
breaks) in the novel and familiar sides were measured during a
20-min preference test by infrared beam disruptions. Chance
performance in this apparatus is indicated by a time of 600 sec
spent in the novel compartment.
Head Dipping on the Hole-Board.Thenumberofheaddipsandtime
spent head dipping into each of four 2.9-cm diameter holes,
spaced equidistant from each other in the corners of a 40 ?
40-cm board, and horizontal activity counts (beam breaks) were
recorded via infrared beam disruption during a 10-min test.
Object Preference on the Hole-Board. Thedayaftertheheaddipping
assessment, mice were placed back onto the same hole-board
apparatus for a second 10-min test. Two small objects (centrifuge
placed into two of the holes at opposite corners. Preference was
calculated as the amount of time spent head dipping in holes that
had objects divided by the total time spent head dipping (percent
preference). Activity was recorded as the number of beam breaks.
Spontaneous Alternation. A two-trial version of the task was used.
of a Y-maze by occluding one arm with an opaque partition. Each
arm was 15 cm tall and 30 cm long ? 5 cm wide clear acrylic, and
to this arm for ?30 sec and placed into a holding cage while the
were then placed back into the stem of the Y-maze and allowed to
An entrance was defined as all four paws being within the arm.
Because of very high performance in B6D2F2?J and B6D2F3?J
mice (the proportion of mice that chose the novel arm was 90%),
a different procedure was used for the selected line experiments.
On trial 1, no partition was used to obstruct one of the arms, so the
mouse was allowed to enter either arm. The mouse was then
restrained in the chosen arm for ?30 sec, the added partition was
removed, and the mouse then entered either the novel arm or the
arm it was placed in at the start of the test. The apparatus was
cleaned between the testing of individual mice.
Statistics. Inbred strain experiments. To assess potential differences
across passes, the responses of C57BL?6J and DBA?2J mice
were compared on each dependent measure by two-way
ANOVA (genotype X pass). Within these two strains, some
relatively small main effects of pass were observed for some
variables, but, with the exception of head dipping on a hole-
board, no significant genotype X pass interactions were ob-
served. Although the difference in head dipping was clearly
unexpected, it is likely that this interaction is stochastic in origin
considering the large number of dependent variables assessed.
Thus, to simplify the analysis and presentation of the data, all
correlations presented ignore any effects of pass. However,
inbred strain-effect sizes are shown separately for each pass in
Table 2 as well as collapsed across pass in Table 1.
Initial analyses for each dependent variable were by two-way
ANOVA with strain and sex as independent variables. Narrow
sense heritability (h2) was estimated as the percentage of total trait
variance accounted for (R2) by inbred strain in a one-way ANOVA
with strain as the only factor. To correct for the varying influence
of locomotion in the tasks, residual scores were obtained from the
regression of the principal dependent variable from each task
(number of rears, number of head dips, percent preference for
objects, or time spent in the novel compartment) on locomotion as
measured in each task. Strain means for this residual score were
then correlated. Scores for spontaneous alternation were not
corrected because activity data were not collected in this task.
Selected line experiments. Differences between HEB and LEB mice
were estimated similarly to the inbred strain experiments as the
percentage of total trait variation in head dipping accounted for by
selected line. Correlated responses to selection were analyzed by
ANOVA according to Crabbe et al. (37) where the strongest
evidence for a genetic correlation is indicated by a significant
difference in a trait between both replicates of the selected lines.
these selected lines were designed for short-term use in the exper-
iments reported here and were not intended as a long-enduring
model for which nonselected control lines would be appropriate
Mackay (38) as the ratio of the cumulative response to selection at
each of the five generations of selection to the cumulative selection
from the previous generation, and the cumulative selection differ-
ential represents the population-normalized deviation of the mice
selected as breeders from the rest of the population.
We thank C. Belzung for suggestions regarding the novel environment
preference tests; A. Cameron, M. Tanchuck, and N. Yoneyama for
conducting the corticosterone experiments; and D. Finn for comments
on the manuscript. This research was supported by a grant from the U.S.
Department of Veterans Affairs and by National Institutes of Health
Grants AA10760, AA015015, AA13519, and AA12714.
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