Behavioral and physiological effects of

social isolation on mice

Michael Bresticker Zachary Gassman Austin Miller Kari Mueller
Introduction
Housing or social conditions have been shown to impact behavioral and physiological
mechanisms in various ways. Social isolation and lack of social support have deleterious effects
on health, thus being regarded as one of the most relevant causes of human diseases (House,
2001). Rodents are studied extensively for their response to hypothesized stress or anxiety
inducing agents, though many other species have been used. Isolated animals are aggressive and
neophobic as well as show an anxiety-like profile in the elevated plus-maze (Parker and Morinin,
1986). This basic knowledge gives support to the use of individual housing/social isolation,
mainly in rodents, as a model for isolation-mediated diseases (Kim and Kirkpatrick, 1996).
The focus of this experiment is to compare the anxiety like behaviors and corticosterone levels of
socially isolated mice to group-housed mice. Isolation has been shown to induce certain
behavioral responses to stress as well as alterations in the regulation of hormones. Isolation
restricted to an early part of development increases anxiety-like and fear behaviors in adulthood,
and also results in depressed levels of plasma corticosterone (Forster et. al, 2009). The impact of
social isolation on cardiovascular disease risk may be mediated through stress-related
dysregulation of cardiovascular, metabolic, and neuroendocrine processes (Grant et. al, 2009).
The hypothesized link between social isolation and stress is positive and direct in that isolation
increases the likelihood of anxiety behaviors and increases corticosterone production. We
predicted the mice housed individually would show significantly more stress induced anxiety
behaviors in both the elevated plus and open field mazes. Consistent with this prediction, we
also expected to see higher corticosterone concentrations in the fur of the post-stressor isolated
mice compared to the pre-stressor and control samples.
Methods
Animals
The subjects used in this experiment were 14 mature female mice. Mice were housed at the
Loras College rodent facility. All the mice lived in the same housing arrangements with 5-10
other female mice prior to the start of the study. Housing consists of a clear plastic cage with
wood shavings for bedding; consistent food and water were available ad libitum. Lighting was
set on a 12h:12h cycle, and air temperature was kept constant throughout the duration of the
study.
Procedure
Housing conditions were manipulated in order to see if and how it affected stressful behavior and
corticosterone levels. The mice were put in two groups: the social group consisted of 7 mice

housed in the same cage which served as the control; the remaining 7 mice were housed in their
own separate cages which served as the isolated or experimental group. Levels of stress were
examined using behavioral testing methods as well as an enzyme immunoassay. Baseline hair
samples were collected from the mice prior to isolation.
Behavioral Trials
Mice were tested for the behavioral expression of anxiety using two standardized testing
apparatuses: the elevated plus maze and the open field maze. The elevated plus maze is a plus
shaped elevated Plexiglas maze in which a mouse can be on either one of two closed projections
with walls or one of two open projections with no walls. The mice were allowed to roam freely
on the apparatus for 120 seconds total and all behavioral trials were video recorded. The open
field apparatus is a flat square of Plexiglas enclosed by walls. Individual mice can either be on
the exterior near the walls or on the interior not near any walls. The subjects roamed the
apparatus for 120 seconds and all behavioral trials were video recorded. In both the elevated
plus maze and the open field maze, video recordings were scored for several anxiety-like
behaviors.
Enzyme Immunoassay
Fur samples were collected from all 14 mice before and after the isolation manipulationa total
of 28 samples were tested in the EIA. The weight of each individual hair sample was recorded.
Samples were washed using 5mL isopropanol were added to each sample then place on the
mutator for 5 minutes, then centrifuged for another 5 minutes at 10,000 rpm at room temperature.
Excess isopropanol was removed and the washing steps were repeated an additional 2 times.
The samples were left uncapped in a fume hood until the hair was completely dry. Hair samples
were transferred to clean 2 mL centrifuge tubes then 1 mL of methanol was added. After the
samples were placed on the mutator for 48 hours there were centrifuged for 5 minutes at 10,000
rpm. 600 L were pipetted from each sample and put into clean individual 1.5 mL tubes and
were placed in the Speedvac for 2 hours to dry. The samples were then stored in a -20C freezer
for later use; upon removal the samples were reconstituted with 1 mL of 5% methanol. Columns
were prepared for the purification of the samples by attaching stopcocks (Supelco Super Select 1
mL) to 12 X 75mm columns. 1 mL methanol was added to the column and allowed to flow
through; this step was repeated with 1 mL distilled water and then again with 1 mL 5% methanol.
Stopcock manifold was then turned to sample collection position and a 1 mL syringe was
attached to the collection outlet. 1 mL methanol was added to the column and was slowly pulled
through with the syringe. The collected samples were transferred into individual 1.5 mL
centrifuge tubes, the syringe was rinsed with 200 L methanol which was then put into the same
tube as the sample. After they were dried in the Speedvac the samples were placed in the freezer.
An Arbor Assays corticosterone EIA kit was used to measure corticosterone levels in the
prepared samples. 400 L of Arbor Assay buffer was added to each sample in order to
reconstitute it. The standards and experimental EIA wells were prepared according to kit
instructions and the plate was run in a spectrometer.
Corticosterone concentration was calculated using the formula:
(x 1mL/103L) (s) (p/q) (r/s)

Y
Where: x = concentration as calculated by spectrometer (in pg/mL), y = weight of fur used in
methanol extraction (in mg, in step IIB), p = amount of methanol used for extraction (in L, in
step IIC1), q = amount of methanol dried down (in L, in step IIC6), r = amount of assay buffer
used to reconstitute dried sample (in L, in step IVA2), s = amount of assay buffer used in assay
(in L, for Arbor Assays this is 50L)
Statistical Analyses
A repeated measures ANOVA was used to compare times for the behavioral tests. Times of each
mouse in the different groups were compared to previous trials in the same experimental group.
A two-way ANOVA compared the baseline assay samples to each other as well as to the final
assay samples.

Results
Behavioral tests showed no statistical difference in the in the amount of stress experienced by the
experimental group as compared to the control. The two different behavioral tests, elevated plus
maze (Figure 1 df=1 F=0.346 p=0.560) and the open field maze (Figure 2 df=1 F=0.339
p=0.572), both showed no statistical behavioral changes induced by the stress experienced by the
mice.

% Time in Closed Arms

80
iso

60

control

40
20
0
0.5

1.5

2.5

3.5

Firgure 1: Behavioral stress demonstrated on elevated plus maze

Mice from each control and isolation groups were placed in the center of the elevated plus and given two
minutes to move freely. Measurements were made on the percent of the time that the mice spent on the
closed arms of the maze as compared to the open arms.

% Time near Perimeter

60
iso

40

control

20
0
0.5

1.5

2.5

3.5

Figure 2: Behavioral stress demonstrated by open field maze

Mice from each group were placed in the center of the open field maze and were allowed to move freely for
two minutes. Measurements were made based on the percent of time the mice spent near the perimeter of
the maze.

Hormone tests that were performed on the hair of the mice showed no significant difference in
the corticosterone difference between the experimental isolation group control group
(Figure 3 df=1 F=1.517 p=0.231).

Hormone Level
20
15

pre
post

10
5
0
iso

control

Figure 3: Corticosterone levels of mice pre and post time spent in isolation
Fur samples were collected before the testing began and at the end of the experiment from the mice and
were measured for the concentrations of corticosterone. The measurements were then compared to both the
results of the control group, as well as to the results of the individual mice before isolation occurred.

Discussion
Our results show a trend that isolation may increase corticostrone but the chance that this
was direct result of isolation is not significant in our study. However in other studies social
isolation on humans have negative health effects that can be attributed to corticosterone (House,

2001). Our study shows a slight trend that could possibly lead to significant findings but, the
actual results indicated no significant correlation however with improved methodology design
more accurate and viable results could be yielded. A change to this study to be made for future
research is increased sample size which would increase the validity of the results. Incorporating
males into the study would aid in the validity of applying the idea that isolation causes stress in
mice for all genders or it could provide evidence that the sexes react to stress differently and the
mechanistic differences should be investigated. Additionally housing of all the subjects in racks
and cages close together may have altered relevant data. Housing conditions that eliminate
outside sight and sound may yield more relevant data.
The technique of measuring Corticosterone via enzyme immunoassay was effective but samples
taken were not consistent or large enough and yielded little variability. A larger sample would
increase the chance of variability and accuracy of the results. The results of the assay would also
be greatly improved by an experiment of longer duration due to the nature of corticosterone and
the time it takes to grow hair. The assay used to measure corticosterone is best used to see stress
over a long period of time.

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