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Burrowing in rodents: a sensitive method for
detecting behavioral dysfunction
Robert M J Deacon
Department of Experimental Psychology, University of Oxford, South Parks Road, Oxford OX1 3UD, UK. Correspondence should be addressed to R.M.J.D.
([email protected]).
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Published online 27 June 2006; doi:10/1038/nprot.2006.19
Virtually all rodents display burrowing behavior, yet measurement of this behavior has not yet been standardized or formalized.
Previously, parameters such as the latency to burrow and the complexity of the burrow systems in substrate-filled boxes in the
laboratory or naturalistic outdoor environments have been assessed. We describe here a simple protocol that can quantitatively
measure burrowing in laboratory rodents, using a simple apparatus that can be placed in the home cage. The test is very cheap
to run and requires minimal experimenter training, yet seems sensitive to a variety of treatments, such as the early stages
of prion disease in mice, mouse strain differences, lesions of the hippocampus and prefrontal cortex in mice, also effects of
lipopolysaccharide and IL-1β in rats. Other species such as hamsters, gerbils and Egyptian spiny mice also burrow in this apparatus,
and with suitable size modification probably almost any burrowing animal could be tested in it. The simplicity, sensitivity and
robustness of burrowing make it ideal for assessing genetically modified animals, which in most cases would be mice. The test is
run from late afternoon until the next morning, but only two measurements need to be taken.
INTRODUCTION
Many animals burrow or use the burrows made by other animals. Rodents in particular are noted for their burrowing ability.
Burrowing is probably an extremely ancient behavior. The first
animals to venture on to land probably sought natural shelters, and
later burrowed their own, to avoid the harmful radiation present
at that time. Later, burrows served as a defense against predators,
as food stores, refuges from cold weather and safe places to rear
young1. Despite the fact that rats and mice, the most commonly
used laboratory species, are well-known burrowers, scientific
investigations seem to have been limited to ethologically oriented
research, and we are not aware of any prior use of burrowing as a
standardized behavioral assay.
The burrowing test evolved in our laboratory from attempts
to measure hoarding in mice2. Since mouse-proof equipment
involving a home cage and an extended alley leading to an external food source was not available at that time, we tried placing the
food source inside the home cage, rather than external to it. It was
observed that, if left overnight, a jar of food pellets placed in the
home cage would generally be empty by the next morning, but
the food was not hoarded in a place away from the source, merely
piled around the mouth of the jar. Later, observation of the behavior of the mice showed that they were digging the pellets out of
the jar with coordinated hind- and forelimb movements and kicking them outside the container, not carrying pellets carefully to a
selected spot as in true hoarding. We therefore termed this behavior “burrowing”. It closely resembles digging3, but the substrate
container may serve as an additional attraction to small rodents, as
it resembles a natural burrow and provides a place to hide.
Burrowing may be related to tunnel maintenance (the container
and its contents representing a blocked tunnel that could be due
to earth fall). It may also reflect elements of defensive behavior;
rodents attempt to prevent predators such as snakes from entering
their burrows by digging earth and kicking it toward the intruder. Defensive burying is a well-known paradigm for measuring
118 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS
anxiety4. Burrowing in mice probably does not wholly represent
an attempt to seek shelter, as mice will burrow a full container that
is next to an empty one. The mouse is, however, often found inside
the container the next morning, and will drag in nesting material if
there is any available. It is also possible to do “reverse burrowing”:
put an empty burrow into a cage with some food pellets on the
cage floor. Pellets will often be found in the container the next day,
suggesting they have been truly hoarded. Again, this differentiates
hoarding from burrowing.
The burrowing paradigm has proved a sensitive assay for monitoring the development of prion disease (scrapie) in mice2,5. It is
sensitive to strain differences; C57BL/6 mice burrow more than
129S2/Sv mice6. Hippocampal cytotoxic lesions produce a large
reduction in burrowing7, and medial prefrontal cortex lesions
induce a smaller reduction8.
Most of our work on burrowing has been on mice, as experience has shown that they will burrow almost anything, including
earth, soiled bedding and the small clay balls sold to cover the soil
on indoor plant pots9 (and food pellets). Clearly burrowing, as
suggested by the different topology of its movements compared
to hoarding, is not appetitively driven. The willingness of mice to
burrow non-food items was useful when food-rationed mice were
being used to detect scrapie disease; burrowing detected disease
onset slightly earlier than a DRL (differential reinforcement of low
rates of responding) operant schedule9.
For a long time, using containers filled with food pellets, we could
not induce burrowing in rats, even if they were on food rationing.
Then it was discovered that although they would not burrow food
pellets, they would burrow earth-like substrates, especially if they
first experienced a burrow in a cage with their cagemates (presumably due to social facilitation). Subsequently, studies (in preparation) have shown that rat burrowing is as sensitive to lipopolysaccharide suppression as is mouse burrowing. It is also decreased by
IL-1β-expressing replication-deficient adenovirus.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
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Other species tested for burrowing include hamsters, gerbils and
Egyptian spiny mice (Acomys cahirinus). Surprisingly, the latter
showed very little burrowing; perhaps they rely on rock crevices
and burrows made by other animals in their desert habitat. Gerbils
are well known for their digging, and our initial studies have shown
that they burrow well, although like rats and hamsters, they seem
to prefer earth-like substrates to food pellets.
A group size of ten is appropriate to detect the effects of treatments on burrowing, although smaller groups could be tested if,
for example, transgenic mice were being used and they were in
short supply. Burrowing is a non-invasive technique and could
well be used as part of the initial assessment of a genetically modified mouse. Several mouse strains are suitable to assess pharmacological treatments; most outbred strains we have tested burrow
reasonably well (CD-1, NMRI, MF-1, NIH-Swiss). Of the inbred
strains tested, C57BL/6, BALB/c, SJL and DBA/2 are good burrowers, while CBA and 129S2/Sv are poor.
The hooded Lister strain of rat is a vigorous burrower of earthlike substrates such as sand or gravel. Although we have not tested
albino strains such as the Sprague-Dawley or Wistar, we suspect
they may be less vigorous. Syrian hamsters and Mongolian gerbils
also burrow well, but like rats they prefer earthy substrates.
MATERIALS
EQUIPMENT
• Burrows (see EQUIPMENT SETUP below for details)
EQUIPMENT SETUP
Burrows can be created as described below. These are only guidelines however;
our first burrow was a beer glass! What is important is that the burrow fits into
the home cage and contains the maximum weight of burrowing material, to
avoid ceiling effects (e.g. both groups empty the burrows completely so no
group difference can be seen). However, many rodents are extremely vigorous
burrowers, so many would completely empty virtually any size burrow
overnight; this is why a 2 h measure is sometimes more sensitive. The vigor
with which many animals burrow suggests it is a rewarding activity, and this is
probably integral to its usefulness and sensitivity as a test.
For mice, hamsters or gerbils:
A 200 mm long, gray or black plastic, 68 mm diameter gray tube (i.e., a section
sawn from a plastic water downpipe). The open end of the tube is raised 30
mm by bolting two 50 mm machine screws through it, each 10 mm in from the
end, spaced just less than a quadrant of the tube apart (Fig. 1). The lower end
of the tube is closed with a plug. This can be cut from mdf (medium density
fiberboard) or plastic and push-fitted in to the tube, sealing with a suitable
adhesive.
! CAUTION Respiratory protection is advised when working with mdf.
For rats:
The plastic tube should be 320 mm long and 100 mm in diameter. The open end
of the tube is raised 60 mm above the cage floor by two 80-mm bolts, placed 25
mm in from the end of the tube, spaced 70 mm apart. The other end is closed by
a plastic or mdf plug, as for the mouse burrow (Fig. 1).
REAGENTS
• Rats, mice, hamsters or gerbils
! CAUTION Experimenters must comply with national regulations concerning
animals and their use. Since animals spontaneously burrow, however, the test is
not aversive and probably represents a form of environmental enrichment.
• Select a suitable burrowing substrate for both the species and the experiment.
Some suggestions are given here, but animals will probably burrow a wide
variety of similar substrates. This can be the food pellets that are the normal
diet of the animals, if you are using mice (note that other species do not burrow
food readily). To avoid neophobia, use the diet that the mice are used to. I use
PCP (mod) P pellets (Special Diet Services, Witham, Essex, UK). All species
readily burrow gravel-like substrates (10 mm pea shingle, B&Q, Chandlers Ford,
Hampshire, UK) and sand (Play Sand; www.BritishPlaySand.co.uk). These latter
two substrates, being very dense, are useful in minimizing ceiling effects. Much
less dense but of proven worth in detecting prion disease9 are clay balls (used
for covering the soil in indoor plant pots) (HYDROLECA, William Sinclair
Horticulture Ltd., Lincoln, UK). The cage floor bedding that the animals are
normally housed on can also be used; my mice are housed on aspen premium
8/20 wood chip bedding (Lillico, Betchworth, Surrey, UK). Note that with
these two latter less dense substrates, only a limited weight can be put into the
burrow; this might result in ceiling effects and a failure to detect the experimental
treatment.
Supplementary Video 1 online shows a hooded Lister rat burrowing colored
granite chips. For artistic purposes, the chips are colored and the burrow is made
out of clear Perspex.
PROCEDURE
Practice runs and baseline testing
1| Mice and rats burrow spontaneously, but their performance generally improves with practice. A practice run in addition to a
baseline test can improve burrowing ability and diminish variability between animals. For practice, simply put a full burrow into
the home cage; with group housed animals social facilitation will further help develop the burrowing behavior. As individual
differences in burrowing behavior occur, if possible run an (individual) baseline test, as described in steps 3–7 below, before
you give the experimental treatment.
2| Assign individuals to groups based on their performance in the baseline tests. Rank the 2 h results from the baseline test
(the 2 h measure is likely to be more sensitive to individual differences than the overnight results) and allocate individuals
sequentially and alternately from this ranked list to the different experimental treatments. For example, mice A, B, C, D and
E burrow 25, 129, 56, 2 and 84 g, respectively, on a baseline test. They can thus be ranked in ascending order: 1 = D (2 g
burrowed), 2 = A (25 g), 3 = C (56 g), 4 = E (84 g) and 5 = B (129 g). Exclude mouse D as an unreliable burrower. Then mice
A and E receive treatment X, and mice C and B receive treatment Y. With n = 10/group, the median burrowing values should
be very similar; as a final check, run a pairwise comparison (Mann-Whitney U test) to confirm this. You may wish to exchange
two mice from the two groups to make final adjustments. Thus the groups will be counterbalanced for burrowing ability and a
treatment effect will be more readily detected with fewer animals.
3|
Fill the burrow with 200 g food pellets for mice, or 2.5 kg of pea shingle for rats, and place the burrow against the longer
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a
Open end
b
Burrowing tube
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Closed end
Supporting screws
Figure 1 | Schematic diagram and photograph of a burrow. (a) Diagram of a burrow made from a section of plastic tubing, sealed at one end with a wooden
(mdf) plug and elevated at the other end by two machine screws to minimize the risk of the animal displacing the contents by non-burrowing movements as
it goes in and out of the tube. (b) Photograph of hooded Lister rats in burrows. Each burrow contained 2,500 g of pea shingle at the start of the test.
wall of a clean cage with a thin layer of bedding. The closed end of the burrow should be against the back wall of the cage.
Provide water but no extra food in the cage hopper for the mice (it may distract them); for rats, have the food hopper of the
cage full as usual.
4| The test is best started 3 h before the dark cycle. Put a single (not food-deprived) mouse or rat into each cage-burrow
setup for 2 h.
5| Measure the amount of material displaced from the burrow (defined as being on the floor of the cage rather than in the
burrow; this could be measured as 200/2,500 g minus the weight left in the tube). It is more practical to measure the weight
of material left in the burrow than to pick up all the material from the cage floor. If there is an animal in the tube, just gently
tip it out into the balance pan and replace it in the cage. It is inadvisable to try to retrieve it from the tube manually.
▲ CRITICAL STEP Replace the material that was left in the burrow for the overnight phase.
6 | Return the animal and burrow, with remaining material in it, to the cage and leave overnight.
7 | The next morning (the time is not critical as the animals will have burrowed all they wanted earlier in the night and many
may be found asleep in the burrows), weigh the amount left in the tube again and calculate the weight burrowed overnight.
Return the animals to their group cages.
? TROUBLESHOOTING
If controls fail to burrow, make sure you are using a suitable strain of animal. If literature reports indicate that they are
relatively slow and inactive in other behavioral tests (e.g., this is true for certain 129 strains) then try a better strain such as
C57BL/6. Young adult mice (2–4 months old) will probably dig more vigorously than mice older than one year old. Give them
extra practice in the home group cages. Try other substrates. Mix the substrate with food or a foraging mix of tasty treats and
remove food from the cage hopper overnight.
Assess whether the laboratory is quiet enough; the mice should be undisturbed. Avoid testing on cage-cleaning days, as the
mice will expend a lot of activity re-exploring their new cages, and subsequently will be less active.
ANTICIPATED RESULTS
Data analysis
The two-hour measurement is generally more sensitive than the overnight one; the latter measurement often suffers from
a ceiling effect, as many animals will burrow the entire tube contents. With sensitivity also comes variability, however,
particularly if this is the first time animals are exposed to the test. Thus, compare the 2 h and overnight measures
separately, using pairwise control vs. treatment group comparisons, or an ANOVA for multi-group comparisons. The data,
particularly for the overnight measure, are likely to be non-parametric, at least partially due to the ceiling effect. Therefore
it may be better, for consistency, to subject both the 2-h and the overnight data to non-parametric tests (the Mann-Whitney
U test for two groups or the Kruskal-Wallis one-way ANOVA for multiple groups) and express the results as medians and the
variability as the interquartile range.
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© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Given a burrow filled with 200 g of food pellets, C57BL/6 mice normally burrow about 60 g over 2 h and 150 g overnight.
Hooded Lister rats with a tube of 2,500 g of pea shingle typically burrow about 1,500 g after 2 h and 2,400 g overnight. As
discussed in the Introduction, the test appears to be very sensitive to many factors, including species, strain, pharmacological
treatments, brain lesions, chemokines and diseases such as scrapie—indeed anything that affects the well-being of the
animal. Burrowing is an excellent way of assessing recovery from surgery; an animal that is even slightly ill will show reduced
burrowing. It may have potential for development as an assay for depression; unfortunately burrowing is decreased by selective
serotonin reuptake inhibitors (SSRIs) such as fluoxetine (unpublished observations), thus a burrowing decrease caused by a
depressive agent would be difficult to reverse using such antidepressants. SSRIs also suppress digging and marble burying,
so it is unsurprising that they also inhibit burrowing, which requires similar physical activity (and, by extrapolation, similar
motivation and neural circuitry)10. Perhaps the clearest application of burrowing, however, given its apparent sensitivity, is in
behavioral toxicology.
Note: Supplementary information is available via the HTML version of this article.
ACKNOWLEDGMENTS This work was supported by grant GR065438MA from the
Wellcome Trust to the Oxford OXION group.
COMPETING INTERESTS STATEMENT The author declares that he has no competing
financial interests.
Published online at http://www.natureprotocols.com/
Reprints and permissions information is available online at http://npg.nature.
com/reprintsandpermissions/
1.
2.
3.
Dudek, B.C., Adams, N., Boice, R. & Abbott, M.E. Genetic influences on
digging behaviours in mice (Mus musculus) in laboratory and seminatural
settings. J. Comp. Psychol. 97, 249–259 (1983).
Deacon, R.M.J., Raley, J.M., Perry, V.H. & Rawlins, J.N.P. Burrowing into
prion disease. Neuroreport 12, 2053–2057 (2001).
Deacon, R.M.J. Digging and marble burying in mice: simple methods for
in vivo identification of biological impacts. Nat. Protocols 1, 122–124
(2006).
4.
Pinel, J.P.J. & Treit, D. Burying as a defensive response in rats. J. Comp.
Physiol. Psychol. 92, 708–712 (1978).
5. Guenther, K., Deacon, R.M.J., Perry, V.H. & Rawlins, J.N.P. Early behavioural
changes in scrapie-affected mice and the influence of dapsone. Eur. J.
Neurosci., 14, 401–409 (2001).
6. Contet, C., Rawlins, J.N.P. & Deacon, R.M.J. A comparison of 129S2/SvHsd
and C57BL/6JOlaHsd mice on a test battery assessing sensorimotor, affective
and cognitive behaviours: implications for the study of genetically modified
mice. Behav. Brain Res. 124, 33–46 (2001).
7. Deacon, R.M.J., Croucher. A. & Rawlins, J.N.P. Hippocampal cytotoxic lesion
effects on species-typical behaviors in mice. Behav. Brain Res. 132, 203–213
(2002).
8. Deacon R.M.J., Penny, C. & Rawlins, J.N.P. Effects of medial prefrontal cortex
cytotoxic lesions in mice. Behav. Brain Res. 139, 139–155 (2003).
9. Deacon R.M.J., Reisel, D., Perry, V.H. & Rawlins, J.N.P. Hippocampal scrapie
infection impairs operant DRL performance in mice. Behav. Brain Res. 157,
99–105 (2005).
10. Shinomiya, K., Fujii, Y., Sugimoto, Y., Azuma, N., Tokunaga, S., Kitazumi,
K. & Kamei, C. Effect of paroxetine on marble-burying behavior in mice.
Methods Find Exp Clin Pharmacol. 10, 685–687 (2005).
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