The Wellcome Trust for providing Open Access funding to Oxford University. Robert Deacon is a member of Oxford OXION group, funded by Wellcome Trust grant WT084655MA.

1. Burrowing
1. Apparatus
Various container designs were tried (for example a metal jug that could be easily cleaned, but mice were reluctant to enter it). Eventually it was discovered that a length of plastic downpipe (as connected to guttering on houses) elevated slightly at one end to prevent accidental (non-deliberate) displacement of food pellets and sealed at the other end, was an efficient piece of apparatus. Burrows are now made from plastic downpipe, 68 mm diameter, cut into 20 cm long lengths (Figure 1). One end is sealed with a plug of 12 mm medium density fibreboard (mdf). (Respiratory protection is advised when working with mdf). For cleaning purposes and waterproofing, a waterproof adhesive mastic compound can be applied to the inside and outside surfaces of the mdf as well as serving to glue it into the burrowing tube. Machine screws (5 cm long) are used to elevate the other end of the tube 3 cm off the floor. Two holes are drilled 1 cm from the open end of the burrowing tube at an angle of 90° and the screws inserted and tightened4.

The diameter of the burrows is probably critical; Schmid-Holmes et al.19 observed that mice in the wild 'burrow cleaned' mainly from burrows similar in size to our artificial ones.
<img src="/files/ftp_upload/2607/2607fig1.jpg" alt="Mouse in PVC tube for animal behavior study; resembling enrichment and habitat preferences." data-altupdatedat="1747911167000" data-alt="Figure 1">
Figure 1. A mouse in a burrowing tube.

2. Procedure
<ol>
 <li>Habituation Burrowing is normally spontaneous, but there is a certain learning element. The learning process is enhanced by social facilitation. Therefore place a full burrow overnight into the group home cage of mice which are later to be tested with a treatment. Each burrow is filled with 200 g of the food pellets normally supplied as diet. (This refers to the dense compressed type of diet; if an 'expanded' diet is in use the burrow will hold less than 200g). Experimenters should take into consideration such factors when selecting a burrowing substrate. 
 If the mice are on food rationing (for example, to motivate them to run a concurrent maze learning task) non-food substances can be used, for example clay balls, as used to line the surface of the soil in indoor plant pots; pea shingle (small stones ~1 cm diameter) and sand. Pea shingle and sand, being three times heavier than food pellets, are useful substitutes for food pellets if the mice are burrowing very vigorously, to avoid a 'ceiling effect'.
 </li>
 
 <li>Baselining Run (preferably two) baseline burrowing tests 48 h apart in the same way as will be done for testing after the proposed treatment. The test is optimally started around 4 pm if the mice are housed on an 0700-1900 h dark-light cycle. This is a sensitive time to test, as they are not normally awake at this time and burrowing starts more slowly than if they were well in to the dark phase and consequently very active. Two hours after the start, a 'snapshot' measurement is taken of the weight of food burrowed; the burrow is emptied into a tared container and weighed and the pellets replaced into the burrow which is put back into the cage. If the mouse is in the burrow, just gently pour it and the contents into the weighing container and replace the mouse in the cage. The weight burrowed at two hours is then calculated by subtraction from the original 200 g. The final reading is taken the next morning. Timing is not critical as the mice will have gone back to sleep (often in the burrow) and are no longer actively burrowing. Use the 2 h measurement taken on the second trial to form groups balanced for baseline performance. As burrowing increases with practice17, baselining also helps the mice to reach more stable asymptotic performance which reveals treatment effects better.</li>
 
 <li>Testing For testing, after treatments are administered the mice are given the burrowing test exactly the same as in the baselining stage. If the burrowing test is to be used chronically, it is recommended that at least 48 h separate the tests to prevent a decline in performance due to over-exposure to the procedure.</li>
 
 <li>Note Do NOT replace the burrowed food on the cage floor into the burrow. Even if the mouse has not burrowed anything, perform the weighing procedure as you would if burrowing had occurred; the disturbance caused by weighing tends to stimulate behaviour, and mice which have already burrowed some of the substrate will often commence burrowing with renewed vigour after the weighing process.</li>
</ol>

2. Expected results
C57~BL/6 mice typically burrow around 70 g in the first two hours, and near 200 g overnight. Hippocampal lesioned mice often burrow less than 5 g11 (Figure 2). Prion disease inhibits burrowing1,2 , as does lipopolysaccharide administration20. Burrowing can detect prion (scrapie) disease at 10-12 weeks after injection of diseased brain homogenate, whereas clinical signs only appear at 22 weeks13. (Figure 3). Burrowing has been shown to be sensitive to strain differences15 and knock-out of potassium ion channel subunits10.
<img src="/files/ftp_upload/2607/2607fig2.jpg" alt="Bar graph comparing burrowed weight; control vs. hippocampal group; neuroscience experiment." data-altupdatedat="1747911167000" data-alt="Figure 2">
Figure 2. Weight of food pellets (medians and interquartile ranges) burrowed in two hours by control and hippocampal-lesioned mice. The latter burrowed significantly less than controls (P = 0.0001). 

<img src="/files/ftp_upload/2607/2607fig3.jpg" alt="Bar chart of food pellets burrowed, comparing control vs. scrapie over weeks post-injection." data-altupdatedat="1747911167000" data-alt="Figure 3">
Figure 3. Weight of food pellets (means) burrowed overnight by control and scrapie-infected mice. Burrowing increases (with practice) in both groups from week 7 to 10 post-injection, but from week 12 burrowing declines in the scrapie group and the difference between the groups becomes statistically significant (exceeds two standard errors of the mean).

3. Discussion 
We have tested burrowing in other species of rodents - rats, gerbils, hamsters and Egyptian spiny mice8. All but the latter could be induced to burrow substantial amounts of terrestrial substrates such as gravel or sand, but burrowing of food pellets was much less, even after extensive experience with the terrestrial materials. The reason for this difference from mice is not known. After this work had been done, it was discovered that Egyptian spiny mice do not in fact make burrows in the wild; they may occupy burrows made by gerbils, also the surface of their natural habitat is often hard rock. Recent observation in Nairobi showed that, within the genus Acomys, Wilson's spiny mouse (Acomys wilsoni) and Percival's spiny mouse (Acomys percivali), which do burrow in the wild, also burrowed in the laboratory. 

4. Nesting 
1. Apparatus 
This test is performed in individual cages; the same ones as used for burrowing are suitable. Normal bedding should cover the floor to a depth of 0.5 cm. (Variation in depth, and very deep bedding, could affect nest construction). Each cage is supplied with a 'Nestlet', a 5 cm square of pressed cotton batting (Ancare).


2. Procedure
Mice are placed individually into the nesting cages about one hour before the dark phase, and the results are assessed the next morning. As with burrowing, and for similar reasons, timing is not critical. 


3. Scoring
The nests are assessed on a 5-point scale (Figures 4-8), and the amount of untorn Nestlet is also weighed5.
<ol>
 <li>The Nestlet is largely untouched (&gt;90% intact).</li>
 <li>The Nestlet is partially torn up (50-90% remaining intact).</li>
 <li>The Nestlet is mostly shredded but often there is no identifiable nest site: &lt; 50% of the Nestlet remains intact but &lt; 90% is within a quarter of the cage floor area, i.e. the cotton is not gathered into a nest but spread around the cage. Note: the material may sometimes be in a broadly defined nest area but the critical definition is that 50-90% has been shredded.</li>
 <li>An identifiable, but flat nest: &gt; 90% of the Nestlet is torn up, the material is gathered into a nest within a quarter of the cage floor area, but the nest is flat, with walls higher than mouse body height (curled up on its side) on less than 50% of its circumference.</li>
 <li>A (near) perfect nest: &gt; 90% of the Nestlet is torn up, the nest is a crater, with walls higher than mouse body height on more than 50% of its circumference.</li>
</ol>
Where criteria do not agree split the difference. For example a perfect nest with an unshredded 0.7g piece would score 4.5.
<img src="/files/ftp_upload/2607/2607fig4.jpg" alt="Rodent behavior observation, bedding substrate with a mouse exploring in a controlled environment." data-altupdatedat="1747911167000" data-alt="Figure 4">
Figure 4. Nest score 1
<img src="/files/ftp_upload/2607/2607fig5.jpg" alt="Rodent bedding analysis; wood shavings and cotton substrate for laboratory animal study." data-altupdatedat="1747911167000" data-alt="Figure 5">
Figure 5. Nest score 2
<img src="/files/ftp_upload/2607/2607fig6.jpg" alt="Rodent bedding setup in an enclosure with wood shavings and nesting material for behavioral study." data-altupdatedat="1747911167000" data-alt="Figure 6">
Figure 6. Nest score 3
<img src="/files/ftp_upload/2607/2607fig7.jpg" alt="Mouse in bedding for laboratory experiment, possibly for behavioral study or genetic research." data-altupdatedat="1747911167000" data-alt="Figure 7">
Figure 7. Nest score 4
<img src="/files/ftp_upload/2607/2607fig8.jpg" alt="Laboratory mouse in bedding; environmental enrichment; animal research; behavioral study concept." data-altupdatedat="1747911167000" data-alt="Figure 8">
Figure 8. Nest score 5

4. Expected results
 Both males and females make nests, as their purpose is thermoregulation as well as being associated with reproduction. For C57BL/6 mice, nest scores for males and females are in the same range, although we have not done a controlled comparison in a single experiment. Most C57BL/6 mice score 4-5 on nest construction, but when the hippocampus is lesioned the median score would be around 1-2; a score of 3 is unlikely to be exceeded (Figure 9). Nesting also proved to be sensitive to prion disease around 10-12 weeks1,2,16.
<img src="/files/ftp_upload/2607/2607fig9.jpg" alt="Bar chart of nest scores comparing Sham, Vent, Dors, and Comp groups; statistical analysis." data-altupdatedat="1747911167000" data-alt="Figure 9">
Figure 9. Nest scores (medians and interquartile ranges) for control mice and mice with lesions of the dorsal, ventral and complete hippocampus. Only the complete lesioned mice show inhibition of nesting, so the effects of selective dorsal and ventral regions appear sub-threshold but additive. 


5. Hoarding
1. Apparatus
A series of 'home bases' are connected to tubes made of wire mesh, sealed permanently at the distal end where the food pellets are placed and temporarily at the proximal end by a wooden plug. This is used to prevent the mice entering the tubes before adaptation to the home base has occurred7. 
The apparatus (Figure 10) can be made in various ways, as long as the basic principles are adhered to. Ours consists of a row of 8 wooden boxes, each 30 x 13 x 15 cm with transparent Perspex lids. Each is supplied with a water bottle, and has a hole in the back into which the hoarding tube is push-fitted. Tubes are made of black plastic, 10 cm long, 40 mm diameter, joined to a wire mesh tube, 45 cm long, 4 cm diameter, to form a total length of 50 cm. The mesh consists of 13 mm squares, and is a double roll with the meshes mis-aligned to make the holes in the mesh smaller and prevent food pellets dropping through. If 6 mm square mesh is available this is a better material as one roll would be sufficient. The distal end of the tube, where the food pellets are placed, is closed. The proximal end is sealed with a removable wooden plug. 
<img src="/files/ftp_upload/2607/2607fig10.jpg" alt="Animal behavior experiment setup with enclosures, food dispensers, tube maze for activity analysis." data-altupdatedat="1747911167000" data-alt="Figure 10">
Figure 10. Mice in the hoarding tubes

2. Procedure
<ol>
 <li>Typically, hoarding is very variable the first time and some mice may not hoard at all. Hoarding, like burrowing, improves with practise. Therefore, where experimental design allows, baselining sessions are best given before a treatment is administered. Clearly, this is not practical if the independent variable is innate, as with a (non-conditional) mutant mouse or when testing different mouse strains. </li>
 <li>A mouse is put into each box early in the day to habituate to it. The box is provided with a cardboard tunnel which has been in the real group home cage for at least one night, also a generous portion of soiled bedding, to make it feel like 'home'. Access to the hoarding tube is prevented until evening with a wooden bung. 100 g food pellets (a mixture of small and large) is placed at the distal end of the hoarding tube (by pouring them in at the other end with the tube held vertically). To make sure the mouse is mildly hungry before testing begins, the home box is not provided with food.</li>
 <li>Just before the start of the dark phase, remove the wooden bung to allow the mouse access to the hoarding tube. The next morning (again, timing is not critical) collect and weigh all the food pellets which have been hoarded into the home base box. </li>
</ol>
6. Expected results 
Using female C57BL/6 mice, the median amount hoarded would be around 50-70 g. Hippocampal lesions strongly suppress hoarding11, whereas prefrontal cortex lesions have only a weak effect12 (Figure 11). 
<img src="/files/ftp_upload/2607/2607fig11.jpg" alt="Weight hoarding comparison, control vs. prefrontal; bar graph showing data variation." data-altupdatedat="1747911167000" data-alt="Figure 11">
Figure 11. Weight of food pellets hoarded by control and medial prefrontal cortex-lesioned mice (medians and interquartile ranges). Although the medians differ considerably, the high variability in the controls means the result is far from statistically significant (P = 0.3).

<ol><li>Cunningham, C., Deacon, R. M. J., Chan, K., Boche, D., Rawlins, J. N. P., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropathologically+distinct+prion+strains+give+rise+to+similar+temporal+profiles+ofbehavioural+deficits%5BTitle%5D)+AND+%22Neurobiol.+Dis%22%5BJournal%5D)">Neuropathologically distinct prion strains give rise to similar temporal profiles ofbehavioural deficits.</a> Neurobiol. Dis. 18, 258-269 (2005).</li>
<li>Cunningham, C., Deacon, R., Wells, H., Boche, D., Waters, S., Picanco Diniz, C., Scott, H., Rawlins, J. N. P., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+changes+characterize+early+behavioural+signs+in+the+ME7+model+of+murine+prion+disease%5BTitle%5D)+AND+%22Eur.+J.+Neurosci%22%5BJournal%5D)">Synaptic changes characterize early behavioural signs in the ME7 model of murine prion disease.</a> Eur. J. Neurosci. 17, 2147-2215 (2003).</li>
<li>Damiani, R., Modesto, S., Yates, A., Neveling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Earliest+evidence+of+cynodont+burrowing%5BTitle%5D)+AND+%22Proc.+R.+Soc.+Lond.+B%22%5BJournal%5D)">Earliest evidence of cynodont burrowing.</a> Proc. R. Soc. Lond. B. 270, 1747-1751 (2003).</li>
<li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burrowing+in+rodents%3A+a+sensitive+method+for+detecting+behavioral+dysfunction%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">Burrowing in rodents: a sensitive method for detecting behavioral dysfunction.</a> Nat. Protocols. 1, 118-121 (2006).</li>
<li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+nest+building+in+mice%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">Assessing nest building in mice.</a> Nat. Protocols. 1, 1117-1119 (2006).</li>
<li>Deacon, R. M. J. Digging in Mice: Marble Burying, Burrowing, and Direct Observation Reveal Changes in Mouse Behavior. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDeacon%2C+RMJ+%22Mood+and+anxiety+related+phenotypes+in+mice%22">Mood and anxiety related phenotypes in mice</a>. Gould, T. D. , Humana Press. New York. (2009).</li>
<li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+hoarding+in+mice%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">Assessing hoarding in mice.</a> Nat. Protocols. 1, 2828-2830 (2006).</li>
<li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burrowing%3A+A+sensitive+behavioural+assay%2C+tested+in+five+species+of+laboratory+rodents%5BTitle%5D)+AND+%22Behav.+Brain.+Res%22%5BJournal%5D)">Burrowing: A sensitive behavioural assay, tested in five species of laboratory rodents.</a> Behav. Brain. Res. 200, 128-133 (2009).</li>
<li>Deacon, R. M. J., Bannerman, D. M., Kirby, B. P., Croucher, A., Rawlins, J. N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+cytotoxic+hippocampal+lesions+in+mice+on+a+cognitive+test+battery%5BTitle%5D)+AND+%22Behav.+Brain.+Res%22%5BJournal%5D)">Effects of cytotoxic hippocampal lesions in mice on a cognitive test battery.</a> Behav. Brain. Res. 133, 57-68 (2002).</li>
<li>Deacon, R. M. J., Brook, R. C., Meyer, D., Haeckel, O., Ashcroft, F. M., Miki, T., Seino, S., Liss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+phenotyping+of+mice+lacking+the+KATP+channel+subunit+Kir6.2%5BTitle%5D)+AND+%22Physiol.+Behav%22%5BJournal%5D)">Behavioral phenotyping of mice lacking the KATP channel subunit Kir6.2.</a> Physiol. Behav. 87, 723-733 (2006).</li>
<li>Deacon, R. M. J., Croucher, A., Rawlins, J. N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hippocampal+cytotoxic+lesion+effects+on+species-typical+behaviors+in+mice%5BTitle%5D)+AND+%22Behav.+Brain.+Res%22%5BJournal%5D)">Hippocampal cytotoxic lesion effects on species-typical behaviors in mice.</a> Behav. Brain. Res. 132, 203-213 (2002).</li>
<li>Deacon, R. M. J., Penny, C., Rawlins, J. N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+medial+prefrontal+cortex+cytotoxic+lesions+in+mice%5BTitle%5D)+AND+%22Behav.+Brain.+Res%22%5BJournal%5D)">Effects of medial prefrontal cortex cytotoxic lesions in mice.</a> Behav. Brain. Res. 139, 139-155 (2003).</li>
<li>Deacon, R. M. J., Raley, J. M., Perry, V. H., Rawlins, J. N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burrowing+into+prion+disease%5BTitle%5D)+AND+%22Neuroreport%22%5BJournal%5D)">Burrowing into prion disease.</a> Neuroreport. 12, 2053-2057 (2001).</li>
<li>Deacon, R. M. J., Rawlins, J. N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hippocampal+lesions%2C+species-typical+behaviours+and+anxiety+in+mice%5BTitle%5D)+AND+%22Behav.+Brain.+Res%22%5BJournal%5D)">Hippocampal lesions, species-typical behaviours and anxiety in mice.</a> Behav. Brain. Res. 156, 241-249 (2005).</li>
<li>Deacon, R. M. J., Thomas, C. L., Rawlins, J. N. P., Morley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+the+behavior+of+C57BL%2F6+and+C57BL%2F10%5BTitle%5D)+AND+%22Behav.+Brain.+Res%22%5BJournal%5D)">A comparison of the behavior of C57BL/6 and C57BL/10.</a> Behav. Brain. Res. 179, 239-247 (2007).</li>
<li>Guenther, K., Deacon, R. M. J., Perry, V. H., Rawlins, J. N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+behavioural+changes+in+scrapie-affected+mice+and+the+influence+of+dapsone%5BTitle%5D)+AND+%22Eur.+J.+Neurosci%22%5BJournal%5D)">Early behavioural changes in scrapie-affected mice and the influence of dapsone.</a> Eur. J. Neurosci. 14, 401-409 (2001).</li>
<li>Mallucci, G. R., White, F. armer, Dickinson, M., Khatun, A., Powell, H., Brandner, A. D., Jefferys, S., R, J. G., Collinge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+Cellular+Prion+Protein+Reverses+Early+Cognitive+Deficits+and+Neurophysiological+Dysfunction+in+Prion-Infected+Mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Targeting Cellular Prion Protein Reverses Early Cognitive Deficits and Neurophysiological Dysfunction in Prion-Infected Mice.</a> Neuron. 53, 325-335 (2007).</li>
<li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRussell%2C+WMS+%22The+principles+of+humane+experimental+technique%22">The principles of humane experimental technique</a>. , Methuen, London. (1959).</li>
<li>Schmid-Holmes, S., Drickamer, L. C., Robinson, A. S., Gillie, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burrows+and+burrow+cleaning+behaviour+of+house+mice%5BTitle%5D)+AND+%22Am.+Mid.+Nat%22%5BJournal%5D)">Burrows and burrow cleaning behaviour of house mice.</a> Am. Mid. Nat. 146, 53-62 (2001).</li>
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No conflicts of interest declared.

These tests are extremely simple to run but the effect sizes are very large, making them extremely sensitive to treatments, strain differences, etc. They conform to the 'Refinement' aspect of Russell and Burch's 3 Rs'18. Indeed, good performance on tests like burrowing seems to be indicative of good general health in animals. Therefore this could be a good test for assessment of animal welfare. 
The importance of environmental enrichment for laboratory rodents is well recognised these days. Provision of nesting material is now standard practice in many animal houses, and this is easy to do. The enthusiasm with which mice burrow makes burrowing a potentially excellent enrichment, but unfortunately a 'self-reloading' burrow which would be a practical way of providing this has not yet been developed. Whether mice find hoarding enriching has not yet been determined.
One drawback is that the data generated by these tests is generally non-parametric (not Gaussian in distribution), so appropriate non-parametric statistics are needed to analyse them, e.g. the Mann-Whitney U test for pairwise comparisons, Kruskal-Wallis ANOVA for multi-group data. If repeated measures designs are used, since there is no suitable ANOVA test for non-parametric data, the results can be transformed, e.g. by square root or log procedures to make them conform to a more normal distribution.
The measurements of the apparatus can be slightly different to those specified in the text (useful for USA experimenters) but wide differences should be avoided. However, in some cases other experimenters may find different constructions work better than those described here.
As for all behavioural measurements, the mice should be tested to see if their activity levels are normal, as inactivity will obviously have a detrimental effect on species-typical behaviours. When scrapie-infected mice were showing impaired burrowing and nesting, their activity levels were normal (tested in an open field apparatus). Indeed, a period of hyperactivity occurs around 2-4 weeks after these species-typical behaviours are first impaired2.
The strong impairment produced by hippocampal lesions on burrowing, nesting and hoarding, all of which are species-typical behaviours, complements the impairments in spatial learning and memory9 which are such well-established effects of these lesions. They appear to be equivalent to the impairments in the activities of daily living so characteristic of AD14.
These species-typical tests may well prove to be of great use in preclinical screening for treatments for Alzheimer's and other neurodegenerative diseases14. In many human brain disorders a loss or reduced ability to perform normal everyday tasks is as important as other features of the disease like cognitive impairment. Particularly, this loss may necessitate involvement of carers and the great socioeconomic burden this so often imposes.

assessing burrowing, nest construction, and hoarding in mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

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Assessing Burrowing, Nest Construction, and Hoarding in Mice

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

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32.0K Views.  University of Oxford. Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute “environmental enrichment” for the mice, and embody the “Refinement” aspect of the “3 Rs”18.

Video: Assessing Burrowing, Nest Construction, and Hoarding in Mice

Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a significant public health concern, particularly in children. Both of these conditions are highlighted by the repeated expression of meaningless behaviors. Individuals with OCD often show checking, frequent hand washing, and counting. Children with ASDs also engage in repetitive tapping, arm or hand flapping, and rocking. These behaviors can vary widely in intensity and frequency of expression. More intense forms of repetitive behaviors can even result in injury (e.g.&#160;excessive grooming, hand washing, and self-stimulation). These behaviors are therefore very disruptive and make normal social discourse difficult. Treatment options for repetitive behaviors in OCD and ASDs are somewhat limited and there is great interest in developing more effective therapies for each condition. Numerous animal models for evaluating compulsive-like behaviors have been developed over the past three decades. Perhaps the animal models with the greatest validity and ease of use are the marble burying test and the nestlet shredding test. Both tests take advantage of the fact that the target behaviors occur spontaneously in mice. In the marble burying test, 20 marbles are arrayed on the surface of clean bedding. The number of marbles buried in a 30 min session is scored by investigators blind to the treatment or status of the subjects. In the nestlet shredding test, a nestlet comprised of pulped cotton fiber is preweighed and placed on top of cage bedding and the amount of the nestlet remaining intact after a 30 min test session is determined. Presently, we describe protocols for and show movie documentation of marble burying and nestlet shredding. Both tests are easily and accurately scored and each is sensitive to small changes in the expression of compulsive-like behaviors that result from genetic manipulations, disease, or head injury.

Disease, head injury, genetic modifications, and treatment of mice with drugs can have profound effects on behavior. Utilizing well-characterized and validated approaches such as marble burying and nestlet shredding, compulsive-like behaviors can be documented accurately in mice as models of human obsessive-compulsive disorder and autism spectrum disorder.

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a ...

Behavior

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

Basic Care Procedures

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Mice and rats account for over 90% of the animals used for biomedical research. The proper care of these research animals is critical to the outcome of experiments. There are general procedures that apply to the majority of these mice and rats, but some of the animals, such as the immunocompromised ones, require additional steps to be taken to sustain them for experimentation. 
Commonly used immunocompromised mice include those that have naturally occurred in inbred mice and those that have been created through genetic engineering. The first immunocompromised mice used in research were "nude" mice. The BALB/c Nude (nu) mouse was discovered in 1966, within a BALB/c colony that was producing mice lacking both hair and a thymus. These athymic mice have an inhibited immune system that is devoid of T cells. The value of this animal was soon discovered for the use in studies of microbial infections, immune deficiencies, and autoimmunity. Although not as commonly used as the nude mouse, there is also a nude rat. The nude rat is T cell deficient and shows depleted cell populations in thymus-dependent areas of peripheral lymphoid organs. Another naturally occurring immune deficient mouse is the severe combined immunodeficiency mouse (SCID). These mice have a defect that impairs the production of functional B and T cells. With the inability to mount an adequate immune response, these mice can serve as host recipients for transplants of human cells. Several genetically-modified mouse strains with immune deficiencies are commercially available.1 The severity of the deficiency varies with the genetic modification; however, all of the immunocompromised mice and rats are handled similarly.
This manuscript will describe the care of both immunocompetent and immunocompromised animals. The housing, diet, environmental requirements, and handling will be discussed for both groups of animals.

Basic Care Procedures for Laboratory Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Mice and rats account for over 90% of the animals ...