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&deg; 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="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="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="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 (>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="Figure 4" />
Figure 4. Nest score 1
<img src="/files/ftp_upload/2607/2607fig5.jpg" alt="Figure 5" />
Figure 5. Nest score 2
<img src="/files/ftp_upload/2607/2607fig6.jpg" alt="Figure 6" />
Figure 6. Nest score 3
<img src="/files/ftp_upload/2607/2607fig7.jpg" alt="Figure 7" />
Figure 7. Nest score 4
<img src="/files/ftp_upload/2607/2607fig8.jpg" 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="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="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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathologically+distinct+prion+strains+give+rise+to+similar+temporal+profiles+ofbehavioural+deficits.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+changes+characterize+early+behavioural+signs+in+the+ME7+model+of+murine+prion+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Earliest+evidence+of+cynodont+burrowing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing+in+rodents:+a+sensitive+method+for+detecting+behavioral+dysfunction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+nest+building+in+mice.">Assessing nest building in mice.</a> Nat. Protocols. 1, 1117-1119 (2006).</li><li>Deacon, R. M. J., Gould, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digging+in+Mice:+Marble+Burying,+Burrowing,+and+Direct+Observation+Reveal+Changes+in+Mouse+Behavior.">Digging in Mice: Marble Burying, Burrowing, and Direct Observation Reveal Changes in Mouse Behavior.</a> Mood and anxiety related phenotypes in mice. , (2009).</li><li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+hoarding+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing:+A+sensitive+behavioural+assay,+tested+in+five+species+of+laboratory+rodents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cytotoxic+hippocampal+lesions+in+mice+on+a+cognitive+test+battery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+mice+lacking+the+KATP+channel+subunit+Kir6.2.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+cytotoxic+lesion+effects+on+species-typical+behaviors+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+medial+prefrontal+cortex+cytotoxic+lesions+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing+into+prion+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+lesions,+species-typical+behaviours+and+anxiety+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+behavior+of+C57BL/6+and+C57BL/10.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+behavioural+changes+in+scrapie-affected+mice+and+the+influence+of+dapsone.">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. a. r. m. e. r., 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Cellular+Prion+Protein+Reverses+Early+Cognitive+Deficits+and+Neurophysiological+Dysfunction+in+Prion-Infected+Mice.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The principles of humane experimental technique. , (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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrows+and+burrow+cleaning+behaviour+of+house+mice.">Burrows and burrow cleaning behaviour of house mice.</a> Am. Mid. Nat. 146, 53-62 (2001).</li><li>Teeling, J. L., Felton, L. M., Deacon, R. M. J., Cunningham, C., Rawlins, J. N. P., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-pyrogenic+systemic+inflammation+impacts+on+brain+and+behavior,+independent+of+cytokines.">Sub-pyrogenic systemic inflammation impacts on brain and behavior, independent of cytokines.</a> Brain Behav. Immun. 21, 836-850 (2007).</li></ol>

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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31.8K 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

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive performance. These effects may be due to widespread connections of the trigeminal system to the ascending reticular activating system (ARAS), to which noradrenergic neurons of the locus coeruleus (LC) belongs. LC neurons contain projections to the whole brain, and it is known that their discharge co-varies with pupil size. LC activation is necessary for eliciting task-related mydriasis. If chewing effects on cognitive performance are mediated by the LC, it is reasonable to expect that changes in cognitive performance are correlated to changes in task-related mydriasis. Two novel protocols are presented here to verify this hypothesis and document that chewing effects are not attributable to aspecific motor activation. In both protocols, performance and pupil size changes observed during specific tasks are recorded before, soon after, and half an hour following a 2 min period of either: a) no activity, b) rhythmic, bilateral handgrip, c) bilateral chewing of soft pellet, and d) bilateral chewing of hard pellet. The first protocol measures level of performance in spotting target numbers displayed within numeric matrices. Since pupil size recordings are recorded by an appropriate pupillometer that impedes vision to ensure constant illumination levels, task-related mydriasis is evaluated during a haptic task. Results from this protocol reveal that 1) chewing-induced changes in performance and task-related mydriasis are correlated and 2) neither performance nor mydriasis are enhanced by handgrip. In the second protocol, use of a wearable pupillometer allows measurement of pupil size changes and performance during the same task, thus allowing even stronger evidence to be obtained regarding LC involvement in the trigeminal effects on cognitive activity. Both protocols have been run in the historical office of Prof. Giuseppe Moruzzi, the discoverer of ARAS, at the University of Pisa.

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

Micro-Drive Implant for Recording Daily Neural Activity in Juvenile Mice

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of particular importance for studying mouse models of human neuropsychiatric disorders. However, such methods often require large cranial implants, which cannot be used in mice at early developmental time points. As such, virtually no studies of in vivo physiology have been performed in freely behaving infant or juvenile mice, despite the fact that a better understanding of neurological development in this critical window would likely provide unique insights into age-dependent developmental disorders such as autism or schizophrenia. Here, a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy are described that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in mice as they age from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond, a time window roughly corresponding to the human ages of 2 years old through to adulthood. The number of recording electrodes and final recording sites can be easily modified and expanded, thus allowing flexible experimental control of the in vivo monitoring of behavior- or disease-relevant brain regions across development.

Author Spotlight: A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice  

Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond.