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. The Oxford Paddling Pools
The paddling pool has been made according to three successive designs, Mk 1-3. All employ shallow (2 cm) water contained in a white (to enhance its aversiveness) base, which is surrounded by transparent walls made of Perspex or clear acrylic plastic. These have true/false exits set in them. The false exits are occluded by black painted wooden plugs, whereas the true exit is open and joined to a black plastic pipe which can be removed with the mouse inside. The mouse is then swiftly and atraumatically returned to its home cage while inside the pipe. After the first publication of the paddling pool, it was reported that return to the home cage was also an effective motivator for learning the Lashley III maze2.
A water temperature of 20-25 &deg;C is used. 20-21 &deg;C is ideal; it has been noted that some mice seem less motivated at higher temperatures.
The first maze5 was circular. However, sometimes mice (particularly those with lesions to the hippocampus) would appear not to notice the open exit tube. An octagonal design, with the exit tubes placed in the corners so as to attract the attention of the mice, greatly ameliorated this problem. However, the error rate at chance performance would be 4/trial, as opposed to 6/trial with the 12-tube circular design. Therefore a dodecagonal pool is now used with 11 false and 1 true exit set in the angles made by the junctions of the walls.
1.1. Evolution and characteristics of the paddling pools
Mk 1: a circular pool 85 cm in diameter with 12 true/false exits (Figure 1).
Mk 2: an octagonal pool 86 cm in diameter with 8 true/false exits set in the corners.
Mk 3: a dodecagonal (12 sided) pool 120 cm in diameter with 12 true/false exit set in the corners (Figure 4).
In all the above (and the paddling Y-maze; see below) the exit tubes were 40 mm diameter. However, it was occasionally sensed that the mice were reluctant to enter these, and control performance in the Y-maze would sometimes dip after they had been responding well. Therefore, and considering how readily they entered the burrowing apparatus (see JOVE publication "Assessing burrowing, nest construction and hoarding in mice"), also that the exit holes in the Barnes maze are 50 mm diameter, it was considered if 40 mm was too small. A test was carried out in the home cage of six C57BL/6 mice. (They seem more ready to enter 40 mm tubes in the home cage as presumably stress is lower here than in a piece of apparatus, so doing the test in the home cage would minimize differences in entry if the smaller tubes were slightly aversive, making the test more conservative). Two 40 mm tubes and two 50 mm tubes were placed on the floor in an alternating pattern. As predicted, there were more entries into 50 mm tubes (28 entries) than 40 mm (9 entries). Therefore pilot tests are planned on a new Y-maze with 50 mm exit tubes; if this is successful the old 40 mm tubes on the dodecagonal pool and paddling Y-maze will be replaced with new 50 mm ones.
As the paddling pool, paddling Y-maze and spatial novelty Y-maze are all spatial tests, relying on the room cues which the mouse sees through the transparent walls of the apparatus, the room should be well furnished with distinctive cues (e.g shelves, cupboards, black plastic shapes on the walls).
2. Running the Paddling Pool
<ol>
	<li>Place a mouse in the center of the pool facing one of four positions on the perimeter (9, 12 or 3 o'clock if the escape tube is at 6 o'clock). Placement is semi-random; a maximum of three consecutive trials can be in the same direction. This can be difficult to achieve in practice, because as learning proceeds the mice tend to orientate themselves towards the correct exit as they are lowered into the pool by the tail. If they do this strongly no attempt should be made to force them into the planned position, since they are simply demonstrating learning.</li>
	<li>Release them when they are just above the water, as then they drop in and instantly know that they are no longer being held. Slowly releasing them results in them struggling to free themselves and this can impair initial orientation. This also applies to the paddling Y-maze.</li>
	<li>Maximum trial length is 60 sec. If the mouse fails to escape by then, manually guide it to the exit using a couple of clear Perspex paddles, each measuring about 30 x 20 cm. The measures taken are the time to find the exit (definition: all of the head within the tube) and errors. Errors were defined as coming within a head's length from a tube (including the real exit). Passing close by the real exit, without entry, generally occurs infrequently after the first few trials. Although a case can be made that this is a problem, an alternative view is that mice impaired in spatial cognition do this because they don't know there is an exit around there. Controls do know, and therefore investigate more fully. A ceiling of 11 errors/trial is imposed on the number of errors in the analysis, to prevent too much skew of the data. This would mitigate against a deficit being found in spatially impaired mice, i.e. it is a conservative measure. Mice which fail to reach the exit within 60 sec are assigned a score of 11 errors for that trial. So 60 sec times always have 11 errors.</li>
</ol>
3. The Paddling Y-maze
To simplify the paddling pool procedure and the apparatus it was decided to experiment with a Y-maze, operated according to the same principles as the paddling pools. The apparatus consists of three arms made of transparent polystyrene or Perspex, each 30 x 8 x 20 cm. This is mounted on a white base (Figure 7). White was chosen to maximize the aversiveness of the floor color and so encourage escape from the shallow (2 cm deep). As in the water maze, there is only one true exit, with the other two arms terminating in false exits which look the same from inside the maze (Figure 8).
Running the paddling Y-maze
Place a mouse at the end of one of the closed arms, facing away from the center. The sequence of arms chosen as the starting position is defined by a semi-random sequence; no more than three consecutive trials with the same position, and equal numbers of the left or right arms. Each trial lasts 60 sec. If the mouse fails to exit (whole head in exit tube) within this time encourage it to enter the arm with the aid of a piece of transparent Perspex. The exit arm's base is blocked by the Perspex and the mouse allowed to finds the exit by itself; this should encourage better learning than pushing it right into the tube. But this may need to be resorted to if the mouse is reluctant to enter the tube after 15 sec. Take measures of the total time to exit and the number of errors (whole body excluding tail enters a blind arm).
Scoring the paddling Y-maze
There are two definitions of a correct trial: firstly one in which the mouse finds the exit in &lt;60 sec and makes no entries into blind arms, with errors being scored if blind arms are entered and/or if the exit is not found within 60 sec. This analysis confounds failures to start moving with errors in arm entries. Secondly, a trial analysis that emphasizes cognitive ability can be performed by excluding, for each mouse, all trials where the mouse did not leave the start arm within 60 sec, (i.e. delete all 0+60 scores) then calculating the % correct of the remaining trials.
4. Two types of Y-maze Combined
1. Apparatus
When building a paddling Y-maze, it is useful to incorporate two doors to enable it to be run as a (dry) spatial novelty memory experiment 6 (Figure 13). A transparent guillotine door covers the exit hole in the end wall of the exit arm, while an opaque guillotine door can be inserted to seal of one arm from the other two during the sampling phase of the test. It is important that the runners for these doors are not within reach of the mice, as otherwise they may divert their attention because they appear potential escape routes. Ideally they should extend 7 cm down from the top of the maze wall.
2. Set-up
Place a thin (0.5 cm) layer of wood chip bedding material on the floor of the Y-maze. This is redistributed between the two test phases for individual mice, and between mice. For best practice, to make the olfactory environment more similar for the first versus subsequent mice, place one or two non-experimental mice in the maze for a few minutes before the experiment starts.
3. Procedure
With the exit arm blocked off by the opaque door and its exit hole blocked by the transparent door, place the mouse in the start arm (one of the two non-exit arms of the maze) and allow it to explore for 5 min. Then remove it, raise the opaque door to allow access to all three arms, replace it in the start arm and observe it for 2 min. Note the number of entries and the time spent in each arm. Control mice should remember that the first two arms, and spend more time in the previously inaccessible arm.

<ol>
	<li>Barnes, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+deficits+associated+with+senescence:+a+neurophysiological+and+behavioral+study+in+the+rat.">Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat.</a> J. Comp. Physiol. Psychol. 93, 74-104 (1979).</li><li>Blizard, D. A., Cousino Klein, L., Cohen, R., McClearn, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Mouse-Friendly+Cognitive+Task+Suitable+for+Use+in+Aging+Studies.">A Novel Mouse-Friendly Cognitive Task Suitable for Use in Aging Studies.</a> Behav. Genetics. 33, 181-189 (2003).</li><li>Deacon, R. M. J., Cholerton, L. L., Talbot, K., Nair-Roberts, R. G., Sanderson, D. J., Romberg, C., Koros, E., Bornemann, K. D., 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=Age-dependent+and+-independent+behavioral+deficits+in+Tg2576+mice.">Age-dependent and -independent behavioral deficits in Tg2576 mice.</a> Behav. Brain Res. 189, 126-138 (2008).</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., 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=Learning+impairments+of+hippocampal+lesioned+mice+in+a+paddling+pool.">Learning impairments of hippocampal lesioned mice in a paddling pool.</a> Behav. Neurosci. 116, 472-478 (2002).</li><li>Dellu, F., Mayo, W., Cherkoaoui, J., Le Moal, M., Simon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-trial+memory+task+with+automated+recording:+study+in+young+and+aged+rats.">A two-trial memory task with automated recording: study in young and aged rats.</a> Brain Res. 558, 132-139 (1992).</li><li>Morris, R. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localization+does+not+require+the+presences+of+local+cues.">Spatial localization does not require the presences of local cues.</a> Learn. Motiv. 12, 239-260 (1981).</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>Sanderson, D. J., Gray, A., Simon, A., Taylor, A. M., Deacon, R. M. J., Seeburg, P. H., Sprengel, R., Good, M. A., Rawlins, J. N. P., Bannerman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+glutamate+receptor-A+(GluR-A)+AMPA+receptor+subunits+impairs+one-trial+spatial+memory.">Deletion of glutamate receptor-A (GluR-A) AMPA receptor subunits impairs one-trial spatial memory.</a> Behav. Neurosci. 121, 559-569 (2007).</li><li>Sunyer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barnes+maze,+a+useful+task+to+assess+spatial+reference+memory+in+the+mice.">Barnes maze, a useful task to assess spatial reference memory in the mice.</a> Protocol Exchange. , (2007).</li><li>Wahlsten, D., Rustay, N. R., Metten, P., Crabbe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+a+better+mouse+test.">In search of a better mouse test.</a> TINS. 26, 132-136 (2003).</li></ol>

No conflicts of interest declared.

In conclusion, paddling seems a generally effective motivator for mice, and avoids the stress associated with deep water swimming. Unlike the path length and escape time measures in the Morris water maze, the error rate in the paddling pool for hippocampal lesioned mice remained constant throughout the training period5, so this represents a pure measure of spatial memory, as opposed to escape time or path length, which both decrease as the mice become acquainted with the non-spatial elements of the task. The errors measure also provides a greater magnitude of difference between impaired (e.g. hippocampal lesioned) mice and controls.
In the original publication, probe tests were conducted to check if the mice were truly using spatial cues5. In Probe 1, the maze was rotated 120 &deg; but the geographical location of the escape tube remained the same as in training. Performance remained virtually unchanged. In Probe 2, similar to the standard extinction test in the Morris maze, the exit tube was blocked. The time spent in the training quadrant of the maze, where the exit tube was previously located, was 50% for control mice, 25% for mice with hippocampal lesions. In a third probe test in which the position of the exit tube was changed, the controls again spent more time in the original training quadrant. These probe tests confirmed that the mice were not using intramaze cues but were guided by the spatial cues in the laboratory external to the maze. This lack of influence of intramaze cues puts the paddling pool at an advantage over the Barnes maze.
Although we have not tried rats in paddling mazes, this might be possible, although it might be an advantage to make the water colder than the 20-25 &deg;C normally used in the Morris maze. However, since their bodies would not be immersed in the water this should not have adverse welfare effects.

The Morris and Barnes Mazes
Since the beginnings of experimental psychology, studies of animal learning have relied heavily on mazes, generally constructed of opaque wood or metal. Inevitably, due to the extraordinary olfactory abilities of rodents, many studies using them were compromised to some extent; when the experimenter thought that he/she had succeeded in teaching an animal a visual or position discrimination, the rat or mouse had in fact been mainly using olfaction to solve the problem. This is epitomised by the controversy over whether rats with lesions of the hippocampus can perform spatial reference memory tasks, discussed in detail in 20024. Essentially, David Olton and colleagues, apart from in early work with the radial maze, do not appear to have always systematically rotated their mazes. This led to hippocampal lesioned animals solving the task, presumably by odor cues that made each arm distinctive. At Oxford, a (naturally) blind mouse was once observed on a six-arm radial maze. It left its start arm and turned right. Then it crossed the center and chose the opposite arm. Afterwards it fell into a stereotyped strategy of always turning right, but when it re-encountered the base of the first arm it had entered, it took a momentary sniff at it (~0.2 sec) then rejected it and moved to the next arm. If olfactory information on a single visit could be remembered, it is clear that testing over repeated trials on a static maze with the same arm always baited would lead to strong olfactory associations with the reward, and learning would easily occur even if the lesion prevented any purely spatial learning.
Problems such as these were the stimulus for Morris to develop his water maze7; water would not provide constant localized olfactory cues.
The paddling pool is essentially a hybrid between the traditional water maze designed by Morris7 and the dry Barnes1 maze. In the Morris water maze, the animal swims in deep water, escape from which is to the shallow water covering a slightly submerged platform located towards the maze center. In the Barnes maze, the animal is placed on a (dry) circular platform with exit holes around the periphery, only one of which offers escape to a box placed underneath.
The use of paddling as an escape motivator arose from problems reported when disproportionate numbers of some strains of transgenic mice failed to swim properly in the Morris maze. They either dived or passively floated11. Since this might represent a stress-related response, it was reasoned that reducing the water to paddling depth might suffice to overcome this problem, which indeed it did. It was also decided to allow escape to a dry tube set in the side of the apparatus, rather than to shallow water covering a platform. This would be a more "natural" escape response, and increase the spatial component of the test, since a lot of the initial learning in the Morris maze is procedural; the animals must first overcome their innate tendency to swim along the walls of the maze before spatial learning can begin.
The Barnes maze also can suffer from animals not being sufficiently motivated to escape the circular platform10,11.

shallow water (paddling) variants of water maze tests in mice

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to the mouse becoming increasingly important. But researchers found that some strains of mutant mice were prone to problems like passively floating or diving when they were tested in the Morris water maze11. This was unsurprising considering their natural habitat; rats swim naturally (classically, the "sewer rat"), whereas mice evolved in the dry areas of central Asia.
To overcome these problems, it was considered whether shallow water would be a sufficient stimulus to provide escape motivation for mice. This would also avoid the problems of drying the small creatures with a towel and then putting them in a heated recovery chamber to avoid hypothermia, which is a much more serious problem than with rats; the large ratio of surface area to volume of a mouse makes it particularly vulnerable to rapid heat loss.
Another consideration was whether a more natural escape strategy could be used, to facilitate learning. Since animals that fall into water and swim away from the safety of the shore are unlikely to pass on their genes, animals have evolved a natural tendency to swim to the edge of a body of water. The Morris water maze, however, requires them to swim to a hidden platform towards the center of the maze - exactly opposite to their evolved behavior. Therefore the paddling maze should incorporate escape to the edge of the apparatus. This feature, coupled with the use of relatively non-aversive shallow water, embodies the "Refinement" aspect of the "3 Rs" of Russell and Burch8.
Various types of maze design were tried; the common feature was that the water was always shallow (2 cm deep) and escape was via a tube piercing the transparent wall of the apparatus. Other tubes ("false exits") were also placed around the walls but these were blocked off. From the inside of the maze all false exits and the single true exit looked the same. Currently a dodecagonal (12-sided) maze is in use in Oxford, with 12 true/false exits set in the corners. In a recent development a transparent paddling Y-maze has been tested successfully.

Hippocampal lesions greatly impair learning in the paddling pool5. Using a paddling Y-maze, an age-dependent deficit has been demonstrated in 10 and 14, but not 3 month old Tg2576 mice (a model of amyloid over-expression). Aged (21 month) control mice also performed poorly3. A spatial novelty memory experiment run in the dry Y-maze revealed impairments in mice with knockout of the glutamate receptor-A (GluR-A) AMPA receptor subunits9.
<img alt="Figure 1" src="/files/ftp_upload/2608/2608fig1.jpg" /> 
 Figure 1. A mouse in the circular Mk 1 paddling pool.
<img alt="Figure 2" src="/files/ftp_upload/2608/2608fig2.jpg" /> 
 Figure 2. The mouse finds the exit tube of the paddling pool.
<img alt="Figure 3" src="/files/ftp_upload/2608/2608fig3.jpg" /> 
 Figure 3. The mouse escaping into the exit tube. The tube is then detached from the maze and the mouse returned to its home cage.
<img alt="Figure 4" src="/files/ftp_upload/2608/2608fig4.jpg" /> 
 Figure 4. A mouse in the dodecagonal paddling pool.
<img alt="Figure 5" src="/files/ftp_upload/2608/2608fig5.jpg" /> 
 Figure 5. A mouse about to commit an error as it approaches a false exit.
<img alt="Figure 6" src="/files/ftp_upload/2608/2608fig6.jpg" /> 
 Figure 6. An error is committed as the mouse's nose comes within a head's distance of a false exit. The real exit is to its right, with the exit tube attached.
<img alt="Figure 7" src="/files/ftp_upload/2608/2608fig7.jpg" /> 
 Figure 7. A paddling Y-maze.
<img alt="Figure 8" src="/files/ftp_upload/2608/2608fig8.jpg" /> 
 Figure 8. Detail of the exit arm end of the Y-maze. A shallow U-shaped trough adjoins the exit hole in the end wall. This supports the exit tube, shown below the support in the picture above. The exit tube can be removed from the support to take the mouse back to its home cage. All arms are fitted with this set-up but only one arm end wall has a circular hole cut into it to allow escape.
<img alt="Figure 9" src="/files/ftp_upload/2608/2608fig9.jpg" /> 
 Figure 9. A mouse in a blind alley of the paddling Y-maze.
<img alt="Figure 10" src="/files/ftp_upload/2608/2608fig10.jpg" /> 
 Figure 10. The mouse finds the exit tube.
<img alt="Figure 11" src="/files/ftp_upload/2608/2608fig11.jpg" /> 
 Figure 11. A mouse, having escaped from the paddling Y-maze, waits to be returned to its home cage.
<img alt="Figure 12" src="/files/ftp_upload/2608/2608fig12.jpg" /> 
 Figure 12. Mice can generally be relied upon to stay in the tube while they are carried back to their nearby home cage.
<img alt="Figure 13" src="/files/ftp_upload/2608/2608fig13.jpg" /> 
 Figure 13. Combining a paddling Y-maze with a spatial novelty Y-maze configuration. An opaque guillotine door is inserted into a slide at the proximal end of one arm, and a transparent guillotine door seals off the exit hole.

Watch this Scientific Journal Video about Shallow Water Paddling Variants of Water Maze Tests in Mice at JoVE.com

Shallow Water Paddling Variants of Water Maze Tests in Mice

Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

Shallow Water (Paddling) Variants of Water Maze Tests in Mice

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to ...

shallow-water-paddling-variants-of-water-maze-tests-in-mice

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23.2K Views.  University of Oxford. Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

Video: Shallow Water Paddling Variants of Water Maze Tests in Mice

Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food deprivation, and may introduce scent cue confounds. The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs. Specifically, subjects are required to avoid the arms previously used for escape during each testing day (working memory) as well as avoid the fixed arms, which never contain escape platforms (reference memory). Re-entries into arms that have already been used for escape during a testing session (and thus the escape platform has been removed) and re-entries into reference memory arms are indicative of working memory deficits. Alternatively, first entries into reference memory arms are indicative of reference memory deficits. We used this maze to compare performance of rats with neonatal brain injury and sham controls following induction of hypoxia-ischemia and show significant deficits in both working and reference memory after eleven days of testing. This protocol could be easily modified to examine many other models of learning impairment.

The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs.

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food ...

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 ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...