How to Use Mice in Behavioral Neuroscience Only after the design of the study and the specific behavioral tests are Chapter 3 - Tests of Mouse Behavior.
Table of contents
In contrast, the calculation of total distance includes constraints that exclude units of activity that are generated by these repetitive beam breaks. Time spent investigating the central region of the chamber can be reported as a percent of total session length for both the short and longer habituation versions of this test. Alternatively, center time can be examined in 5-min bins over the duration of the min session to examine changing patterns of anxiety-related behavior. Behavioral measures reported include total distance traveled, horizontal beam breaks, and time spent exploring the center area of the chamber.
Effect of GalR2 mutation on open field exploration. Males were significantly more active than females on horizontal activity and distance more This well-established paradigm has a long and successful history in assessing anxiety-like behavior in mice [ 22 , 42 , 54—56 ]. The test takes advantage of the natural tendency of mice to explore novel environments.
The mouse is given the choice of spending time in open, unprotected maze arms or enclosed, protected arms, all elevated approximately 1 m from the floor. Mice tend to avoid the open areas, especially when they are brightly lit, favoring darker, more enclosed spaces. This approach—avoidance conflict results in behaviors that have been correlated with increases in physiological stress indicators [ 52 ]. In contrast, administration of benzodiazepines and other anxiolytic treatments results in increased exploration of the open arms, without affecting general motivation or locomotion [ 42 , 55 , 57 , 58 ].
The primary requirements for subjects performing this test are normal ambulatory ability and average levels of exploratory drive. Mice that spend prolonged time in the center start area, enter only partially into one arm of the maze without transitioning through, or do not explore the entire maze, may confound the interpretation of behavioral data for a group. In these cases, data may primarily reflect physical motor abilities that are minimally relevant to anxiogenic or anxiolytic traits. It is important to note this type of behavior during the test session, as it may be necessary for later identification of outliers.
Strains that consistently demonstrate very low levels of exploratory behavior e. Conceptually the equipment design has remained virtually unchanged for mice since its introduction [ 59—61 ]. However, there have been substantial alterations and modifications in the materials and specific details of the maze construction.
Mouse Behavioral Testing | ScienceDirect
Two arms are enclosed with cm high walls. The remaining two arms are open. Differences in maze construction include wood construction versus Perspex or other similarly smooth material. Some researchers have provided a slightly raised lip 0. Walls of the enclosed arms may be transparent, opaque, or dark. While a consensus has not been reached about the advantages or limitations of wall transparency, researchers may want to consider the impact of these different materials on light levels within the arm [ 62 ]. Ideally, minimizing variability in external factors e.
The elevated zero-maze offers a conceptually identical behavioral test that eliminates the ambiguous center start area of the elevated plus-maze EPM [ 63 ]. In the plus-maze, test subjects will often remain in the central start area, or return to it regularly, thereby spending considerable amounts of time in a region of the maze that is considered ambiguous in the evaluation of anxiety-related behavior.
The elevated circular runway alternates equally sized, open, brightly lit areas and enclosed, dark arc areas. The uninterrupted nature of the open versus closed segments of the circular runway mitigates the concerns surrounding the central start area of the plus-maze.
Similar behavioral measures are scored for this version of the test during the 5-min session. Scoring from a videotaped session minimizes environmental variables introduced by the presence of the investigator that may impact anxiety-related behaviors. Technological advances have been introduced as a means of standardizing the EPM paradigm, including automated tracking and scoring software e.
Concerns have been raised [ 10 , 42 ] about the sensitivity of automated systems for detecting measures of ethologically relevant risk assessment behaviors and the utility of scoring many additional behavioral indices as factors to explain anxiety-related behavior of animals [ 64 , 65 ]. Inconsistent results with anxiolytic compounds and a desire for more targeted therapeutic treatments suggests that scoring additional, ethologically relevant behavioral indicators e.
It remains to be determined whether current tracking and scoring software can accurately and consistently detect these additional behavioral indices in the wide variety of inbred strains and transgenic and knockout KO lines currently being studied. Subjects are generally group-housed four to five per cage in same-sex home cages. Home cages are brought to the testing room or a common staging area 1 hr prior to testing.
Transport mice singly in clean cages to the apparatus or testing room. Room level lighting should be consistent for all subjects.
Mice generally avoid brightly lit areas, therefore high illumination levels would be expected to increase anxiety-like behaviors. Care is taken to avoid light levels that are high enough to restrict the natural exploratory tendency of mice. Pilot studies will assist in determining the most appropriate illumination level from those reported in the literature [ 67—70 ].
Each subject is placed in the central area of the maze with open access to any arm. Mice are allowed to freely explore the maze for 5 min. The number of arm entries and the amount of time spent in the open and closed arms are recorded. These can be recorded manually by a highly trained observer, or by an automated photo beam sensor recording system. The session can also be recorded using any one of the currently available video tracking systems for subsequent scoring. There are advantages and limitations to each of these methods. The obvious advantage to scoring from a recorded test session is the ability to minimize errors and recheck the reliability of the scoring at a later time.
Similarly, photo beam recording systems remove the subjective interpretations by the experimenter. For researchers with limited resources, however, these systems may be cost prohibitive. Two advantages of manual scoring by highly trained observers are lower equipment costs and identifying unusual or ethologically relevant behaviors that might go undetected by automated systems.
There are several factors that should be considered when interpreting EPM results. Strains or treatment groups that show unusually high or low time spent in the open arms may do so for reasons other than anxiety-related behavior. For instance, extensive time spent in the open arms may reflect a group that displays very low levels of exploratory activity. The arm they first transition into, closed or open, is where they remain.
In addition, specific pharmacological treatments, background strain differences, genetic mutations, or environmental factors can impact locomotor activity, exploratory behavior, or behavioral motivation for novelty [ 25 , 38 , 39 , 44 , 71 , 72 ]. Finally, behavior in the EPM is influenced by prior handling, exposure to previous behavioral testing paradigms, or repeated experience in the plus-maze [ 10 , 54 , 56 , 73—77 ].
Repeated testing was thought to have no significant impact on measures of anxiety behavior [ 59 , 61 ]. However, recent studies suggest that prior test experience increases open arm avoidance behavior and alters the effectiveness of anxiolytic drugs in reducing open arm avoidance [ 57 , 75 , 77—84 ].
For situations in which retest in the EPM is necessary, Adamec and Shallow [ 86 ] have developed a test—retest protocol that appears to prevent the increase in open arm avoidance generally exhibited in trial 2. They suggest a 3-wk interval between test sessions and moving the maze to a novel test room for the second session. In this experiment mice with a null mutation in the galanin receptor subtype GalR2 were tested in several complementary approach—avoidance paradigms designed to assess anxiety-like traits.
Previous research has implicated the neuropeptide galanin in rodent emotionality [ 52 , 88—91 ]. The results from two independent cohorts of mice missing the galanin subtype-2 receptor indicate an anxiogenic phenotype in the EPM. Both cohorts spent significantly less time exploring the open arms Figure 5. Importantly, the number of overall arm transitions Figure 5.
Although not shown in the figure, some labs are now reporting additional ethologically relevant behaviors in their experimental results, including head dips and stretch-attend postures. These behaviors have been described as a means for the animal to actively assess dangers within the specific testing environment and are characterized as risk assessment behaviors [ 6 , 42 ]. Anxiogenic-like phenotype of GalR2 knockout mice on the elevated plus-maze. As with the EPM, the subject is exposed to a novel environment with protected dark compartment and unprotected light compartment areas.
The inherent conflict between exploratory drive and risk avoidance is thought to inhibit exploration [ 16 , 92 , 93 ]. Most mice naturally demonstrate a preference for the dark, protected compartment. The key measure for assessing anxiety-related behavior in this design is a change in willingness to explore the illuminated, unprotected area, reflected in increases or decreases in the number of transitions between the compartments, and in time spent in each compartment, during a min test session.
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Treatment with anxiolytic drugs increased the number of transitions between the two compartments, without altering the preference of the mice to spend more time in the dark compartment [ 16 , 93 ]. This increase in exploratory activity is interpreted as a release of exploratory inhibition [ 16 ]. Similar to the EPM, careful consideration should be given to testing specific inbred strains of mice and mice with genetic manipulations that inhibit locomotor activity or interfere with novelty-seeking behavior. The smaller compartment 14 cm is painted black and covered by a hinged lid. The larger compartment 28 cm is uncovered with transparent sides and is brightly lit from above by fluorescent room lighting.
Transitions between the compartments are electronically recorded by four sets of photocells mounted in the partition opening. Entry into the dark compartment triggers a timer that records the duration of time spent in the dark compartment. Transport acclimated mice to the test room or test apparatus singly, in clean cages. The mouse is placed centrally into the larger, brightly illuminated compartment facing away from the partition.
Mice are allowed to freely explore the chamber for 10 min while transitions and time spent in the dark compartment are recorded. After completion of the test, return mice to the home cage. Unlike the EPM, some previous testing experience with this, or other behavioral tests, does not appear to alter behavioral performance [ 40 , 92 , 94 ]. The number of transitions and the time spent in the dark compartment are analyzed using one-way ANOVAs and Newman-Keuls post-hoc comparisons when indicated.
Mice exhibiting higher levels of anxiogenic-like behavior will make fewer transitions between the brightly illuminated, open area and the dark, enclosed compartment. Many laboratories also use time in the dark or, reciprocally, time in the light as a measure of anxiogenic-like behavior [ 51 , 95—97 ]. Recently some laboratories have included time spent in risk assessment as another measure of anxiety-related behavior [ 97 ]. Risk assessment includes a stretch-attend posture in which the head and fore-paws extend into the lighted area but the remainder of the body stays in the dark compartment.
This test has been shown to be very sensitive to the anti-anxiety—like effects of benzodiazepines. Benzodiazepines increase transitions between the compartments without affecting general locomotor activity. Investigators should use caution when interpreting results of new compounds on anxiety-like behavior until they have been screened for nonspecific locomotor effects in a separate apparatus such as an automated open field.
The study [ 97 ] illustrated in Figure 5. Mice receiving two different doses of NPY made more transitions between the two chambers and spent more time in the light compartment than controls or galanin-treated mice. In addition, Figure 5. Thus, neuropeptide Y, but not galanin, produced an anxiolytic-like action when centrally administered to mice. Mice treated with NPY at an icv dose of 0. The social interaction test, developed by File and Hyde [ 98 ], provided the first test of anxiety-like behavior that focused on ethologically relevant concepts.
The test eliminated the need to introduce aversive or appetitive conditions. Pairs of male rats are allowed to freely interact in an arena while time spent interacting is recorded as the dependent measure. Interaction time for each of the rats in the pair is directly impacted by the behavior of the partner animal.
Therefore, the pair counts as one unit for data collection purposes. If the design of the experiment involves one rat receiving treatment while the other serves as a control, then interaction time initiated by the treated rat is the appropriate dependent measure. Anxiolytic-like behavior is inferred if social interaction time increases and general motor activity remains unaffected.
Conversely, decreased time spent engaging in social behavior would indicate anxiogenic-like behavior. Manipulating environmental conditions allows the researcher to induce varying levels of anxiety in the test subject. The arena is either familiar or novel and illumination levels can range from bright to dim. These conditions can be characterized as low anxiety inducing when the environment is familiar F and illumination levels are low L , versus high anxiety inducing when lighting conditions are bright and the test arena is unfamiliar U.
The remaining two conditions, low illumination, novel arena and high H illumination, familiar arena, result in moderate baseline anxiety levels [ 99 ]. The ability to systematically increase or decrease baseline anxiety levels has proven especially useful for screening novel pharmaceutical compounds developed for treating anxiety. Higher baseline anxiety levels, induced by the HU condition, are well suited for detecting the effects of anxiolytic compounds.
Conversely, robust anxiogenic effects can be detected in the LF condition, when it is expected that the greatest amount of time would be spent in social interaction [ 99 ]. It should be noted that adult female rats failed to increase time spent in social interaction as a function of increasing familiarity of the test environment [ ], suggesting that some environmental manipulations have different salience for the social behaviors of each sex [ ]. Although originally designed for rats, modified versions of this test have been used relatively successfully to evaluate anxiety-like behavior in mice [ , ].
It should be noted that effects demonstrated in mice are less consistent than those exhibited by rats. Of the manipulated variables, light level appears to have the greatest impact on anxiety in mice [ , ], while familiarity of the test arena, similar to the response of female rats, does not provide consistent changes in anxiety level in mice. In singly housed mice, similar to the effect seen in rats, anxiolytics reverse the inhibition of social interaction induced by brighter lighting [ , ].
Inconsistent findings with female mice would indicate that this test is more suitable for testing male social behavior. Noticeably aggressive, dominant, group-housed mice should not be used, as this could significantly impact the sociability of the isolate mouse. The novel cage environment can be a standard polypropylene rat cage or clear Plexiglas chamber that is unfamiliar to the subjects before acclimation.
Recording equipment is mounted above the cage at a distance that provides complete coverage of the arena but does not interfere with the test environment. Social interaction is tested between pairs of mice that are either singly housed for 3—6 wk or group housed. Test pairs can involve one group-housed and one isolate mouse, or two unfamiliar, group-housed mice.
Singly housing mice has been demonstrated to increase social investigation [ , ]. At the end of the acclimation period a group-housed mouse is introduced for a 4-min test period. In the case of pairs of unfamiliar, group-housed mice, each mouse is given a min acclimation session in the test cage on the two days prior to the experiment. On day 3 the pair of mice is placed into the test cage for the min test session [ ].
Test sessions are recorded and scored at a later time. As the test was originally developed, the mean total time engaged in social behaviors is scored, analyzed, and reported [ 17 ]. An alternative to this method is to score categories of behavior for each treatment group including aggressive attack, aggressive unrest , fearful vigilant posture, escape and defense activity , social following, social sniffing, over-under climbing , and locomotion rearing, walking during cage investigation and report the mean number of events in each category [ ].
Scorers should be blind to any experimental treatment. Inter-rater reliability values are determined for a sampling of the tested mice by multiple scorers. If the experimental design includes evaluating the effects of anxiolytic ligands, illumination levels can be increased to inhibit baseline social investigation. Mean time spent in social interaction is the most reported parameter of social behavior.
Active social behavior of the subject mouse is scored, including following, sniffing, and climbing on or under the other mouse. If lighting levels have been manipulated, or more than two groups are included, then analyze the means using an ANOVA followed by Neuman-Keuls post-hoc test when indicated. Time spent interacting with an unfamiliar partner is the primary measure reported as a measure of sociability in mice. Previous findings had implicated this receptor in modulating social recognition memory. The mean amount of time spent engaged in social behavior is shown in Figure 5.
Time spent in social investigation in the social interaction test of V1aR knockout KO mice and wild type mice. Rodents encountering a desirable food in a novel environment will consume very limited quantities after considerable investigation. Mice tend to avoid exploration of novel open environments, yet are motivated to approach and consume palatable food.
This inhibition of feeding behavior has been termed hyponeophagia and is robust in both rats and mice. The response is unconditioned, requires no training, and can be elicited in food-deprived or satiated animals by substituting a highly palatable food source for regular chow. Treatment with a variety of drugs used to manage anxiety in humans reliably reverses this decrement in feeding, reducing the latency to the first taste and increasing the total amount of food consumed for review see [ , ].
Several factors have been found to influence baseline levels of hyponeophagia in mice, including the genetic background of inbred strains, long durations of isolate housing, and specific genetic mutations that affect anxiety-related behaviors [ — ]. Several methodological concerns have been raised with hyponeophagia-based testing. One is the failure of many designs to include a comparison of food consumption in the home cage environment [ ]. Investigators should report equivalent assessment measures of feeding behavior latency and total consumed in both the novel and home cage environments to determine the contribution of the independent variable to any observed differences [ ].
Another possible confound is the potential impact of drug treatments or genetic manipulations on factors unrelated to anxiety.
5.1. INTRODUCTION
Drugs targeting serotonergic function selectively decrease feeding behavior and alter macronutrient intake [ , ]. Experimental protocols that incorporate food deprivation may compound these appetite-related effects, potentially masking or exacerbating anxiety-like measures. Substituting a familiar, highly palatable food in the home cage and unfamiliar environment minimizes some of these methodological problems [ ]. In the home cage mice quickly approach and ingest the food. In the novel environment they show a marked increase in latency to first taste the familiar food [ ].
In addition, Dulawa and Hen [ ] suggest using higher illumination levels for the novel environment to optimize hyponeophagia levels. In their modified model, Dulawa, Holick, Gundersen, and Hen [ ] propose reporting measures of both latency and total food consumed in the novel and home cage environments. When latency alone is reported, home cage scores may be very low, making it extremely difficult to detect manipulations expected to enhance appetite.
This modified model provides some advantages over older versions, including improved sensitivity and reliability of the test results by assessing two behavioral measures, and increasing the likelihood that the test will discriminate treatments that enhance, as well as decrease, feeding behavior. However, as with most designs, there are a few limitations to note, including training the mice to consume the highly palatable novel food and single housing animals immediately prior to testing. Mice ranging in age from juvenile to older adult can be tested in this paradigm. As mentioned above, attention should be given to the background strain, housing arrangements, and genetic mutations designed to influence emotionality, as these may alter baseline levels of feeding behavior.
Depending on the independent variables of interest in the experimental design, group-housed mice should be singly housed for at least 5 days prior to testing. Standard mouse cages of identical size can be used for the home cage and novel cage environments. In the novel environment condition, cages can be either free of bedding or have new bedding.
One option for a highly palatable food source is diluted 3—1 sweetened condensed milk Carnation , although other food may be substituted. Singly housed mice are trained to consume the palatable food source by introducing it to them in their home cage for 30 min daily over three consecutive days. Diluted condensed milk in plastic serological pipettes 10 mL with attached sippers and rubber stoppers are mounted to the wire cage lid. Mice are allowed access for 30 min daily. On the fourth day mice are tested in the home cage condition.
Remove mice from the cage while the pipette is installed on the cage lid. This maintains a consistency in the handling procedure for the two home versus novel cage experimental conditions. Commence testing as soon as mice are returned to the cage. Record the latency to the first lick and the total volume consumed in 5-min intervals across the min session. Note any mice that do not consume any condensed milk. They should be excluded from further testing as they failed the training protocol.
On day 5, position the pipette in the wire lid of the novel cage and place the mouse into the novel cage environment. Record latency and total volume consumed as previously described. Comparison of the total volume of food consumed across the 30 min can be analyzed using a between-subjects repeated measures ANOVA. Although the initial 5-min period may provide sufficient information for assessing anxiolytic effects of treatment, the sensitivity of this initial period for distinguishing anxiogenic effects is less certain [ ].
Latency data generally violate several assumptions of the ANOVA test; therefore, violations of these tenets should be examined. It may be necessary to transform or truncate the data, according to statistical convention, prior to analysis [ ]. The graphs presented in Figure 5. Fifteen mice per group received tap water laced with one of three fluoxetine doses or tap water only.
Fluoxetine decreased the latency to the first lick at all doses in the novel cage, but had no effect on latency to drink in the home cage Figure 5. In addition, in the novel environment fluoxetine decreased overall food consumption compared to the home environment. The difference in latency to consume A in the home cage, B in a novel cage, and C more Several ethologically relevant tests of anxiety-like behavior have been presented as a representative sampling of the broader collection of assays designed to assess anxiety-related behavior in mice in the field of behavioral neuroscience.
Space limitations and methodological specificity necessitated limiting the scope of the present work to this smaller subset of anxiety-related behavioral tests. The interested researcher seeking additional tests that directly assess anxiety-like behavior may wish to explore the following excellent paradigms: Investigators seeking an in-depth characterization of anxiety-related behaviors in a mutant line of mice are encouraged to conduct two or more of these well-validated assays to strengthen the interpretation of their findings.
Turn recording back on. National Center for Biotechnology Information , U. Show details Buccafusco JJ, editor. Open Field Exploration Test Originally introduced as a measure of emotion00al behavior in rats [ 8 ], open field exploration has proven to be equally successful with mice [ 47 ]. Procedure Transport acclimated mice to the test room singly, if only one test chamber is available, or as a group in the home cage, if several automated chambers are available for testing. Analysis and Interpretation Open field exploration results are generally analyzed using repeated-measures analysis of variance ANOVA for longer sessions when the researcher is interested in comparing levels of exploratory activity over the duration of the session.
Sample Results Figure 5. Subjects The primary requirements for subjects performing this test are normal ambulatory ability and average levels of exploratory drive. Equipment Conceptually the equipment design has remained virtually unchanged for mice since its introduction [ 59—61 ]. Procedure Subjects are generally group-housed four to five per cage in same-sex home cages.
Mouse Behavioral Testing
Subjects Similar to the EPM, careful consideration should be given to testing specific inbred strains of mice and mice with genetic manipulations that inhibit locomotor activity or interfere with novelty-seeking behavior. Procedure Transport acclimated mice to the test room or test apparatus singly, in clean cages. Analysis and Interpretation The number of transitions and the time spent in the dark compartment are analyzed using one-way ANOVAs and Newman-Keuls post-hoc comparisons when indicated.
The Social Interaction Test The social interaction test, developed by File and Hyde [ 98 ], provided the first test of anxiety-like behavior that focused on ethologically relevant concepts. Subjects Inconsistent findings with female mice would indicate that this test is more suitable for testing male social behavior. Equipment The novel cage environment can be a standard polypropylene rat cage or clear Plexiglas chamber that is unfamiliar to the subjects before acclimation.
Procedure Social interaction is tested between pairs of mice that are either singly housed for 3—6 wk or group housed. Analysis and Interpretation Mean time spent in social interaction is the most reported parameter of social behavior. Sample Results Time spent interacting with an unfamiliar partner is the primary measure reported as a measure of sociability in mice.
Novelty-Inducedhy Pophagia Rodents encountering a desirable food in a novel environment will consume very limited quantities after considerable investigation. Subjects Mice ranging in age from juvenile to older adult can be tested in this paradigm. Equipment Standard mouse cages of identical size can be used for the home cage and novel cage environments. Procedure Singly housed mice are trained to consume the palatable food source by introducing it to them in their home cage for 30 min daily over three consecutive days.
Analysis and Interpretation Comparison of the total volume of food consumed across the 30 min can be analyzed using a between-subjects repeated measures ANOVA. Sample Results The graphs presented in Figure 5. The pharmacology of human anxiety. Neurobiological alterations associated with traumatic stress. Perspectives in Psychiatric Care. Diagnostic and statistical manual of mental disorders: American Psychiatric Association; Attack and defense in rodents as ethoexperimental models for the study of emotion.
Differentiation of anxiolytic and panicolytic drugs by effects on rat and mouse defense test batteries. The mouse defensive test battery: Pharmacological and behavioral assays for anxiety and panic. European Journal of Neuroscience. Cryan JF, Holmes A. The ascent of mouse: Advances in modelling human depression and anxiety. Emotional behavior in the rat. Defecation and urination as measures of individual differences in emotionality.
The book discusses the practical matters Access Online via Elsevier Bolero Ozon. How to Use Mice in Behavioral Neuroscience. A guide to running a behavioral testing lab, including the many aspects of mouse research beyond the confines of the specific test Diagrams and photographs are shown for many kinds of apparatus and test situations with sufficient details such as dimensions to enable building of replicas Provides step-by-step instructions on planning and executing behavioral experiments in order to run them successfully.
Chapter 1 Introduction to the Research Process.
Chapter 3 Tests of Mouse Behavior. Chapter 5 Sample Size. Chapter 6 Ethics Approval. Chapter 10 Domains and Test Batteries. Chapter 11 Motivating Mice. Chapter 12 Qualities of Behavioral Tests. Chapter 13 Task Refinement and Standardization. Chapter 14 Video Tracking. Chapter 15 The Laboratory Environment. Appendix Utilities in Excel. Chapter 8 Getting Ready for Testing.