5.1. INTRODUCTION

Human anxiety disorders are broadly grouped according to symptomology and responsiveness to pharmacological and psychological treatment [1,2]. Generalized anxiety disorder and panic disorder are the two primary classifications of pathological anxiety in humans. The distinguishing feature of generalized anxiety disorder is a pervading sense of unrealistic worry about everyday life situations. In contrast, panic attacks constitute the primary symptom of panic disorder. These events are characterized as sudden, extreme fear accompanied by autonomic nervous system arousal [3].

Similar changes in physiological indicators and behavioral responses to fear and painful stimuli in humans and other animals suggest the possibility of homologous or analogous, ethologically motivated defensive responses [4–10] In the description of human anxiety disorders, the concepts of “state” and “trait” anxiety have a long history. However, it is only recently that these concepts have been suggested as a means of differentiating situational anxiety-like behavior in rodents from anxiety that transcends the situation and is an enduring condition in the animal [11]. The former is the focus of the rodent behavioral tests reviewed in this chapter. Procedures are designed to trigger ethologically relevant conflict or conditioned behaviors. The latter is most often associated with selective breeding, e.g., the high versus low anxiety-related traits in the high anxiety-related behavior (HAB) versus low anxiety-related behavior (LAB) rats [12], inbred mouse strains such as BALB/c, and mice with relevant targeted gene mutations [13,14].

In an attempt to model human pathological anxiety in rodents, a wide range of behavioral testing paradigms have been developed [8,15–19]. Many of these tests induce a fearful response through an aversive event or anticipated aversive event. Others integrate an approach–avoidance conflict designed to inhibit an ongoing behavior that is characteristic for the animal, such as contrasting the tendency of mice to engage in exploratory activity or social investigation against the aversive properties of an open, brightly lit, or elevated space. The premise that basic physiological mechanisms underlying fear in rodents can be equated to similar mechanisms operating in humans provides a degree of face validity for these paradigms [7,9,10]. In rodents, these responses are deemed appropriate and adaptive for the current conditions, whereas in humans, anxiety disorders constitute maladaptive or pathological responses to the existing situation. Further exploration of rodent neuroanatomy and neurochemistry involved in fear extinction and inhibition of conditioned fear could offer important insights into effective targets for novel pharmacological treatment of pathological human anxiety [20].

Although rats have been the rodent of choice for much of the preclinical research on anxiety-like behavior, recent technical advances in molecular genetics have placed the mouse in the forefront of neuropsychiatric research [7,13,21–26]. This has resulted in the adaptation of many well-validated behavioral tests of anxiety from rats to mice, with varying degrees of success. This chapter offers a sampling of well-established tests of anxiety-like behavior in mice that use an ethological conflict. The interested researcher is directed to the classic source literature for more information on other types of anxiety-based tests, including conditioning paradigms [27–29], punishment-induced conflict tests [18,19,30,31], developmental models [32,33], and aversive tests [5,34].

5.2. GENERAL METHODOLOGICAL CONSIDERATIONS

Several excellent papers have identified important factors that careful researchers will want to consider when designing experiments to assess anxiety-like behaviors in mice [35–37]. The behavioral paradigms described in this chapter are suitable for most inbred mouse strains. In addition, mice with targeted genetic mutations that do not alter exploratory drive, motor ability, or recognition memory are also suitable test subjects for these paradigms. Other factors to consider when designing experiments assessing anxiety-like behavior include, but are not limited to, the experimental history of test subjects, prior test exposure, differences in exploratory motivation, and whether the test is to be conducted as part of a test battery or administered as a single behavioral assessment [9,38–42]. Mice are social animals and are typically group-housed (four to five per cage) in same-sex home cages. Special circumstances (e.g., aggressive strains or mice with head mounts or other surgical interventions) may require single-housing prior to testing. Environmental conditions in the animal housing rooms should be as quiet as possible and consistent across experiments to control for extraneous variables that can significantly alter physiological and behavioral indicators of stress [37,43]. Food and water are typically ad libitum unless the experimental design requires restriction. Avoid behavioral testing on days when, as a part of normal animal husbandry, home cages are scheduled for changing. Cage change typically causes an increase in general activity and stress levels [44–46]. Researchers should fully describe any special circumstances pertaining to housing and care of test subjects in their experimental methods. To ensure consistency of experience prior to the test session, subjects are brought to the testing room or a common staging area, in their home cages, at least 1 hr prior to the start of behavioral testing. Individual mice can then be transported singly, in clean cages, into the testing apparatus. Test room lighting, temperature, and noise levels should be consistent for all subjects. Behavioral testing equipment described in this chapter is usually cleaned thoroughly with a solution of mild soapy water at the end of a test session. Prior to running the first subject on a day of testing, the experimenter wipes the behavioral equipment with 70% ethanol. After each subject completes its test session, fecal boli and urine are removed, surfaces are wiped with 70% ethanol, and the test chamber is allowed to dry completely before starting another subject. Most studies use a minimum of 12–15 animals per experimental group to insure sufficient power for statistical analysis [22].

5.3. PARADIGMS

5.3.1. 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]. The test provides a unique opportunity to systematically assess novel environment exploration, general locomotor activity, and provide an initial screen for anxiety-related behavior in rodents [48]. In addition, repeated exposure or extended session length provides a method for assessing habituation to the increasingly familiar chamber environment. It has been suggested that two factors influence anxiety-like behavior in the open field. The first is social isolation resulting from the physical separation from cage mates when performing the test. The second is the stress created by the brightly lit, unprotected, novel test environment [17,48].

5.3.1.1. Equipment

Although several different shapes have been used as rodent open fleld arenas [49,50], the most common design for mice is a large square chamber ranging in size from 28 × 28 cm to 56 × 56 cm. Chamber walls and floor can be plastic or wood but many automated systems use transparent Plexiglas. The open fleld arena is divided into a grid of equally sized areas by infrared photocell beams or lines drawn on the chamber floor for visual scoring of activity by the experimenter. Automated systems such as the VersaMax Animal Activity Monitoring System with Analyzer software (AccuScan Instruments, Inc., Columbus, Ohio, USA), SmartFrame open field system with Motor Monitor control and software (Lafayette Instruments, Lafayette, Indiana, USA), Open Field Activity System MED–OFA-MS (Med Associates, Inc., St. Albans, Vermont, USA), and Photobeam activity system–open field (San Diego Instruments, San Diego, California, USA), record each beam break as one unit of exploratory activity, similar to manual scoring of each line crossed.

5.3.1.2. 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. Place each mouse in the center of a chamber. If the experimenter intends to remain in the testing room, care should be taken to be as distant and unmoving as possible once the test session has started. Sudden motion or noise can greatly affect exploratory activity. Mice are allowed to freely explore the chamber for the duration of the test session. Each line crossed or photocell beam break is scored as one unit of activity. For assessing novel environment exploration, a 5-min test length is typical. If the researcher is interested in examining habituation to an increasingly familiar environment, a 30-min test session is recommended. Mice are allowed to freely explore the test arena for the entire session duration. Upon completion of the test, return the mouse to the home cage. In addition to horizontal units of activity, rearing behavior, defecation, and grooming activity can also be scored. These parameters provide measures of general physical motor abilities and level of interest in the novelty of the environment.

Rodents will typically spend a significantly greater amount of time exploring the periphery of the arena, usually in contact with the walls (thigmotaxis), than the unprotected center area. Mice that spend significantly more time exploring the unprotected center area demonstrate anxiolytic-like baseline behavior. The center area of the chamber can be defined by the experimenter as a proportion of the overall test arena size. Many software systems allow the researcher to designate this center area, as well as multiple other regions of the test chamber, to track exploratory activity. When the open field arena size is 40 × 40 cm², the center region size is often designated as 20 × 20 cm² [51,52].

5.3.1.3. 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. The session can be divided into time bins (e.g., 5 min) and changes in exploratory behavior can be compared across the length of the session. By contrast, if the experimenter’s only interest is novel environment exploration, then a one-way ANOVA can be run using the total scores across the test session for each behavioral measure (e.g., vertical activity, horizontal activity, total distance, and center time). A 5-min test session is often sufficient to capture the critical components of general exploratory locomotion. The most commonly used measure of overall exploratory/locomotor activity is currently the total distance traveled. Although horizontal activity appears to be recording a similar measure, in fact, the equipment records every beam break including those not associated with ambulatory activity (e.g., repetitive head movements). 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 30-min session to examine changing patterns of anxiety-related behavior.

5.3.1.4. Sample Results

Figure 5.1A–C illustrates open field activity for galanin receptor subtype 2 (GalR2) null mutant mice [53]. Behavioral measures reported include total distance traveled, horizontal beam breaks, and time spent exploring the center area of the chamber. There were no effects of genotype on horizontal activity or total distance traveled (all p comparisons > 0.05). Males were significantly more active than females on horizontal activity and total distance traveled in the arena (p = 0.0009 and p = 0.0042, respectively). Galanin null mutant mice spent less time exploring the central area of the chamber compared to wild type (WT) littermates, but this did not reach significance (p = 0.0714).

FIGURE 5.1

Effect of GalR2 mutation on open field exploration. There were no significant genotype differences on horizontal activity or total distance traveled (p > 0.05). Males were significantly more active than females on horizontal activity and distance (more...)

5.3.2. Elevated Plus-Maze/Elevated Zero-Maze

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].

5.3.2.1. Subjects

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.g., AJ, some 129 substrains) should be avoided.

5.3.2.2. Equipment

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. The apparatus consists of two sets of opposing arms approximately 30 × 5 cm extending from a central (5 × 5 cm) region. Two arms are enclosed with 15-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 (0.25 cm) on three sides of the open arms to minimize falls. 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.g., light level differences) will increase replication across labs and simplify interpretation of behavioral findings.

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.g., Noldus Ethovision video tracking, Hamilton-Kinder infrared photobeam tracking). 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.g., head dipping, stretch-attend postures) may provide more sensitive measures of the effects of new anxiolytic compounds [10,65]. 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.

5.3.2.3. Procedure

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.

5.3.2.4. Analysis and Interpretation

EPM results are generally analyzed using between-subjects ANOVAs followed by Newman-Keuls post-hoc comparisons when a significant ANOVA value is obtained. 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]. The change in pharmacological efficacy has been termed “one-trial tolerance” [78] and has been alternately explained as reflecting a change in the state of the benzodiazepine receptor or the gamma-aminobutyric acid (GABA)_A receptor complex,58,83 a change in the anxiety state manifested in trial 1 compared to trial 2 [84], or a change in the underlying mechanism triggering the behavior from one of unconditioned fear avoidance to learned avoidance based on prior exposure to the situation [85]. These concerns can best be addressed by ensuring that test subjects are experimentally naïve and receive minimal handling. 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.

5.3.2.5. Sample Results

Figure 5.2A–D provides an example of the behavioral measures most commonly reported in the literature [60,61,87]. 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 5.2A) and made fewer open arm entries (Figure 5.2B). Importantly, the number of overall arm transitions (Figure 5.2D) did not significantly differ compared with WT littermates [53]. 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].

FIGURE 5.2

Anxiogenic-like phenotype of GalR2 knockout mice on the elevated plus-maze. Two independent cohorts of GalR2 −/− displayed an anxiogenic-like phenotype compared to their +/+ littermates in the elevated plus-maze. GalR2 −/− (more...)

5.3.3. Light ↔ Dark Exploration Test

The light ↔ dark exploration test, developed by Crawley and Goodwin [16], was a precursor to the EPM and provides another means of examining anxiety-like behavior in rodents. 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 10-min test session. 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].

5.3.3.1. 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.

5.3.3.2. Equipment

The chamber is constructed from a standard polypropylene rat cage (44 × 21 × 21 cm) divided into two unequal compartments by a dark partition with a small aperture (13 × 5 cm) located in the bottom center. 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.

5.3.3.3. Procedure

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].

5.3.3.4. 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. 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.

5.3.3.5. Sample Results

Figure 5.3A and B illustrate the two most commonly reported behavioral measures from the light ↔ dark exploration test. Figure 5.3C illustrates risk assessment, a measure appearing more often in recent literature as another indicator of anxiety-related behavior in rodents [5,6,42]. The study [97] illustrated in Figure 5.3 examined the effects of two centrally administered neuropeptides, NPY and galanin, on anxiety-related behavior in C57BL/6J mice. 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 5.3C illustrates that NPY-treated mice spent significantly less time engaged in risk assessment than controls or galanin-treated mice [97]. Thus, neuropeptide Y, but not galanin, produced an anxiolytic-like action when centrally administered to mice.

FIGURE 5.3

Light ↔ dark exploration. Mice treated with NPY at an icv dose of 0.5 and 1.0 nmol spent significantly more time in the brightly lit open area (A) and made significantly more transitions (B) between the two compartments than the vehicle-treated (more...)

5.3.4. 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. The test eliminated the need to introduce aversive or appetitive conditions. In addition, the design of the social interaction test is suitable for use with naïve animals. 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 [100], suggesting that some environmental manipulations have different salience for the social behaviors of each sex [101].

Although originally designed for rats, modified versions of this test have been used relatively successfully to evaluate anxiety-like behavior in mice [102,103]. 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 [104,105], 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 [102,106].

5.3.4.1. Subjects

Inconsistent findings with female mice would indicate that this test is more suitable for testing male social behavior. Young male mice of approximately the same weight (< 4 g difference) are the preferred subjects. Noticeably aggressive, dominant, group-housed mice should not be used, as this could significantly impact the sociability of the isolate mouse.

5.3.4.2. Equipment

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.

5.3.4.3. Procedure

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 [105,106]. Isolate mice are acclimated to the testing cage (size ranges 30 × 25 × 17 cm, 20 × 30 × 20 cm) for 30 min prior to testing. 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 10-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 10-min test session [103]. 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 [102]. 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. Conversely, low illumination levels (< 20 lux) may enhance social investigation, providing a method for exploring anxiogenic effects on baseline social interactions.

5.3.4.4. Analysis and Interpretation

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. The means for two groups are analyzed using an unpaired Student’s t-test. 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.

5.3.4.5. Sample Results

Time spent interacting with an unfamiliar partner is the primary measure reported as a measure of sociability in mice. Figure 5.4 illustrates a study that examined social investigation in vasopressin 1a receptor (V1aR) KO and WT control mice [103]. 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 5.4 for pairs of mice with a null mutation in the V1aR KO compared to WT pairs. V1aR KO pairs spent significantly less time in social interaction than WT pairs.

FIGURE 5.4

Time spent in social investigation in the social interaction test of V1aR knockout (KO) mice and wild type mice. V1aR KO mice spent significantly less time in social interactions (***p < 0.0001) compared to wild type mice. N = 8 pairs of each (more...)

5.3.5. Novelty-Inducedhy Pophagia

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 [107,108]). 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 [109–112].

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 [107]. 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 [107]. Another possible confound is the potential impact of drug treatments or genetic manipulations on factors unrelated to anxiety. Drugs targeting serotonergic function selectively decrease feeding behavior and alter macronutrient intake [113,114]. 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 [107]. 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 [108]. In addition, Dulawa and Hen [107] suggest using higher illumination levels for the novel environment to optimize hyponeophagia levels.

In their modified model, Dulawa, Holick, Gundersen, and Hen (2004) [115] 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.

5.3.5.1. Subjects

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.

5.3.5.2. Equipment

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. Lighting in the home cage condition is dim (~50 lux). The illumination level for novel cage testing is very bright (~1200 lux) and the table area under the test cage is lined with white paper.

5.3.5.3. 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. 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 30-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.

5.3.5.4. 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. 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 [115]. One-way ANOVA may be used for comparing means for total food consumed. 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 [116].

5.3.5.5. Sample Results

The graphs presented in Figure 5.5A and B illustrate the behavioral measures generally reported in novelty-induced hypophagia testing, latency to the first lick, and total volume of food consumed in the initial 5-min period [115]. This study examined the effect of chronic fluoxetine treatment (29 days) on the latency and volume of food consumed in a familiar home cage environment versus a novel environment for BALB/cJ mice, a highly anxious strain. 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 5.5A). In addition, in the novel environment fluoxetine decreased overall food consumption compared to the home environment. However, in both the home and novel cages, the 18 mg/kg dose increased food consumption over that of the 0 and 10 mg/kg doses. Although 25 mg/kg did increase food consumption in both conditions, serum levels of mice in the home cage were more than twice that observed in humans. In this study the 10 and 18 mg/kg doses produced anxiolytic effects in the novelty-induced hypophagia test [115].

FIGURE 5.5

Novelty-induced hypophagia. The effects of a novel cage on latency to consume, and the amount consumed, of a familiar and palatable snack are shown for BALB/c mice. The difference in latency to consume (A) in the home cage, (B) in a novel cage, and (C) (more...)

5.4. CONCLUSION

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: stress-induced hyperthermia, a measure of the effect of stress (handling, temperature measurement) on body temperature; [117] the mouse marble-burying test, a modification of the shock-probe burying test for rats; [118,119] the open field emergence test; [52] fear conditioned startle and light enhanced startle; [27,120,121] and the Vogel conflict test [19]. 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.

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