Novel Object Recognition Test |Barnes Maze |Spontaneous Alternation Mazes (T and Y mazes) |Stereotypic Behavior in Rodents

Rodent Behavior and Behavioral Testing

Many different standardized behavior tests exist in rodent research. For best results, investigators should familiarize themselves with the intent and methodology of a test before committing to using it in their reseach protocols. Behavioral tests must be adequately described and justified in your IACUC protocol prior to approval for use. 

A short description and basic guidelines for some commonly used behavioral tests are included on this page. PI's may use this information to assist them in completing their IACUC protocol and for guidance in planning their experimental procedures.

Novel Object Recognition (NOR) Test 

A type of spontaneous preference test in which recognition memory (i.e., ability to distinguish novel from familiar stimuli) is inferred by the greater lengths of time spent exploring novel versus familiar objects in an open field or maze4. This test takes advantage of normal, spontaneous exploratory behavior and has been used in many species including rats and mice. However, in rodents the context in which a novel object is encountered affects the level of exploration. In rats and mice, a novel object in an unfamiliar environment (e.g., open field) is readily explored but a novel object in a familiar environment (i.e., home cage) may be avoided and buried1.

Various forms of the NOR test are used to assess short- and long-term memory, memory consolidation/reconsolidation, spatial and episodic memory, pattern separation and cross-modal recognition3. Advantages of the NOR include the spontaneous nature of animal responses (i.e., no requirement for food deprivation or other aversive stimuli or appetitive rewards that may influence results); no required training other than habituation to the test arena; task difficulty can be varied by changing the interval between habituation and test sessions; and testing can be completed in few sessions. Disadvantages include reliance on locomotor activity for test accuracy; individual/strain variability in behavior and preference for specific types of objects (requires counterbalance of novel and familiar object choice); and a lack of standardization of pre-test habituation procedures and objects used as test stimuli. In addition, running the test in an open field requires repeated animal handling which increases animal stress. A less stressful alternative may be to run the test in a bow-tie maze in which multiple trials within one session are completed without handling in between4.

Commonly used variations of the NOR include2,3:

  • Novel object recognition (NOR) – Test animals must recognize a novel object (what). Primarily evaluates perirhinal cortex function.
  • Object location (OL) – Test animals must recognize a familiar object in a novel spatial location (where). Primarily evaluates hippocampal function.
  • Temporal order recognition (TOR) – Test animals are exposed to objects they have interacted with at different points in time (when) and must recognize the least familiar object. Primarily evaluates connections between hippocampus, perirhinal and prefrontal cortex.

 

Troubleshooting:

Tests may be run in an open field or a maze (e.g., bow tie maze4). Large differences in test object size may result in recognition performance differences when using mazes3.

Features such as shape, size, color, brightness, texture and odor can determine how an animal recognizes a test object. There is limited knowledge of which physical features rats and mice use when discriminating between test objects; however, recognition memory performance often varies inversely with similarity (i.e., increasing number of shared features) between test objects3.

Exploration parameters should be of test object exploration not overall exploration of the open field [e.g., snout contact or orientation toward object within a specific distance from the object, crossing a line surrounding the location of an object or head placement within a specific distance from the perimeter of an object (zone entries)]. However, zone entries (often used by automated video tracking systems) may not be an accurate measure of object exploration and can fail to reveal discrimination between novel and familiar objects3.

The number of pre-test habituation sessions used varies but Ennaceur3 recommends five (1/day/10 minutes each) to decrease emotional reactivity to the open field or maze. However, rodent strains vary in emotional reactivity to novelty, so the number of habituation sessions may be influenced by strain (e.g., C57BL/6J and CD-1 = 1-2 sessions; BALB/c = 4-5 sessions; Wistar rats = 4 sessions3). Pre-test habituation sessions can also be used to evaluate spontaneous locomotor and exploratory activity3.

References:

  1. Blaser R, Heyser C. Spontaneous object recognition: a promising approach to the comparative study of memory. 2015. Frontiers in Behavioral Neuroscience 9: 183.
  2. Cruz-Sanchez, A., Dematagoda, S., Ahmed, R. et al. Developmental onset distinguishes three types of spontaneous recognition memory in mice. 2020. Sci Rep 10. https://doi.org/10.1038/s41598-020-67619-w
  3. Ennaceur A. Object Novelty Recognition Memory Test. Ed: Ennaceur A, de Souza Silva MA, Handbook of Behavioral Neuroscience. 2018. 27, 1-22. Elsevier. https://doi.org/10.1016/B978-0-12-812012-5.00001-X.
  4. Kinnavane L, Albasser MM, Aggleton JP.  Advances in the behavioural testing and network imaging of rodent recognition memory. 2015. Behavioural Brain Research 285, 67-78.

 

Barnes Maze

Mazes, such as the Morris water maze (MWM), Barnes maze and t maze, are commonly used to evaluate spatial learning and memory in laboratory rodents. These mazes assume that animals use visual-spatial signals to learn and remember a location that provides safety, food, water or some other reward. However, the complexity of animal behavior required to complete these tests means that other cognitive or non-cognitive processes cannot be excluded. In addition, the diversity of mazes used and the large number of variables that influence animal performance means that not all mazes evaluate the same cognitive process. Mice have been shown to use three different search strategies: random, spatial and serial, in the Barnes maze (Sunyer B et al.) The Barnes maze tests learning, memory and cognitive flexibility. It was developed for use in rats, then modified for mice. C57BL mouse strains have shown good learning and memory in the Barnes maze while other strains have shown poor performance.

The maze consists of a circular table with circular holes around the circumference. Visual cues placed by the experimenter (e.g., colored shapes) and as part of the room (e.g., door, furniture) are located near the maze. The rodent must search for and find a target box positioned beneath one of the holes to escape an aversive stimulus such as loud noise or bright light. Training is required as the rodent must learn the position of the target box before being tested.

Various published training protocols are available. All consist of the following phases:

  • Habituation - allows animals to learn that the target box is a rewarding place to be.
  • Acquisition - allows animals to learn how to find the target box as quickly as possible.
  • Probe trial - probe trial(s) are conducted 24 hours (short-term) and/or several days (long-term, e.g., 10-12 days) after the last training (acquisition) day to assess short- and long-term memory.

Advantages (versus MWM):

  1. Does not involve swimming so is less stressful than the MWM. In addition, there is no reduction in core body temperature and mice cannot float to avoid performing.
  2. Allow clear delineation of the three possible search strategies used by mice during each trial.

Potential problems:

  1. Lack of stressful stimuli in the Barnes maze may slow learning. White noise of loud buzzers can be used to increase stress (and motivation to find the target box).
  2. Requires more training than the MWM (~15-20 acquisition trials versus ~12 for MWM).
  3. Most published literature on Barnes maze in mice used C57BL/6 mice (3-5 months old). Rates of learning vary substantially between individuals so group size must be at least 10-12.
  4. Performance in both MWM and Barnes maze are highly sensitive to anxiety in rodents (e.g., pharmacological and genetic manipulations) although this appears to be more of a concern in the MWM. Anxiety may be reduced by lowering light intensity.

References:

Pitts MW. Barnes Maze Procedure for Spatial Learning and Memory in Mice. Bio Protoc 2018 Mar 5; 8(5): e2744. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891830/

Sunyer B, Patil S, Hoger H et al. Barnes maze, a useful task to assess spatial reference memory in the mice. 2007 Oct 4, PROTOCOL (Version 1) available at Protocol Exchange https://doi.org/10.1038/nprot.2007.390

Spontaneous Alternation Mazes (T and Y mazes)

Information provided for this description taken from: d’Isa R, Comi G & Leocani L.  d'Isa R, Comi G, Leocani L. Apparatus design and behavioural testing protocol for the evaluation of spatial working memory in mice through the spontaneous alternation T-maze. Sci Rep. 2021 Oct 27;11(1):21177. doi: 10.1038/s41598-021-00402-7. PMID: 34707108; PMCID: PMC8551159.

Well established test of spatial working memory in rodents that, along with the novel object recognition test, is considered among the least stressful and animal friendly options for assessment of cognitive function. The T maze is a T-shaped apparatus that provides a choice between two opposite arms. Spontaneous alternation refers to the natural tendency of rodents to prefer exploring a novel maze arm over a familiar one, which leads them to alternate the choice of the goal arm in the T maze test. The animal must remember which arm was visited in the previous trial to correctly choose the less familiar one. The T maze is considered more sensitive in evaluating hippocampal function than the Morris water maze.

In mice, a series of test trials are conducted (~5-12) with an intertrial interval (ITI) of up to 60 seconds. No pre-test training is required. The maze is placed in one location (only) in a dimly lit testing room (to ensure consistency of extra-maze visual cues). Percentages of alternation across the test trials are calculated as an index of working memory. Normal wild-type mice typically show an index of 70-75%, well above chance level (50%). Spontaneous alternation rates are inversely proportional to ITI and task difficulty can be increased by increasing ITI. The T maze test may not be suitable for mice with severe motor impairments because they may not be able to complete test trials within the fixed cut-off time (before working memory fades).

Published variations of the basic T-maze protocol include:

Rewarded alternation: Correct alternations are reinforced by the experimenter using a food reward. This version of the t maze test does not appear to be as sensitive in detecting cognitive impairment as the spontaneous t maze and requires food deprivation (increases stress for the subject animals).

Continuous trials: The test is performed in a Y-maze (equal 120° angles between arms) and animals are allowed to freely explore the maze for a specific amount of time or number of arm visits. Correct alternation is scored when the animal chooses to enter one of the two arms not visited in the previous trial, or alternatively, when all three arms are explored consecutively. This test version avoids stress from handling in between trials but may not detect hippocampal dysfunction. In addition, ITI is uncontrollable in this test version. Fixed ITI is important because spatial working memory lasts for only a short time.

Enclosed arms (versus open): Less anxiety producing in rodents (may require less time to habituate) and prevents escape by jumping off arms.

Tips for success:

  • When possible, house mice in pairs or groups to lessen stress associated with individual housing. Published studies have shown that isolation stress in mice can result in increased anxiety, neophobia, aggression, and locomotor activity and impair short- and long-term memory, alter sensorimotor gating and decrease cognitive flexibility. However, male mice in strains with a high incidence of territorial aggression may need to be housed individually as repeated stress and injuries from fighting also affect welfare and behavior.
  • Mice are traditionally tested during the dark phase so housing under a reversed light cycle is recommended.
  • Environmental enrichment of home caging is encouraged to maximize animal welfare.
  • Do not change home cages on the day before testing (ensure at least 48 hours of habituation to a new cage prior to testing).
  • Do not handle mice by the tail for placement and retrieval in the maze as this is highly stressful. Tunnel handling or cupping within the hands is preferred (requires pre-test addition of tunnel in home cage or training to habituate to handling within cupped hands).

Troubleshooting:

  1. If test results indicate a side preference (left or right arm of maze), the experimental room environment and maze design/set up should be reviewed to eliminate unexpected stimuli such as non-uniform lighting or odor from nearby mice.
  2. Normal mice spontaneously alternate above chance level (between 70 and 80%). If control mice show chance level alteration check the following:
    • No auditory, visual or olfactory distractions in the experimental room during testing.
    • Verify that housing conditions are normal with no unexpected stressful events or situations.
    • Check that the home cage has not been changed within 48 hours of the test start.
    • Make sure mice have not undergone stressful manipulations in the previous days (e.g., anesthesia and surgery). At least five days of recovery are recommended before behavioral testing.
  3. Choice latencies should not be too high (i.e., group average T0 <30 s; T1-T6 mean <60 s). Latencies commonly increase over trials (T0 to T6) within one test session. In addition, repeating the entire test (i.e., multiple test sessions) will result in the exclusion of increasing numbers of mice because they will exceed the time limit for task completion. No more than three test replications should be used unless sample sizes are great enough to compensate for this loss. Gentle auditory or tactile stimulation may be used to encourage mice to finish a test trial if required but must be used consistently and carefully to avoid influencing results.

Reference:

d’Isa R, Comi G & Leocani L.  d'Isa R, Comi G, Leocani L. Apparatus design and behavioural testing protocol for the evaluation of spatial working memory in mice through the spontaneous alternation T-maze. Sci Rep. 2021 Oct 27;11(1):21177. doi: 10.1038/s41598-021-00402-7. PMID: 34707108; PMCID: PMC8551159.

 

Social, Maternal and Aggressive Behaviors in Rodents

Many standard behavioral tests exist for the study of interactive behavior in mice and rats. In order to choose the most appropriate test for a research study it is important to understand something about the range of rodent social behaviors and what, specifically, behavioral tests are attempting to measure. Rodent social behavior may be classified into general categories such as aggression and social dominance behavior; parental and maternal behavior; and social recognition and approach behavior. Specific tests are designed to investigate behavioral differences in each of these categories.

Rats and mice used in research are considered social species, meaning, in general, they prefer some form of group living. Species that live together must interact and so have evolved various behaviors that allow and facilitate group living. Environmental conditions and individual characteristics (e.g., sex, age, reproductive status, genetic background, etc…) are important in determining the form and amount of social interaction that occur within a group. In addition, sensory and motor abilities and health status can influence the expression of social behavior in individual animals. For example, an animal may be less willing to interact with others if it is ill or in pain. In another example, the sense of smell (olfaction) is extremely important in mouse communication and mice with olfactory deficiencies may behave quite differently than normal mice.

Before performing behavioral tests on rodents, especially when using unfamiliar strains or mutants, investigators must evaluate overall health and specific sensory and motor capabilities of the animals to avoid biased and inaccurate interpretations of the role of genetics in behavior.

Aggression and Social Dominance Behavior

Specific tests include the standard opponent test, isolation-induced fighting, resident-intruder test, and tube-test for social dominance. These tests are described below.

Aggressive behaviors are usually related to either territorial or maternal defensive actions or the establishment and maintenance of social status within a group. Males tend to show more territorial and social dominance behaviors than females but there are exceptions. Predatory behaviors (behavior oriented toward catching and killing of prey) are not included in this category. Rodents who bite humans are also not displaying true aggressive behavior but rather, fear induced defensive behavior.

Rats and mice differ in their social organization and use of aggressive behaviors. Male mice are territorial and do not tolerate unfamiliar males within their home range (or cage). Females may establish territories but tend not to defend them with aggressive behavior. Male (and female) mice mark territorial boundaries with urine; this is an important method of avoiding unnecessary aggression and its consequences in this species. In contrast, rats have evolved to live in multi-male/multi-female groups and tend to coexist peacefully if group composition is stable.

Although both mice and rats establish social dominance hierarchies within groups, they differ in important characteristics. Male social hierarchies in stable rat groups tend to stay the same despite changes in weight and/or size of individuals. In these types of groups, age may be the best predictor of social status. Male mice also establish social dominance hierarchies in a group but they will continuously compete for dominance. This often results in fighting and subsequent injuries. Changes in group composition, the presence of female mice in the room (olfactory stimulation) or manipulation of the mice (e.g., cage changing, temporary removal for experimental procedures) may increase fighting. If multiple mice are in the cage, removal of the dominant mouse will not necessarily stop the injuries, as the remaining mice will fight to reestablish a social order. Female mice and rats also establish social dominance hierarchies but tend not to fight. This makes it easier to group house them but harder to study social organization.

Rat and Mouse Parental and Maternal Behavior

Parental behaviors can be classified as direct (having an immediate physical impact on offspring and their survival) or indirect ((behaviors that do not involve physical contact but still affect offspring survival). Examples of direct behaviors include nursing, grooming or licking, retrieving and huddling. Some direct behaviors may be performed by males (i.e., the sire). Examples of indirect behaviors include nest building, defense against conspecifics or predators, acquiring and defending critical resources and care for pregnant or lactating females. Indirect behaviors may be performed by either parent and by other (non-parent) adults, which is referred to as alloparental care.

Although some laboratory studies indicate that adult male mice and rats are capable of parental behaviors, these occur at a low level and care of the young is primarily left to the female. Studies of wild mice and rats have shown that males are not involved in care of the young and will kill young that are not their own. Males will also kill unrelated or unfamiliar young under laboratory conditions. While the presence of the male sire in the breeding cage is generally not harmful to pups, there is no evidence that the male benefits pup growth and development. Adult males other than the sire, however, should not have access to young other than their own. See references 1 and 2 below for more information.

Maternal behavior typically refers to all aspects of behavior of the dam between parturition and weaning of the offspring and includes both direct and indirect behaviors. Some aspects of maternal behavior (e.g., nestbuilding) may begin prior to birth of the young. Laboratory studies with rodents have shown that hormonal changes (e.g., oxytocin) are important triggers for onset of maternal behavior. As hormonal influence decreases after parturition, infant stimulation increases in importance in this regard. Stimuli from pups, including ultrasonic vocalizations (USV), are needed to maintain maternal care after about 5 days postpartum (2, 3, 4). Infant rats and mice emit a variety of sonic and ultrasonic vocalizations that attract the dam’s attention. In mice, inbred strain differences in hearing ability and the number of USV emitted by pups have been found. USV have been extensively studied in rodents and various protocols for are available for experimental research (2,4).

In rats and mice, a postpartum estrus occurs within 24 hours after parturition. Laboratory studies have suggested that postpartum mating activities are shorter in duration than during normal estrus periods and do not significantly reduce maternal time spent with the litter (2). After the postpartum estrus period the female will not come into estrus again until after the pups are weaned. If she mated and conceived during the postpartum estrus, the second gestation may be prolonged by a week or more.

Young rats and mice are altricial, which means they are born in a relatively undeveloped state and cannot move, maintain body heat, see or hear on their own. Extensive maternal care is required for the young to survive. Rats and mice have evolved specific behaviors that contribute to the survival of altricial offspring. Both rats and mice will actively build nests in which to rear their young. These nests are built by the female and may be complex, multi-entrance enclosures if the dams are provided with appropriate building material. Significant strain differences in nest building skills have been shown in mice.

Both rats and mice will nest communally (multiple females rear their young in the same nest) and nurse offspring that are not their own. Laboratory studies have indicated that pup survival to weaning is higher for rats who rear their litters alone rather than in a communal nest. The opposite may be true in mice. Multiple studies have shown that mouse pups reared in communal nests had higher growth rates and better survival than pups reared alone with their dam (4). However, communal nesting/nursing may not be successful if the age difference between litters is greater than 5-7 days. In this situation, dams may be aggressive toward pups that are not their own.

Lactating females will display aggressive behavior to defend their offspring from others of their own species. The presence of pups appears to be the primary trigger for female postpartum aggression. The presence of unfamiliar male or female conspecifics will provoke maternal aggression although the likelihood and expression of maternal aggression varies with strain, individual and location (e.g., home cage versus test arena) (2,4).

There are a number of events and experiences that will influence the behavior of both the mother and the pups. These include the effects of handling of the dam and/or pups and disturbance of the cage environment by the researcher. Depending on the experimental objective these could be confounding factors and must be considered. Maternal behavior during lactation will also be affected by changes in the pups as they grow and mature and by the evolving physiological state of the dam.

Laboratory studies have shown that the main components of rodent maternal behavior (nursing, licking and grooming, pup retrieval and nest building) are present at high levels in almost all rats and mice after giving birth (2,4). Time spent in these behaviors typically declines gradually during the first two weeks of lactation and then decreases further or disappears during the third or fourth week after parturition. Consumption of food and water by the dam increases dramatically over the first two weeks of lactation and may influence the amount of time spent on maternal behaviors. Although commonly used as experimental measures of maternal behavior, nest building and pup retrieval do not normally occur at high frequencies in undisturbed conditions. Mice and rats build nests if material is available but once made, the nest is not rebuilt from scratch unless disturbed. Pup retrieval is also infrequently necessary under normal conditions.

Rat and mouse pups start eating solid food around 15-17 days of age and nursing by the dam ends by four weeks after gestation. Weaning of a litter is normally a gradual process that can stretch well beyond the third week. The typical abrupt weaning that takes place in the laboratory when the pups are 3-4 weeks of age provides another example of experimental manipulation influencing normal behavior.

References:

  1. Brown, R. E. (1986). Paternal behavior in the male long-evans rat (rattus norvegicus). Journal of Comparative Psychology, 100(2), 162-172. doi:http://dx.doi.org/10.1037/0735-7036.100.2.162
  2. Elwood, R.W. (Ed.). (1983). Parental Behaviour of Rodents. Chichester: Wiley and Sons.
  3. Kazutaka, M., Nagasawa, M., Kikusui, T. (2011). Developmental consequences and biological significance of mother-infant bonding. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 35, 1232-1241.
  4. Weber, E.M. & Olsson, A.S. (2008). Maternal Behaviour in Mus musculus sp.: An ethological review. Applied Animal Behaviour Science, 114, 1-22.

 

Stereoptypic Behavior in Rodents

Captive environments (e.g., cages) often reduce an animal’s ability to control and modify its environment, which can be stressful. Stress may lead to abnormal behavior and physiology. Abnormal repetitive behaviors (ARBs) are a type of abnormal behavior and are often referred to as stereotypies. ARBs are found in many species, including laboratory rodents, and are thought to occur as a result of brain malfunction. Therefore, once ARBs have developed, environmental enrichment may not eliminate them due to permanent developmental changes in brain physiology. There is evidence that the physiologic mechanisms (in the brain) that produce ARBs will also affect measures in behavioral experiments. This means that animals with ARBs may display other types of abnormal behavior that can influence results in behavioral testing. Even though environmental enrichment may not reverse established stereotypic behaviors, the provision of enrichment may prevent the development of abnormal behaviors such as ARBs, leading to improved validity, reliability, and replicability in behavioral experiments.

Reference: Garner JP. 2005. Stereotypies and Other Abnormal Repetitive Behaviors: Potential Impact on Validity, Reliability, and Replicability of Scientific Outcomes. ILAR Journal 46 (2): 106-117.