Morris Water Maze | Porsolt Forced Swim Test | Tail Suspension Test | Use of Electric Shock in Research Animals
Social, Maternal and Aggressive Behaviors in Rodents

Rodent 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.

Morris Water Maze

Synonyms: (submerged platform) water escape task, Morris water escape task, Morris water navigation task.

The purpose of this test is to evaluate spatial memory.  In this test the animal is required to swim in a tank of opaque water until it finds a submerged platform that it can mount to escape the water. Presumably, the animal uses specific visual cues placed around the outside of the tank to learn where the platform is located. An animal that is able to remember the cues will find the platform more quickly each time it completes a trial swim. Animals are initially given a series of “learning trials” in which they are allowed to swim in the tank until they find the platform. Each learning trial lasts a specific amount of time and the time between trials must also be specified. Following this, a “probe trial” is run in which the submerged platform is removed and the time the animal spends swimming in the quadrant of the tank where the platform was previously located is measured. Animals that have learned the position of the platform will spend most of their time in the quadrant where the platform was previously located. Animals that are poor learners will spend time searching other areas of the tank.

See this link for an illustration1 of the Morris water maze.

Species used:  Rats and mice. This task was developed for use in rats (generally good swimmers). In mice, performance in this test is highly dependent on genetic background. Special consideration must be given to the use of this test in mouse strains or genotypes with reduced ability to navigate using spatial cues or to swim, e.g., visual or musculoskeletal impairments. In addition, mice with other physiological or behavioral traits such as impaired thermogenesis or high anxiety levels may also perform poorly in this test1.

Important considerations:

Water Tank

  1. Size (diameter) and depth of the tank (varies with species). Water depth of 15-20 cm is adequate for mice. Rats are larger and may dive to the bottom so require deeper water.
  2. Size of platform in relation to the diameter of the tank (task difficulty increases with decreasing size of the platform).
  3. A round shape is recommended for the escape platform (provides the same tactile cues on all sides)3. The platform surface should be textured so the animal can maintain a secure grip and close enough to the water surface so the majority of the animal’s body is out of the water when on top of the platform (e.g., mice ≤0.5 cm below water surface).
  4. Mice are more susceptible to hypothermia than rats. Hypothermia risk can be lessened by increasing the water temperature and/or increasing the inter-trial interval. Water temperatures < 20⁰C can lead to hypothermia. Temperatures that are too warm may discourage active swimming/searching. Animals should be allowed to dry in a warm environment after removal from the tank. Absorbent towel(s) may be placed in the holding cage to collect water dripping off the animal and a heating source directed over or underneath the cage may provide warmth. Do not attempt to towel- or blow -dry animals as this is stressful and rough handling can cause injury.
  5. Substances used to make the water opaque must be edible and nontoxic because animals will consume the substance when grooming after each trial. Tempera paint is recommended3. Milk supports bacterial growth, especially in warm water and lime or chalk may be toxic. Alternatives that may allow the use of clear water include using a clear Plexiglas platform or using a platform the same color as the tank surface.
  6. Cleaning schedule for the tank (water changes). Urine and fecal material will accumulate in the water and contribute to bacterial contamination and growth. The tank should be drained and disinfected after each day’s trials. Partial water changes between mice can reduce the accumulation of urine/fecal material. Fecal material may be removed after each animal with a small mesh net3.

 

Test Procedures 

  1. Trials should not exceed 2 minutes.
  2. Inter-trial intervals should be long enough to prevent the development of hypothermia and fatigue over repeated trials. It is recommended that intervals be at least 10-15 minutes, especially in mice5.
  3. Animals must be observed continuously while in the tank and removed from the water if their head sinks below the surface4.
  4. Two to four trials per day are generally adequate for training.

 

USDA category: E

Alternative tests for spatial learning and memory:  Radial arm maze, Barnes circular platform maze

References: 

  1. Terry AV Jr. Spatial Navigation (Water Maze) Tasks. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press; 2009. Chapter 13. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5217/
  2. Crawley JN. What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice, 2nd ed. Hoboken (NJ): Wiley; 2007.
  3. Wahlsten D. Mouse Behavioral Testing: How to use Mice in Behavioral Neuroscience. Amsterdam: Elsevier; 2011.
  4. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press, 2003.
  5. Iivonen H, Nurminen L, Harri M, Tanila H, Puolivali J (2003). Hypothermia in mice tested in Morris Water Maze. Behav Brain Res 141: 207-213.

Porsolt Forced Swim Test

Purpose: The Porsolt swim test (PST) was developed as a rodent screening test for potential (human) antidepressant drugs. It is based on the assumption that an animal will try to escape an aversive (stressful) stimulus. If escape is impossible, the animal eventually stops trying and gives up. In the PST, the animal is placed in a cylindrical container of water from which it cannot escape. Most animals will attempt to escape by actively swimming. When the animal stops swimming and floats on the surface of the water it is considered to have “given up”.  An animal that gives up relatively quickly is thought to be displaying characteristics similar to human depression.  The validity of this test stems from the finding that physical or psychological stress (which can induce depression in humans) administered prior to the test causes animals to give up sooner and treatment with an antidepressant drug will increase the time an animal spends in escape attempts.

Species used: Rats and mice. Impaired swimming ability due to musculoskeletal or other abnormalities will affect performance in this test.

Important considerations

  1. The water must be deep enough so the animal cannot touch the bottom with its tail or feet. A depth of 30 cm is commonly recommended, although less depth may be adequate for mice. Water temperature should be 24-30⁰C2.
  2. Animals should be allowed to dry in a warm environment after removal from the water. Absorbent towel(s) may be placed in the holding cage to collect water dripping off the animal and a heating source directed over or underneath the cage may provide warmth. Do not attempt to towel- or blow-dry animals as this is stressful and rough handling can cause injury.
  3. Water changes:  Urine and fecal material will accumulate in the water and contribute to bacterial contamination and growth. The container should be emptied and disinfected after each day’s tests. Fecal material may be removed after each animal with a small mesh net.
  4. Test procedures:  A wide range of test session durations have been reported (4-20 minutes)1Animals must be observed continuously during the swim test. Any animal that sinks below the surface should be removed from the water immediately2.

 

USDA category: E

Alternative tests: Tail-suspension test and others1,2,3.

References: 

  1. Crawley JN. What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice, 2nd ed. Hoboken (NJ): Wiley; 2007.
  2. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, DC: The National Academies Press; 2003. Available from: http://www.ncbi.nlm.nih.gov/books/NBK43327/
  3. Castagne V, Moser P, Porsolt RD. Behavioral Assessment of Antidepressant Activity in Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press, 2009. Chapter 6. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5222/

Tail Suspension Test

Purpose: The tail suspension test (TST) was developed as a rodent screening test for potential (human) antidepressant drugs. It is based on the assumption that an animal will actively try to escape an aversive (stressful) stimulus. If escape is impossible, the animal will eventually stop trying ("give up"). In the TST a mouse is suspended by the tail so that its body dangles in the air, facing downward. The test lasts for six or more minutes and may be repeated multiple times. Mice initially struggle to face upward and climb to a solid surface. When the animal stops struggling and hangs immobile it is considered to have “given up”.  Longer periods of immobility are characteristic of a depressive-like state.  The validity of this test stems from the finding that treatment with an antidepressant drug will decrease the time the animal spends immobile.

Species used: mice

Important Considerations:

  1. Mice are suspended (a variable distance) above a solid surface by the use of adhesive tape applied to the tail. If the tape is incorrectly applied or fails, the mouse will fall. The use of a “cushioned” surface below the TST may be needed to help prevent injury to the animal. Mice that experience a fall should be removed from the experiment1.
  2. Vinyl or medical adhesive tape is recommended. Duct tape is too adhesive and will tear hair and skin when removed1. The tape should be applied in a consistent position ¾ of the distance from the base of the mouse’s tail2. If the tape is applied too near the tip of the tail it may pull off the skin of the tail tip and the mouse will fall.
  3. Some strains (e.g., C57BL/6J) may not perform well in the TST due to tail climbing behavior. Strains with vestibular deficits may show an abnormal spinning phenotype and should not be used in the TST. Other mouse phenotypes that display neurological abnormalities that lead to unusual leg clasping behavior or that influence immobility times may also not be appropriate models for this test1.

 

USDA Category: E

Alternative tests: Porsolt swim test and others2.

References:

  1. CL Bergner, AN Smolinsky, PC Hart, BD Dufour, RJ Egan, JL LaPorte, AV Kalueff. 2010. Mouse Models for Studying Depression-Like States and Antidepressant Drugs.  In: Mouse Models for Drug Discovery, Methods in Molecular Biology 602: 267-282.
  2. Castagné V, Moser P, Porsolt RD. Behavioral Assessment of Antidepressant Activity in Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press; 2009. Chapter 6. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5222/
  3. B Thierry, L Steru, P Simon, RD Porsolt. 1986. The tail suspension test: Ethical considerations. Psychopharmacology 90: 284-285.

Use of Electric Shock in Research Animals

Purpose: Electric shock is used as an aversive stimulus in behavioral testing with humans and other animals, including invertebrates. Aversive stimuli function as a type of negative reinforcement: The frequency of a measured behavior increases in order to end or avoid the aversive stimulus. Electric shock is favored as an aversive stimulus because it is easily quantifiable; can be manipulated to have discrete or gradual onset and offset; and (at levels typically used in research) does not cause physical damage to the subject. The disadvantage of electric shock includes the fact that it can be painful and is an “unnatural” stimulus (i.e., not normally experienced outside the laboratory). Electric shock stimulates uncontrolled muscle contractions and will result in (increasing) pain as intensity increases.

Species used: Many species although rodents are most commonly used. Impaired motor coordination due to musculoskeletal or other abnormalities will affect performance if animals are expected to coordinate movements to escape the electric shock.

Important considerations

Shock Intensity

  1. The level of shock intensity used must be sufficient to elicit a reaction in the animal but not enough to injure or create unnecessary pain or distress.
  2. The investigator must be familiar with the capacity of their equipment and the shock levels typically applied in the species under study. Devices designed for larger animals (e.g., rats) may not be suitable for mice.
  3. Some authors recommend that the shock intensity being used be evaluated daily by placing a hand onto the electric grid while the shock is being delivered. No more than a “mild tingling” should be felt1.
  4. Shock delivered in pulses provides for “shock-free intervals” that allow more effective escape attempts by the animal2.
  5. Water decreases the electrical resistance of skin and other tissues. The presence of urine or other sources of moisture will increase the shock intensity experienced by the animal.
  6. Electric current delivered to a small area of skin is perceived as more aversive than the same current applied to a larger area2. An animal standing on a rough surface may perceive greater shock intensity than one standing on a smooth surface.
  7. Species, genetic background and other intrinsic variables may influence an animal’s degree of sensitivity and type of response to shock and must be considered2. Please consult the references listed at the end of this section for additional information on how to design and set up experiments using electric shock (in rodents).

 

Test procedures

  1. Do not require animals to perform complex or skilled maneuvers to escape shock2.
  2. Mice show two primary reactions to electric shock: Jumping and running. Genotype will influence which reaction predominates in a strain. Investigators may want to consider the typical reaction pattern of the strain(s) they are using when planning what type of escape response will be required by the animal (e.g., a strain that responds to shock by running may have difficulty learning to escape if jumping is required to leave the shock chamber)2.
  3. If animals can retreat to non-electrified areas within the apparatus (e.g., chamber edges) they may be able to avoid the shock. This is more likely to occur when tasks are too difficult and cannot be learned quickly2.

 

USDA category: E

Alternative types of aversive stimuli or methodology:  Air puffs, loud noises, bright lights or ultrasonic tones. Alternative training methods include the use of a reward (e.g., preferred food) for correct responses instead of punishment (electric shock) for incorrect responses.

For more information on test procedures and experimental design please consult the following references: 

  1. Graham JH and Buccafusco JJ (2001). Inhibitory Avoidance Behavior and Memory Assessment. In Buccafusco JJ (ed.), Methods of Behavior Analysis in Neuroscience, p.141-151. Boca Raton: CRC Press.
  2. Wahlsten D (2011). Mouse Behavioral Testing: How to Use Mice in Behavioral Neuroscience. London: Academic Press.

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.

Standard Opponent Test

This test evaluates male aggression and social dominance in a test animal placed with an unfamiliar conspecific in a neutral area. The test subject is confined with a ‘standard opponent’ partner for a specific time in an unfamiliar cage or other defined space.

Important considerations for this test:

  1. The standard opponent(s) males are selected for highly replicable behavior as either submissive or dominant males in repeated tests with other males. Standard opponent partners are usually chosen from mouse strains known for either high or low levels of aggression. The selected mice are then used as standard opponent test partners in pairings with experimental mice.
  2. Differences in weight, age and size between the test mouse and the standard opponent may also influence test results.
  3. Keep in mind that it will be necessary to keep track of which mouse is the test subject and which is the ‘standard opponent’ during the test session. Mice with different coat colors will make this easier to do.
  4. Test sessions are often 5 minutes in length but are terminated early if attacks and biting are severe.
  5. The presence of humans can influence animal behavior during the test. The observer should be screened from the animals and/or the mice may be videotaped for scoring later.
  6. The frequencies of specific (predetermined) behaviors are scored. Examples of behaviors include body and anogenital sniffing sniffing, following and chasing, number and location of bites and tail rattling.

More information on standard opponent testing may be found in this and other references:

Crawley, JN. What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice, 2nd ed. Wiley-Interscience, 2007.

Isolation-Induced Fighting

This is a modification of the standard opponent test in which male mice are singly housed for a specific time period (e.g., four weeks) prior to placement with an unfamiliar male mouse into a test arena or cage.  Isolation of male mice tends to increase the frequency of fighting and attack behaviors.

Resident-Intruder Test

Another modification of the standard opponent test, the resident-intruder test is conducted in the home cage of the test mouse. The unfamiliar male mouse is the ‘intruder’. The test mouse (‘resident’) will attempt to defend its home cage from the intruder. Isolation is not needed prior to this test. Aggression in the resident mouse will be higher if he is living with a female and her litter (sired by him). However, the female and her pups must be separated from the fighting area, as the female will also display aggression toward the intruder (maternal defense).

Tube-Test for Social Dominance

This test measures dominant/submissive behavior in mice without allowing them to fight and injure each other. Both male and female mice may be tested with the Tube-Test. In the test, two mice of the same gender are placed at opposite ends of a clear, cylindrical tube and allowed to explore toward the tube center. At the point where the mice meet, the submissive mouse will tend to back up as the dominant mouse continues moving forward. The mouse that leaves the tube first (‘pushed out’) is the loser and the other mouse (dominant) is the winner. Automated equipment for this test exists that can measure additional parameters such as duration of match, latency to enter the tube, etc… This test can be used for determining social dominance relationships within a group of mice.

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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.