Behavioral tests and equipment

Acoustic startle and pre-pulse inhibition

To check for their general excitability, we can place mice in a restrainer so that they can move only minimally, present them with a loud sound (e.g., 110 dB) and measure the vibrations caused by their startling reaction. This test can be combined with a protocol for pre-pulse inhibition, which requires proper functioning of the limbic and cortico-pallido-striato-thalamic circuitry. In this pre-pulse inhibition test, another loud sound (e.g., 85 dB) is presented before the test sound (e.g., 110 dB). If sensory gating works properly, the startling response to the test sound is inhibited. This pre-pulse inhibition is impaired in several neurological diseases (e.g., in schizophrenia).

Barnes maze

In this spatial learning test, mice are trained over multiple sessions to find a dark escape box, which is placed underneath one of many holes inserted in a table placed in a brightly illuminated room. The mouse uses visual cues in the room to orient itself and is motivated to escape the aversive bright light. We evaluate the latency until the escape box is found. Memory extinction and behavioral flexibility can be checked by moving the escape box to a new location and measuring the latency to find the new location.

Beam walk test

In this test for motor function and balance, mice are trained over several days to walk across a round beam 20 mm in diameter. On the test day, the same task is repeated on round beams with decreasing diameters (down to 10 mm). Videos are recorded and analyzed using Noldus Observer software and we score various motoric parameters such as the distance reached until falling, number of slips, time needed to cross 1 m of beam, and number of stops.

CatWalk test

The Noldus CatWalk system allows for high-throughput gait analysis under non-stressful conditions (in the dark), as well as detailed footprint analysis. Mice learn to walk across a glass plate along a dark corridor. A light beam crosses the glass plate and its refraction changes at the place of the footprint of the mouse. A detailed footprint analysis with intensities of body weight distribution can give indications about the motoric functions of the mice and can detect e.g., pain-related weight shifts. In addition, the gait pattern can be analyzed from below.

CODA monitor

The CODA monitor by Kent Scientific (USA) allows for non-invasive heart rate and blood pressure measurements by placing mice into a restrainer and applying a tail cuff.

Elevated plus maze

This anxiety test is based on the unconditioned response of mice to a potentially aversive environment. The apparatus consists of two closed arms with walls and two open arms without walls. The mouse is placed in the center zone where the four arms meet and allowed to explore freely for 5–20 min. The mouse experiences a conflict between the tendency to explore a new environment (however, with aversive properties – open arms), and the tendency to remain in an environment that is perceived as safe (closed arms). Increased anxiety leads to a shorter duration of time spent in the open arms.

Dynamic plantar (von Frey) test

Mechanical sensitivity can be tested using a “dynamic plantar aesthesiometer” (Ugo Basile SRL, Italy) in an automated von Frey test. Mice are placed into the testing chambers on a metal grid for 2 h to acclimatize. Then each hind paw is tested for touch sensitivity by pricking the plantar surface with a von Frey filament of a certain diameter with a force that increases from 0 to 10 g within 20 s. As soon as the prick gets unpleasant for the mouse, it will lift its leg, the filament gets retracted immediately and the applied force is recorded.

Fear conditioning

In this classical conditioning test, mice are trained to associate the context of the test box and a specific sound with an electric foot shock stimulus. Mice are then re-exposed to the same test box and contextual memory is evaluated by the percentage of time spent “freezing” (no movement except breathing, evaluated using FreezeFrame software (Actimetrics)). Short- and long-term memory can be evaluated, depending on the time interval between the training and testing. By changing the appearance and the smell of the test box and re-exposing mice to the sound, we are able to evaluate amygdala-dependent cued memory.

Forced swim test (Porsolt test)

We measure the amount of time that a mouse spends swimming or struggling in a beaker filled with water at room temperature as opposed to the amount of time that the mouse spends in an immobile or floating posture in a session of 6 min. Staying in an immobile posture is interpreted as giving up and the latency until this posture is taken is lower in mice with a depression-like phenotype, while anti-depressants produce the opposite effect.

Grip strength test

With this test for muscle strength, we can measure the grip strength of all four limbs or just the forelimbs as the mouse is gripping onto a wired mesh and pulled by the tail by the experimenter at a 45° angle. The power at which the mouse loses the grip is recorded by the grip strength meter.

Hot plate test

Thermal nociception of mice is tested on a plate at 52–55°C. This is a temperature range that is unpleasant for a mouse but does not cause serious damage within a maximum duration of 20 s. The following parameters are measured: forepaw licking, hind paw licking or shaking, and jumping. The latencies until these reactions occur are recorded and the test is stopped after jumping occurs or at the latest after 20 s. This test can also be performed at 4°C (cold plate) or in a plate preference set-up where the mouse gets to choose between two plates of different temperatures.

Inhibitory (passive) avoidance

In this fear learning task, mice are first placed in a brightly illuminated compartment and then given the choice to enter a dark compartment. Naturally, mice prefer the dark compartment and will go there after a short latency. However, after entering the dark compartment mice receive an electric foot shock and learn to associate this aversive event with the dark compartment. Successful learning will lead to longer latencies to enter the dark compartment during subsequent trials.

IntelliCage

The IntelliCage allows assessment of the cognitive functions and decision-making behavior in operant conditioning tasks, as well as depression-like and emotional behavior and sucrose preference in an automated experimenter-independent manner. Mice are automatically identified by the system via subcutaneously implanted RFID chips. The access to water (or e.g., sucrose solution) can be computer-controlled by a gate mechanism and can be coupled with light stimuli and aversive air puffs. In this way, a broad spectrum of conditioning paradigms can be programmed and automatically performed on socially-grouped mice (up to 16 mice simultaneously) in one large IntelliCage.

Latency to find buried food test

This test evaluates the olfactory abilities of a mouse by placing a food-deprived mouse into a large new cage with a food pellet buried underneath fresh pellet material. The amount of time required for the mouse to find the food pellet is an indicator of their ability to smell.

Light-dark transition test

The light-dark transition test is a standardized test for anxiety-like behavior. Mice are placed in a dark box with a door that gives access to a brightly lit arena. The mice are video tracked for 10–20 min and the latency until the mouse enters the bright arena for the first time, the duration and path in the bright arena, and the number of visits to the bright arena are evaluated. A reduced number of visits to the bright arena indicates increased anxiety levels.

Marble burying test

To check whether mice are stuck in a repetitive behavior, we can perform a marble burying test where mice are placed inside a large cage with 20 marbles evenly distributed on the surface of the bedding material. We evaluate how many marbles the mice bury within 30 min. Marble burying is a natural behavior of mice, but mice that tend towards repetitive behavior (as e.g., in models for autism) bury more marbles in a given time than controls.

Morris water maze

In this spatial learning test, mice are trained over multiple sessions to swim and find a submerged platform in opaque water, each time releasing the mouse from a different starting position. The mouse uses visual cues in the room to orient itself. When the platform is removed, we check in which quadrant of the pool the mouse looks for the platform. This test can be repeated after some days to evaluate long-term memory. Memory extinction and behavioral flexibility can be checked by moving the platform to a new location and measuring the latency to find the new location.

TSE-MotoRater System

The MotoRater system allows tracking of points of interest of the mouse body in 2D-space as the mouse is walking across a plexiglass runway or a ladder, or as it is wading through knee-deep water or swimming. Using the SimiMotion software to track each point (e.g., the knee, hip, ankle, shoulder, elbow, nose, tailbase, or tip of the tail) allows for gold-standard gait analysis, measurement of anatomic distances and angles across the gait cycle, and creation of stick-figure images and videos for presentation purposes. The movement is recorded from both body sides of the mouse, as well as from below.

Nest-building test

For the nest building test, mice are single-housed for one night in a cage containing a standardized cotton nestlet that can be used for nest building. The next morning the quality of the nest and the amount of nestlet material used is evaluated. This is an indication of the fine motor skills of the mouse, but also the emotional state, since stressed mice or mice with a depression-like phenotype tend to show reduced nest-building activity. This test is also influenced by the body temperature of the mouse, as mice that feel cold will build better nests.

Nestlet shredding test

This test evaluates repetitive behavior by placing mice inside a big cage with a standardized nestlet and evaluating the amount of time spent shredding the nestlet and the amount of nestlet used during a 30-min test session. Repetitive behavior is one of the hallmarks of e.g., autism-spectrum disorders.

Novel object recognition test

In this non-forced, voluntary learning and memory test, mice are first familiarized with two identical small objects inside an arena under non-aversive light settings. Then, one of the two objects is replaced by a novel object of similar size. Object recognition and memory is evaluated by measuring the latency to explore the novel object versus the familiar one as well as total time spent investigating the objects and number of approaches to the novel versus familiar object.

Olfactory habituation/dishabituation test

Odors are presented on a cotton swab to a mouse in a novel standard cage and the latency to sniff and the time spent sniffing at the swab are scored and recorded using Noldus Observer software. The odors can be defined concentrations of non-social odors (e.g., vanilla extract, peanut butter solution, 2-methyl-butyric acid) or social odors collected by swiping the cotton stick through the soiled bedding of an unfamiliar mouse cage. When the same odors are presented repeatedly, we can observe habituation and loss of interest for the odorant, which is enhanced again when changing to a different odor.

Open field test

The open field test is a standardized test for the examination of the following parameters: exploratory drive, response to novelty, emotionality, locomotor function, and spontaneous activity. The mouse is placed in an open arena (50 × 50 cm) and observed for 30 min via video tracking, so that the path of the mouse is tracked and the rearing is recorded. A reduction of exploration of the center zone can indicate increased anxiety levels.

TSE-PhenoMaster-System

Up to 12 mice can be single-housed in metabolic cages that record the following parameters during the day and night cycle: amount of food consumed, amount of liquid consumed, amount of oxygen consumed, amount of CO2 produced, locomotion activity in the cage plus rearing activity, and running wheel measurements. Optionally, the access to food/liquid can be controlled (allowing e.g., for paired feeding experiments). The PhenoMaster cages are located inside a climate chamber allowing for user-defined light-cycle, temperature, and humidity.

The metabolic measurements in the PhenoMaster can be combined with simultaneous telemetry recordings of core body temperature, ECG, and blood pressure.

RotaRod test

In this test for motor coordination, balance, muscle strength, and fitness, up to five mice can be placed simultaneously on a rotating rod and the latency until the mice fall from the rod at either constant or accelerating speed can be detected. Motor learning can be observed when this test is repeated over several testing sessions and several days.

SHIRPA screening

SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment

This is a screening procedure that is recommended for new types of mouse lines or to assess the health of individual mice. We check for several factors in a quick screening procedure, such as appearance of the coat and whiskers, absence/presence of lacrimation, tremor, pinna and cornea reflex, position righting reflex, startle response, locomotion in a quick open field procedure, appearance of skin and tail, gait abnormalities, limb clasping, vocalization, and many more.

Three-chamber social interaction test

The test mouse is placed in the middle chamber. One of the outer chambers contains an empty restrainer. The other outer chamber contains a restrainer containing an unfamiliar mouse. We record the time the test mouse spends in each chamber and additional parameters of social interaction. Mice with normal social behavior show more interest in the unfamiliar mouse than the empty restrainer, while mice with disturbed sociability show no preference. Then a second unfamiliar mouse is placed in the so far empty restrainer. Now, mice with healthy social memory should prefer contact with the new unfamiliar mouse.

Social interaction in a novel environment

In this test for sociability, we use the TSE open field equipment and place the test mouse together with an unfamiliar mouse (from another cage) into the same arena and evaluate various parameters of social interaction, such as the number of approaches initiated by each mouse, the latency until the first approach, the time spent in direct contact, and many more.

Sucrose preference test

Mice, like humans, naturally tend to prefer sucrose solutions over water. However, this preference can be enhanced or diminished and allows the study of addictive behavior and calorie-seeking behavior on the one hand, and anhedonia and depression-like symptoms on the other hand. We measure how much sucrose versus water the mice consume when given the choice, either in their home cage, or in the PhenoMaster system, so that metabolic measurements can be recorded, and consumption can be monitored during the day and night cycle. This test can also be performed in our IntelliCage equipment on socially grouped mice.

Tail suspension test

The tail suspension test is a widely-used test for depression-like phenotypes. Mice are taped to a rod by their tails and left dangling for 6 min. The latency until the mouse ceases its efforts to get to an upright position is measured. A reduced latency is indicative of a depression-like phenotype.

Telemetry measurements of core body temperature

The core body temperature of the mice can be measured during the day and night cycle as they move freely in their home cages (non-stressed), by intraperitoneally implanting telemetry transmitters (TA-F10) from Data Sciences International. These transmitters record both the core body temperature and the locomotor activity in the home cage in a continuous fashion and transmit these recordings as a radio signal to receiver plates that are located underneath the home cage. These recordings can also be performed inside the PhenoMaster equipment so that metabolic parameters are measured at the same time.

Telemetry measurements of ECG and blood pressure

The ECG and blood pressure of mice can be measured during the day and night cycle as they move freely in their home cages (non-stressed), by implanting telemetry transmitters (HD-X11) from Data Sciences International. These transmitters record both the cardiovascular parameters and the locomotor activity in the home cage in a continuous fashion and transmit these recordings as a radio signal to receiver plates that are located underneath the home cage. These recordings can also be performed inside the PhenoMaster equipment so that metabolic parameters are measured at the same time.

Wire hanging test

In this test for muscle strength, we place the mouse on a wired mesh, carefully turn the mesh around and shake it gently, measuring the time until the mouse lets go and falls onto a soft surface.

Y-maze

In the Y-maze test, mice can explore three equally-sized arms with opaque walls and the sequence of entries into the three arms is observed. Healthy exploratory drive will lead to uniform visits of all arms, while working memory and attention deficits lead to more frequent returns to the same arm or alternations between two of the arms.