Rodent Behavioral Phenotyping Core

The Barnes maze consists of an elevated circular platform with twenty holes, where a nesting chamber is placed underneath a single hole, and the space around the maze contains environmental cues. This test assesses spatial learning and memory, since animal subjects learn which hole has the nesting chamber beneath based on nearby environmental cues.

Barnes maze is an alternative to the Morris Water Maze that does not require swimming, and is recommended for investigators who are looking to evaluate spatial learning and memory with animals that exhibit motor and/or neurological (e.g., seizure prone) deficits.

The RBPC currently has three Barnes mazes from Maze Engineers, which are all made of opaque blue acrylic. A Barnes maze for mice is available at both the Downtown and South campus locations, and the Downtown campus also has a Barnes maze for rats.

Conditioned place preference (CPP) or conditioned place aversion (CPA) is a multi-day classical conditioning experiment, where animals learn to associate an environment with a rewarding or aversive stimulus. A chamber with two compartments is used, where each compartment has different walls and flooring. One compartment is paired with a drug treatment, and the other compartment is paired with a vehicle treatment.

After multiple sessions where the animal is treated and confined to one compartment, a final test is performed where the animal receives no treatment and is allowed to traverse both compartments. During the test, an animal may spend more time on the drug-paired side if the drug has rewarding effects, or may spend more time on the vehicle-paired side if the drug has aversive effects.

The RBPC currently has six automated CPP/CPA chambers from Omnitech Electronics. Four CPP/CPA chambers for mice are available Downtown, and two mouse or two rat CPP/CPA chambers are available at the South campus location.

Electronic von Frey is a means of measuring sensitivity to mechanical stimulation, where a probe is typically applied to a hindpaw, and the amount of force needed to elicit a behavioral response (e.g., lifting or licking the hindpaw) is recorded. These measurements may be used to evaluate mechanical sensation, and to assess candidate analgesics using pain models.

The RBPC currently has two MouseMet and two RatMet instruments from Topcat Metrology. A MouseMet and a RatMet device are available at both Downtown and South campus locations.

Elevated zero maze and elevated plus maze are used to evaluate anxiety-like behavior, and consist of a raised platform – shaped like a zero or a plus sign – with areas with walls (enclosed) and without walls (open). Since rodents prefer enclosed spaces, a drug that increases the amount of time rodents spend in the open areas is said to be anxiolytic.

The RBPC currently has two elevated zero mazes with black walls and a gray floor from Maze Engineers, and one elevated plus maze with dark gray walls and a dark gray floor from Stoelting. An elevated zero maze for mice is available at both the Downtown and South campus locations, and the Downtown campus location has an elevated plus maze for rats.

Fiber photometry is a technique that enables neural activity to be captured in freely moving animals in real-time. These setups include two- or three-color light sources, typically an isosbestic control with a red or green wavelength, or an isosbestic control with both red and green wavelengths. These light sources excite fluorescent sensors (red: RCaMP, tdTomato; green: GCaMP, dLight) that are genetically or virally expressed in specific neuronal populations, and these sensors then emit their own light in response, which allows neuronal response patterns to be recorded across time.

The RBPC currently has four fiber photometry setups from Tucker Davis Technologies. Two fiber photometry setups are available at both the Downtown and South campus locations. Each setup can be equipped with one or two cameras to simultaneously record behavior of the animal. Each campus also has the following behavioral modules available for use alongside fiber photometry: a high voltage I/O interface for operant experiments (iH16 – compatible with Med Associates, Lafayette, and Coulbourn), an infrared driver interface (iR10) for locomotor activity experiments involving beam break technology, a multi-function interface with a speaker (iM9) for auditory behavioral experiments, and an aversive stimulator (iS9) for fear/startle experiments.

Hot/cold plate is a means of measuring sensitivity to thermal stimulation, where an animal is placed on a heated/cooled platform, and the amount of time it takes to elicit a behavioral response (e.g., lifting or licking the hindpaw) is recorded. These measurements may be used to evaluate thermal sensation, and to assess candidate analgesics using pain models.

The RBPC currently has two hot/cold plates from IITC. A hot/cold plate is available at both Downtown and South campus locations.

A light/dark box is divided into an open light and an enclosed dark compartment, where an animal can cross to the other side through a doorway. This equipment is used to assess anxiety-like behavior, and capitalizes on rodents innate preference for dark spaces and their natural tendency to explore novel environments. An anxiolytic drug should increase the amount of time an animal spends in the light compartment.

The light/dark box test should generally be performed after an open field test has been conducted to determine whether increased or decreased locomotor activity could be a potential confound.

The RBPC currently has four light/dark boxes for mice from Stoelting. These four light/dark boxes are available at the Downtown campus.

The Morris Water Maze consists of a circular pool with a submerged platform, where the space around the maze contains environmental cues. This test assesses spatial learning and memory, since animals learn where the submerged platform is located based on nearby environmental cues.

Barnes maze is an alternative to the Morris Water Maze that does not require swimming, and is recommended for investigators who are looking to evaluate spatial learning and memory with animals that exhibit motor and/or neurological (e.g., seizure prone) deficits.

The RBPC currently has three Morris Water Mazes from Maze Engineers, which are all made of opaque blue acrylic. A water maze for mice is available at both the Downtown and South campus locations, and the Downtown campus also has a water maze for rats.

The open field test is commonly used to evaluate locomotor activity, anxiety and exploratory behavior. Typically rodents are tested in an open field only once, since they are less likely to explore when the environment is no longer novel. However, repeated tests in an open field can be performed to evaluate whether locomotor sensitization develops to a drug after repeated treatment (may predict abuse liability), or if a therapeutic intervention has an effect on locomotor sensitization induced by drugs of abuse.

Rodents often stay close to the walls of an open field (thigmotaxis), so a treatment that increases the amount of time an animal spends in the center may be anxiolytic. That said, since rodents have a natural tendency to explore, open field is generally performed alongside other behavioral tests such as elevated zero maze or light/dark box to determine whether a treatment reliably demonstrates anti-anxiety effects across tests.

Open field can be an important test to perform to establish that the effects of a potential therapeutic are specific and not due to generalized behavioral suppression. For example, if a potential therapeutic has analgesic effects in electronic von Frey or hot/cold plate tests, performing an open field test with a group of naive, pain-free animals is recommended to rule out the possibility that the drug is actually sedative. 

The RBPC currently has twenty four open field chambers from Maze Engineers, which have opaque black walls and a clear floor. These chambers can be placed on either a black or white surface to maximize contrast between the animal and the floor. The Downtown campus location has eight open fields for mice and four open fields for rats, whereas the South campus location has four open fields for mice and eight open fields for rats.

Rotarod is used to assess motor coordination and balance, and involves placing a rodent onto a rotating rod and measuring how long an animal is able to stay on the rod without falling. This test can be used to evaluate motor function as a phenotype, to determine whether a drug treatment causes sedation or motor impairment, or to determine whether a drug treatment improves performance in rodent models of neurological disease.

The RBPC currently has a rotarod that can be used for either mice or rats. A Rotamex (Columbus Instruments) is available at the Downtown campus location.

The three-chamber sociability test can be used to evaluate social behavior, whether it be to investigate social preference between unfamiliar and familiar conspecifics (social recognition or memory), or to determine whether a subject spends more time with an object compared to a conspecific (deficit in social behavior). Sociability tests are particularly useful for investigating potential treatments using rodent models of neurodevelopmental (e.g., autism spectrum disorder) and psychiatric disorders (e.g., schizophrenia).

The RBPC currently has four automated sociability chambers from Omnitech Electronics. A sociability chamber for mice and a sociability chamber for rats is available at both the Downtown and South campus locations.

Y-maze is used to assess short-term memory in rodents, since rodents tend to explore different stimuli when they are placed in an apparatus for consecutive trials (spotaneous alternation). That is, when rodents are placed in a Y-shaped maze, they choose to explore one arm of the apparatus, and will likely explore the other arm in a subsequent trial. If a drug treatment (such as a benzodiazepine) decreases the frequency of alternation, it is considered to impair spatial working memory or cause short-term cognitive impairment.

The RBPC currently has two Y-mazes from Maze Engineers, which have opaque black walls and a clear floor. These mazes can be placed on either a black or white surface to maximize contrast between the mouse and the floor. A Y-maze for mice is available at both the Downtown and South campus locations.

Body temperature measurements can provide useful information about estrus phase, circadian rhythm, or hypothermic/hyperthermic effects of drug treatments. These measurements can also be used to investigate interactions between drugs, if at least one drug being tested has established effects on body temperature (e.g., clonidine-induced hypothermia).

The RBPC currently has one body temperature meter from Physitemp Instruments, which includes a mouse- and a rat-sized rectal probe. This instrument is portable and is therefore available to reserve at either the Downtown or South campus location.

A Feeding Experimentation Device (FED) is a stand-alone pellet dispensing device for training mice on operant tasks within their homecage. These devices are battery-powered and do not need to be connected to a computer to operate. Originally developed by Kravitz Lab at Washington University, these devices are open-source and can be programmed to achieve new functionality. Fixed-ratio (FR1, FR3, FR5) and progressive-ratio (PR) training protocols are pre-installed.

The RBPC currently has four FED3.2 instruments from open ephys. These are portable and are therefore available to reserve at either the Downtown or South campus location.

The use of a grip strength meter enables an investigator to measure the muscle strength of a rodent's front and hindlimbs. Testing grip strength is particularly useful for evaluating motor function in aging animals and animal models of neurodegenerative disease. Grip strength can also be used to assess potential analgesics, or to determine whether a drug may have neurotoxic effects.

The RBPC currently has one grip strength meter from Bioseb, which includes a mouse and a rat T-shaped bar. This instrument is portable and is therefore available to reserve at either the Downtown or South campus location.

The forced swim test is used to assess depression-like behavior, or more specifically despair, by determining how long it takes for an animal to give up when presented with an inescapable stressor. This test has predictive validity for monoaminergic antidepressants, but its construct validity is under debate. Animals are placed into a tube or a jar of water and the latency to immobility is recorded. Video recordings can be analyzed using ANY-maze software.

Novel object recognition (NOR) is used to assess learning and short- or long-term memory. This test is predicated on the tendency of rodents to explore novel environments and objects. NOR is conducted over two or more sessions using a standard open field chamber. Animals may first undergo a habituation session or sessions in an empty open field chamber, and are later placed in an open field with two identical objects in opposite quadrants of the arena for a familiarization session. Later the animal undergoes a recorded test session, where it is placed into an open field chamber with one familiar and one novel object in those same quadrants. The amount of time the animal explores the novel object is used to determine the novelty preference or discrimination index, and is interpreted as recognition memory.

NOR experiments have to be performed carefully and with more consideration than other memory tests such as Y-maze. A group of naive animals should be tested for any innate preference if the objects being used have not previously been validated with that particular strain of mouse or rat. Pairs of objects that are about equally preferred by the strain should be used for NOR testing. During the test session, it is important that the familiar and novel objects be counterbalanced across subjects. Furthermore, sanitation needs to be consistently and routinely performed for the chambers and the objects, since the scent of other animals or unevaporated alcohol can be confounds.

The RBPC currently has twenty four open field chambers from Maze Engineers, which have opaque black walls and a clear floor and can be used for performing NOR. These chambers can be placed on either a black or white surface to maximize contrast between the animal and the floor. The Downtown campus location has eight open fields for mice and four open fields for rats, whereas the South campus location has four open fields for mice and eight open fields for rats.

The sucrose preference test is used to assess depression-like behavior, or more specifically anhedonia, which is a decreased ability to experience pleasure from positive stimuli. For this test, rodents are generally individually housed and are habituated to having two bottles on their homecage. During the testing period, one bottle contains 1-2% sucrose and the other bottle contains plain water. Intake is calculated by measuring the weight of the bottle before and after the test period,  and sucrose preference is calculated using the following formula:

SP = [sweet solution consumed (g) / (sweet solution consumed (g) + plain water consumed (g))] * 100

Values <65% are considered anhedonic. This test can be performed with or without the use of a stress procedure. Most sucrose preference tests have been performed using C57BL/6 mice and Sprague Dawley rats, so these may be useful reference strains to use if sucrose preference is being used for phenotyping and not as a test for potential anti-depressants.

The sucrose splash test is used to assess depression-like behavior, or more specifically apathy, by evaluating whether an animal has decreased motivation to groom itself. For this test, the back of a rodent is sprayed with 10% sucrose solution, and grooming behavior is recorded for a short period of time. These video recordings can be analyzed using ANY-maze software or deep learning pipelines.

ANY-maze is a commercial video tracking software for setting up, performing, and analyzing behavioral experiments. This software has a user-friendly interface, allowing users to easily build protocols to automate the testing process. 

The RBPC currently has two full licenses for ANY-maze from Stoelting. End-users can install ANY-maze software on a personal or laboratory Windows PC for performing video analysis. However, setting up and running a designed protocol can only be done at a computer station with a full license.

The RBPC has six desktop computers equipped with ANY-maze software for video collection and analysis. These desktops have the following configuration:

• CPU: Intel Core Ultra 7 265 (20 core)
• Memory: 32 GB DDR5
• Storage: 2 TB NVMe SSD

The RBPC has three high-performance desktop computers with dedicated graphics processing units (GPUs), which are primarily intended for use with open-source deep learning software. One desktop is available for end-users to reserve for performing video analysis at both the Downtown and South campus locations. These desktops have the following configuration:

• CPU: Intel Core i9-14900KF (24 core)
• GPU: GeForce RTX 5070 Ti
• Memory: 32 GB DDR5
• Storage: 2 TB NVMe SSD

It is recommended that end-users understand how to activate a conda environment in order to open a dedicated graphical user interface (GUI) for navigating DeepLabCut, LabGym, SLEAP, etc.

We are able to customize a comprehensive phenotyping package for end-users who need a mouse line evaluated in a behavioral test battery. These experiments are performed by core facility personnel, making this service ideal for investigators who are not looking to maintain a mouse colony for a long-term project.

Our recommended test battery covers the following behavioral domains:

• Anxiety-Like: Elevated Zero Maze, Open Field, Light/Dark Box
• Learning & Memory: Y-Maze, Social Preference, Morris Water Maze*
• Motor Function: Open Field, Grip Strength, Rotarod
• Sensation & Perception: Hot/Cold Plate, Sucrose Preference

*Barnes Maze will be performed instead of Morris Water Maze if mice exhibit signs of motor or neurological deficits.

Please contact the Facility Manager for more information and we can provide an estimate.

Metabolic cages provide 24/7 readouts of locomotor activity, feeding/drinking behavior, and voluntary exercise, and can supply an investigator with a wealth of information about rodent behavior. These cages can run continuously for up to ten days without experimenter intervention, allowing for behavioral measures to be recorded in a uniquely low-stress environment. Capture circadian patterns of behavior, liquid intake in an intermittent two-bottle choice paradigm, and more in a precisely temperature- and humidity-controlled environment.

PhenoMaster metabolic cages from TSE Systems are located in Park Hall on North Campus. Designated personnel can run an experiment for up to ten consecutive days on an investigator's behalf. The system includes eight rat cages, enabling up to eight rats to be tested simultaneously. Alternatively, one mouse cage is available. Each cage can be equipped with a running wheel, water and food hopper(s), and a glass tube that doubles as a means to measure a subject's body weight. Access to water and food hopper(s) can be continuous or scheduled, and water/food can be restricted to a specified amount. Please contact the Facility Director for more information.