Acoustic Startle / PPI

The Acoustic Startle Response (ASR) and Tactile Startle Reflex (TSR) are established tests for evaluating complex brainstem-mediated reflex pathways.

Pre-pulse inhibition (PPI), an established method for testing sensorimotor gating. PPI is abnormal in some human conditions, such as schizophrenia and major depression. ASR and PPI responses are similar in humans and rodents, offering homologous cross species comparability.

Active Avoidance

Active/two-way/shuttle box avoidance tests the ability of animals to learn to respond to a warning signal (light, tone, or both) that signals impending (foot-shock) unless they move to the lighted side when the light switches sides. If the animal crosses to the opposite side during the warning period, they avoid shock; if they wait until the warning period is over, the shock is activated and then they can escape by crossing to the opposite side. Two-way active avoidance is a classically conditioned response. A typical assessment requires 20-25 trials/day for 5 days.

Activity with Pharmacological Challenge

This is done in our automated open-field activity test chambers. The test involves familiarizing the animal with the apparatus, and then assessing changes in activity after the rat or mouse is injected with a drug challenge. We often test animals using indirect dopaminergic agonists, selective dopamine D1 and D2 agonists and antagonists, cholinergic-muscarinic antagonists, and glutamatergic-NMDA receptor antagonists. We also have experience using a variety of other agents for this test.

Cincinnati Water Maze

This is a labyrinthine maze with ten T-shaped cul-de-sacs branching from a central, circuitous channel leading from the start to the goal. The test is conducted in darkness under infrared light to eliminate distal cues, thus testing the animal’s “sense of direction” or ability to navigate using internal, route-based egocentric cues of self-movement (speed and direction).

We record latency to find the goal and errors of commission (entry into blind alleys). Recent studies demonstrate that learning in this maze depends on neostriatal dopamine but not prefrontal cortex dopamine. The test is affected by dopamine in the striatum and nucleus accumbens but not the prefrontal cortex. This test is only suitable for rats.

Conditioned Freezing

The test evaluates amygdala using cued conditioning, and hippocampus using contextual conditioning. The test can be modified, but a common method is to use a four-day procedure. Day-1 the animal is placed in the apparatus and allowed to explore for 10 min. Day-2 the animal can explore for another 6 min and then is exposed to 9 light-tone stimuli for 1s at 30 s intervals with a foot-shock during the last 1 s of each stimulus presentation. Day-3 the animal is put back in the same test chamber and inactivity (freezing) recorded for 6 min for memory of the context in which they were shocked on Day-2. Day-4 they are placed in a different test chamber (different in shapes color, and texture) for 6 min to explore followed by alternating periods of light-tone presentations and no signal intervals. Again, freezing is recorded. Freezing in response to the conditioning cues is mediated by the amygdala.

Elevated Zero Maze

The elevated zero (EZP) maze is used to test anxiety-like behavior. The maze is circular with two open and two closed quadrants (closed quadrants have high, black acrylic walls). Animals are tested for five minutes; the time spent in the open areas provides an index of anxiety. Behavior is video tracked and computer scored using AnyMaze software.

Forced Swim Test

The Forced Swim Test (FST) is a well-known test for assessing stress responses including HPA axis markers such as ACTH, corticosterone, and others. It also serves as a test of adaptive behavior (the Persolt test of swimming despair). The Persolt test evaluates swimming immobility after a training session in which the animal is placed in a cylinder half-filled with water and scored for how vigorously they try to escape.

Light-Dark Box

This test evaluates the time it takes the animal to move from the lighted side to the dark side in a divided test chamber, and measures anxiety-related behavior. The test uses photocell-equipped locomotor activity test chambers with the light-dark compartment inserted into the open-field.

Locomotor Activity

This automated open-field test is conducted for various lengths of time, but typically 60 minutes. The system records horizontal activity, distance traveled (cm), central versus peripheral movement or time, repetitive movements, and time the animal spends exploring different zones. The test measures exploration of a novel environment, habituation rate (as the environment becomes familiar with its surroundings) and baseline ambulation. Locomotor activity test assesses basal ganglia (caudate putamen) function.

Morris Water Maze

This is the most widely used test for measuring spatial navigation and reference memory. The animal is placed in an open, circular pool of room temperature water with a submerged platform. Over a series of trials, the animal learns to use distal cues located outside the maze to spatially navigate progressively more efficient paths to the platform despite being placed in the maze at a different position around the perimeter on every trial. Mice require a one-day training session to swim to a visible platform, then 5 days of learning to navigate to a hidden platform. Rats do not require the training day. A day after hidden platform trials, animals are given a probe trial to assess reference memory with no platform and their search pattern is mapped. This procedure is repeated but with the platform moved to the opposite quadrant of the pool. This reversal phase assesses cognitive flexibility. The day after the last set of reversal trials, animals are given an additional probe trial. Rats are given yet a third phase, the same as the first two, but with the platform in one of the other quadrants. Last, animals receive 2 days of proximal cue learning in which the platform is made visible, curtains are closed around the pool to block sight of distal cues, and the platform and start positions are randomized. This is a control procedure to ensure that rats can see and swim comparably among the groups. Tests are video tracked using AnyMaze. The test relies on an intact hippocampus and entorhinal cortex.

Novel Object Recognition

The test uses the animal’s reaction to a novel object within the context of familiar objects as a test of recognition memory. First, the animal is familiarized with two or four objects. After a predetermined interval it is placed back in the test chamber with identical copies of the original objects and one new object. Time spent exploring the novel object in preference to the familiar objects reflects memory of what has changed. It is mediated by the dorsal hippocampus.

Object Burying

This test places an array of foreign objects (marbles) on top of a pre-measured amount of bedding and the animal placed in the cage for 30 minutes. Rodents reliably push bedding over the marbles.  The the number of marbles buried is recorded and reflects anxiety and compulsive tendencies.

Passive/Inhibitory Avoidance

Passive avoidance uses the Gemini apparatus. For this test, one side is lighted and the other side dark with a door in between. The animal is placed on the lighted side for 3 min. Typically, animals cross to the dark side. When they do, the door is closed, and a brief food-shock given and then they are removed for some interval (ranging from 1-24 h). After the delay interval they are placed back on the lighted side and the divider door is opened. They are given 3 min to crossover again or remain on the lighted side as a test of working memory. Animals can be assessed by the one-trial method or given daily trials until they reach a predetermined criterion for staying on the lighted side.

Rotor-Rod

A San Diego Instruments automated rotating rod (or Roto-Rod). The apparatus consists of a rotating drum, the speed of which is computer controlled. Animals are taught to walk on the surface at a slow speed. They are then tested in a series of trials in which the speed is increased at regular intervals until they cannot keep up and fall to a padded surface. How long and at what speed they remain on the drum is a measure of motor coordination.

Schedule Controlled Operant Behavior

Animals are trained to perform complex tasks by responding to different visual cues to obtain rewards. Procedures include delayed matching to sample, DRL (differential reinforcement of low rates) and others including 5-choice serial reaction time procedures that assess attention, impulsivity, executive function, and other abilities.

Social Preference

This test places each animal in a three-compartment apparatus with connections between them. The two end compartments contain either a small cage with a conspecific in it or an inanimate object. After familiarization with the apparatus with no objects, the test animal is placed in the apparatus with a “stranger” mouse or rat in one of the small cages in one of the end compartments and the other small cage containing an inanimate object. The animal is placed in the middle compartment and allowed to explore. The time the animal spends with Stranger-1 versus the opposite end compartment reflects social preference. Later, the test subject is placed back in the center but this time a second new animal, Stranger-2, is placed in the small cage that held the inanimate object before. How the test animal divides its time between Stranger-1 and Stranger-2 is recorded and hypothesized to reflect sociability and social novelty and used as a marker for abnormal social interactions in autism spectrum disorder (ASD) models. Most of the data available come from studies in which candidate genes associated with ASD have been genetically altered in a mouse model.

Tail Suspension

The test measures immobility in a mouse (the test is not suitable for rats) by suspending it by the tail. Once the mouse determines that escape is impossible, it stops struggling. This test can show the effect of antidepressants and sedatives and is used to provide converging evidence for results from Forced Swim Testing (FST). Animals are suspended for no more than six minutes, and the time spent immobile is recorded. This test is available only for use with mice.